J . Phys. Chem. 1984, 88, 1077-1083
1077
Analysis of the Pulsed-Laser-Induced Fluorescence Spectrum of the Chlorophyll a Dimer in Solution A. C. de Wilton, L. V. Haley, and J. A. Koningstein* The Ottawa-Carleton Institute for Research and Graduate Studies in Chemistry, Department of Chemistry, Carleton University, Ottawa, Canada K l S 586 (Received: May 10, 1983)
Fluorescence spectra for chlorophyll a (Chl a ) in solution in benzene, carbon tetrachloride, and hexane are reported. In addition to the strong monomeric fluorescence at -670 nm, red emission bands are observed which are assigned to the Chi a dimer and larger oligomers. Selective excitation of fluorescence of dimeric and monomeric species of Chl a is achieved with a pulsed tunable dye laser operating at wavelengths in resonance with either the blue (Soret) or red absorption bands of the respective species of Chl a. The fluorescence spectrum of the dimer is characterized by a weak band at -625 nm with a radiative lifetime T N 5 ns, and a stronger band at -685 nm (T < 5 ns). Wavelength-dependent photoquenching of the fluorescence of both the Chl a dimer and monomer is observed when the sample is irradiated with the focused laser beam. Together with a difference in the excitation spectra of the two dimeric fluorescence bands, these results suggest that there are specific channels for energy relaxation resulting in fluorescence from two levels of the Chl a dimer in solution. Several alternative assignments for the origin of the dimer fluorescence bands arise from consideration of both absorption and fluorescence data for the Chl a dimer and their comparison with the predictions of simple theoretical models for the absorption spectrum of the Chl a dimer
I. Introduction The application of tunable pulsed lasers to the study of chlorophyll a (Chl a ) in vitro is a recent innovation in the history of chlorophyll research.'-4 Over the last three decades, the coordination properties and aggregation behavior of Chl a in solution have been established by a number of techniques including vapor-phase o ~ m o m e t r y ,nuclear ~ magnetic resonance: infrared absorption,6 Raman scattering,' and visible absorption6S8-" and fluorescence ~ t u d i e s . ~ J ' - Investigation ~~ of the spectroscopic properties of dimeric, oligomeric, and aggregated Chl a in vitro is now directed toward an understanding of the nature of the interaction of two or more Chl a molecules and the effects of aggregation on the electronic states of Chl u in relation to the characteristics of Chl a in vitro.8*12 There have been a number of recent investigations of laserinduced fluorescence from monomeric Chl a4,I4-l6and aggregated species of Chl a involving waterI2 or another nucleophilic species? (1) J. Baugher, J. C. Hindman, and J. J. Katz, Chem. Phys. Lett., 63, 159-62 (1979). (2) M. J. Yuen, L. L. Shipman, J. J. Katz, and J. C. Hindman, Photochem. Photobiol., 36, 21 1-22 (1982). (3) M. Asano and J. A. Koningstein, Chem. Phys., 57, 1-10 (1981). (4) A. de Wilton, L. V. Haley, and J. A. Koningstein, Can. J. Chem., 60, 2198-206 (1982). (51 K. Ballschmiter, K. Truesdell. and J. J. Katz, Eiochem. Eiophvs. . . Acta, 184, 604-13 (1969). (6) J. J. Katz, L. L. Shipman, T. M. Cotton, and T. R. Janson, "The Porphyrins", Vol. 5C, D. Dolphin, Ed., Academic Press, New York, 1978, pp 401-58. (7) M. Lutz, J. Raman Spectrosc., 2, 497-516 (1974). (8) L. L. Shipman, T. M. Cotton, J. R. Norris, and J. J. Katz, J . Am. Chem. SOC.,98, 8222-30 (1976). (9) K. Sauer, J. R. Lindsay-Smith, and A. J. Schultz, J. Am. Chem. SOC., 88, 2681-8 (1966). (10) T. M. Cotton, P. A. Loach, J. J. Katz, and K. Ballschmiter, Photochem. Photobiol.. 27. 6735-749 (19781. (11) V. J. Koester, J. S. Polles,'J. G.'Koren, L. Galloway, R. A. Andrews, and F. K. Fong, J. Lumin., 12/13, 781-6 (1976). (12) F. K.Fong, M. Kusunoki, L. Galloway, T. G. Matthews, F. E. Lytle, A. J. Hoff, and F. A. Brinkman, J . Am. Chem. Soc., 104,2759-767 (1982). (13) R. P. H. Kooyman, T. J. Schaafsma, G. Jansen, R. H. Clarke, D. R. Hobart, and W. R. Leenstra, Chem. Phys. Lett., 68, 65-9 (1979). (14) M. J. Yuen, L. L. Shipman, J. J. Katz, and J. C. Hindman, Photochem. Phorobiol., 32, 281-96 (1980). (15) J. C. Hindman, R. Kugel, A. P. Svirmickas, and J. J. Katz, Chem. Phys. Lett., 53, 197-200 (1978). (16) N. E. Geacintov, D. Husiak, T. Kolubayev, J. Breton, A. J. Campillo, S. L. Shapiro, K. R. Winn, and P. K. Woodbridge, Chem. Phys. Lett., 66, 154-8 (1979).
0022-3654/84/2088-1077$01.50/0
Fluorescence from several covalently linked chlorophyll dimers has also been reported.I7J8 However, the Chl a dimer, (Chl a)2, and the oligomer, (Chl a)2n,which exist in dry nonpolar solvents, J~~~~ are generally considered to be essentially n o n f l ~ o r e s c e n t . ~This belief is based on the observation by Livingston et aL2' that the fluorescence yield from a solution of Chl a in a dry hydrocarbon was less than 10% of that of a solution in which the monomeric fluorescence was activated by addition of water or other nucleophilic species. At that time, the aggregation of Chl a in nonpolar solvents had not been established, and Livingstonzl dismissed the hypothesis of the presence of a nonfluorescent dimer in favor of a "nonactivated" monomeric species of Chl a in order to explain the weak fluorescence of the dry solutions. More recently, however, it has been inferred2~'9~20 from the observations of Livingston*' that the Chl a dimer is nonfluorescent, and, despite the introduction of laser sources and superior detection systems in fluorescence spectroscopy,the investigation of weak fluorescence from the Chl a dimer in solution appears to have been neglected. We have employed a pulsed (3.2 ns) tunable dye laser and a detection system which is capable of detecting Raman scattered light to obtain fluorescence spectra from solutions of Chl a in benzene, carbon tetrachloride, and hedane. The fluorescence spectra presented in this work demonstrate that the Chl a dimer present in these solutions is indeed fluorescent. Two fluorescence bands which are assigned to the Chl a dimer may be observed by selective excitation of the Chl a solutions at a wavelength in resonance with either the blue or the red absorption band of the Chl a dimer in solution. Comparison of the absorption spectra of monomeric and dimeric Chl a in solution shows that the main red absorption band of the Chl a dimer is broadened and red shifted with respect to that of the monomer.6 The dimeric absorption band has previously been interpreted to be due to two exciton transitions of the dimer which correspond to the Qytransition of the Chl a monomer, and which are split by -300 cm-1.899 Our analysis of the absorption spectra of Chl a in dry benzene, carbon tetrachloride, and hexane, together with the fluorescence data for the Chl a dimer and some theoretical considerations, leads to alternative assignments. (17) J. C. Hindman, R. Kugel, M. R. Wasielewski, and J. J. Katz, Proc. Natl. Acad. Sci. U.S.A. 75, 2076-9 (1978). (18) R. R. Bucks and S. G. Boxer, J . Am. Chem. Soc., 104,340-3 (1982). (19) J. Goedheer, Annu. Reu. Plant Physiol., 23, 87-112 (1972). (20) R. Livingston, Q.Rev. Chem. Soc., 14, 174-99 (1960). (21) R. Livingston, W. F. Watson, and J. McArdle, J. Am. Chem. Soc., 71, 1542-50 (1949).
0 1984 American Chemical Society
1078 The Journal of Physical Chemistry, Vol. 88, No. 6,1984
de Wilton et al.
Analysis of the absorption spectra of Chl a in dry benzene, carbon tetrachloride, and hexane is discussed in section IIIA. Results of selective excitation of fluorescence of monomeric and dimeric Chl a and time-resolved fluorescence studies to obtain the fluorescence lifetimes of the dimer emission bands are presented in section IIIB. A difference in the excitation profiles of the two fluorescence bands of the dimer, in addition to photoquenching of the dimer fluorescence observed when the solutions of Chl a are irradiated with high photon flux densities, indicates that there are specific energy relaxation pathways resulting in fluorescence from two levels of the Chl a dimer (section IIIC).
XI. Experimental Section A . Sample Preparation. Chl a was extracted from spinach by the method of Omata and Murata22and purified by chromatography on Sepharose CL-6B. The purified Chl a was dried by codistillation with dried benzene or carbon tetrachloride. Benzene and carbon tetrachloride (Baker Photrex) and hexane (Caledon Labs, HPLC grade) were dried by passing through a column of neutral activated alumina (Woelm, activity 1). Sample preparation was carried out in a dry nitrogen-purged glovebox to exclude both water and oxygen from the solutions. The fluorescbnce cells were sealed with vacuum-tight stopcocks. The Chl a concentration of the stock solutions was determined spectrophotometricallyafter evaporation of the solvent and solution of the Chl a in a known volume of diethyl ether (Mallinckrodt, anhydrous). The molar absorptivity of Chl a in diethyl ether was taken from Seely and J e n ~ e n(e660 ~ ~= 8.51 X lo4 L mol-' cm-'). The Chl a concentrations given are therefore the total Chl a concentration measured as monomer. The sample solutions were M stock solution. prepared by dilution of a The effectiveness of solvent drying with activated alumina24 was checked by monitoring the near-infrared absorption of H 2 0 at 19600 8, (Cary 14 spectrophotometer). The water content of the solvents after drying was estimated to be less than M. B. Visible Absorption Spectra. Visible absorption spectra of solutions of Chl a in dry hexane, benzene, and carbon tetrachloride were recorded on a Varian DMS 90 ultraviolet/visible absorption spectrophotometer. Spectra were recorded for Chl a solutions of concentrations in the range 10-3-10-6 M. An analysis was made of absorption difference spectra for solutions of different concentrations of Chl a in cells of path lengths inversely proportional to the Chl a concentrations of the solutions. The absorption spectrum of monomeric Chl a in dry hexane was obtained by titration of the dry Chl a solution with pyridine to disaggregate the Chl a dimer to the monomeric chlorophyll a pyridinate. C. Fluorescence Spectra. A detailed description of the pulsed-laser Raman and fluorescence spectrophotometer has been given in an earlier publication.25 This system is capable of detecting Raman signals from weakly scattering liquids and solids. A discussion of the use of photomultiplier tubes as detectors in time-resolved pulsed-laser-induced fluorescence spectroscopy has also been pre~ented.~ It is emphasized that these detectors have a limited dynamic range when operated in the pulsed mode; at higher light levels saturation effects may cause nonlinear response in the intensity of the signal as a function of time, leading to erroneous interpretation of fluorescence decays. The fluorescence spectra were excited with 3.2-11slight pulses, at a repetition rate of 25 Hz, produced by a tunable dye laser which was pumped by a N2laser (Lambda Physik Models FL2000 and M2000, respectively). A PTR tunable grating filter was used to eliminate superradiance from the dye laser radiation. The average laser power at 430 mm was -0.5 mW. The unfocuped laser beam (beam diameter 2 mm) was incident on the sample solution in a sealed quartz cell ( 10 mm X 10 mm) . Fluorescence
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(22) T. Omata and N. Murata, Photochem. Photobiol., 31, 183-5 (19fjO). (23) G. R. Seely and R. G. Jensen, Spectrochim. Acta, 21, 1835-45 11965). \ -
(24) D. R. Burfield, Kim-Her Lee, and R. H. Smithers, J . Org. Chem., 42, 3060-5 (1977). ( 2 5 ) D. Nicollin, P. Bertels, and J. A. Koningstein, Can. J . Chem., 58, 1334-43 (1980).
I t I
I
\-c
Figure 1. (A) Absorption spectrum of M solution of Chi a in dry hexane (2-mm path length cell); (B) calculated absorption spectrum of the Chl a dimer in dry hexane; (C) calculated absorption spectrum of the Chl a monomer in dry hexane. It is estimated that 19% of the Chi a in M solution (spectrum A) is present as the monomer. a
was collected at 90" to the laser beam and focused into the entrance slit of a Jarrell-Ash 1-m monochromator. The detection system included a cooled RCA 7102 photomultiplier tube, fast pulsed preamplifiers (le Croy 101A) with XlOO gain, and a PAR boxcar integrator, Model 162 main frame, equipped with Model 165 gated integrator, which has a 2-ns gate. 111. Experimental Results A. Analysis of Absorption Spectra. It is well documented that in dry nonpolar solvents such as benzene and carbon tetrachloride Chl a exists predominantly as a dimer over a wide range of concentrations:*s-'o whereas the aggregation number of Chl a in hexane is highly concentration dependent.* Both the Soret band and the Q band of the visible absorption spectrum of a solution of dry Chl a in dry benzene or carbon tetrachloride show marked broadening to longer wavelength compared to the absorption spectrum of monomeric Chl a in solution, which is characteristic of the presence of the dimer.6t8-10 Analysis of the absorption spectrum and the fluorescence spectrum (see section IIIB) of a M solution of Chl a in dry hexane shows that the solution has spectral features similar to those of Chl a in dry benzene and carbon tetrachloride solutions (Figure 1A). It is therefore concluded that the majority species of Chl a in the solution in dry hexane at this concentration and at room temperature is also the dimer. Although the Chl a dimer is found to be the majority species in the lo4 M solutions of Chl a in benzene, carbon tetrachloride, and hexane solutions, the dimer always exists in equilibrium with smaller amounts of monomer and possibly larger oligomers. The absorption spectrum of the dimer cannot be obtained directly for solutions of Chl a in the concentration range 10d-104 M for which significant amounts of monomer in the solution contribute to the absorption spectrum. The absorption spectra of the dimer in hexane, benzene, and carbon tetrachloride were therefore calculated from analysis of the difference absorption spectra of solutions of Chl a over a range of concentrations (10-6-10-3 M). In dry nonpolar solvents, any monomeric Chl a present should arise from small amounts of residual water or adventitious nu-
The Journal of Physical Chemistry, Vol. 88, No. 6,1984 1079
Fluorescence Spectrum of Chlorophyll a Dimer
TABLE 1: Visible Absorption Data for Chla Monomer and Dimer in Hexane red absorption band
chlorophyll a pyridinate (measured) Chl a monomer (calcd) Chl a dimer (calcd)
max, nm
fwhm, cm-’
10-4e,ed, L mol-’ cm-’
66 1 659 669
460 460 630
8.5 8.5“ 4.1b
blue absorption max, nm 429 421 432
1O-4~blue, L mol” cm-I 10.7 10.4 5.1
a Based on the assumption that the molar absorptivity of Chl a in hexane is a good approximation for the residual monomer in hexane.
Measured per mole of Chl a monomer as dimer.-
cleophiles which are not removed during purification of the Chl a sample. As a first approximation to the absorption spectrum of the residual monomer in dry hexane, the absorption spectrum of monomeric chlorophyll a pydridinate in hexane was obtained from a solution of Chl a in dry bexane which had been titrated with pyridine to disaggregate the dimer. The absorption spectra of the dry solutions and of the monomeric chlorophyll a pyridinate were digitized. The absorption spectrum of the dimer was then calculated by subtraction of increasing amounts of monomer absorption from that of the dry solution until the bandwidth of the main red band of the dimer absorption spectrum conformed approximately with the bandwidth of the corresponding fluorescence band (see section IIIB). From these calculations it M solution of Chl a in hexane conwas estimated that the tained 19% Chl a as monomer. The relative amounts of monomer and dimer in solutions of different concentrations were calculated from the amount of monomeric absorption and the known total concentration of Chl a in the solutions. The absorption spectrum of the dimer was then obtained over the wavelength range 800-350 nm by recording the difference spectrum between two solutions of different Chl a concentrations in different path length cells such that the amounts (concentration X cell path length) of the monomer in each cell were equal, and the difference spectrum would represent purely dimeric absorption. (For example, the 1.9 X 10“ M solution was estimated to contain 6.48 X lo-’ M Chl a as monomer, while the 3.89 X M solution M monomer. The difference absorption contained 6.20 X spectrum of the 1.9 X 10” M solution in a IO-mm path length cell and the 3.89 X IO-” M solution in a 0.1-mm path length cell represents the absorption of the equivalent of 2.02 X lo6 M Chl a as dimer and 2.8 X M Chl a as monomer. The contribution of the monomer to this spectrum is therefore small (-2%)). The monomeric absorption spectrum in dry hexane was obtained in a similar manner. The calculated concentrations of the monomer and dimer were refined to give a good fit of the calculated spectra to the absorption spectra of the dry solutions over the concentration range 2 X 104-1 X M total Chl a concentration. The calculated absorption spectra of the Chl a dimer and monomer in dry hexane are shown in Figure 1, B and C, respectively. Table I lists the main absorption maxima and the calculated molar absorptivities of the monomer and the dimer in dry hexane. The absorptivities are calculated on the assumption that the value of the molar absorptivity of the monomeric chlorophyll a pyridinate at the red absorption maximum is a good approximation to the molar absorptivity of the residual monomer in hexane, although the calculated absorption maxima of the residual monomer are blue shifted (2 nm) relative to those of the chlorophyll a pyridinate in hexane (see Table I). It is also assumed that there is negligible contribution to the absorption spectra from oligomeric species of Chl a, because there is a significant amount of monomeric Chl a present, which indicates that the equilibrium is not in favor of the larger aggregates. Similar results were obtained from the analysis of the absorption spectra of Chl a in dry carbon tetrachloride and benzene. Several features of the calculated dimer spectra should be noted. The main red absorption band of the Chl a dimer in hexane exhibits a single maximum at 659 nm. The full width at half-maximum (fwhm) of the dimeric absorption band (-630 cm-I) is significantly broader than that of the monomer (-430 cm-I). The corresponding maxima of the monomer and dimer absorption bands in dry carbon tetrachloride are 663 and 672 nm, respectively. The molar absorptivities of the dimer at the maxima of the blue
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and the red absorption band measured per mole of Chl a monomer as dimer are significantly smaller than those of the monomer (Table I). It is known from infrared absorption and nuclear magnetic resonance studies that the interaction between two or more Chl a molecules in a dry nonpolar solvent occurs via a C=O. .Mg linkage, thought to involve the C9 keto carbonyl group,6 although a novel structure for the dimer involving the C l oester carbonyl group has recently been proposed.26 There is also evidence that a greater than 1:l mole ratio of water to Chl a is required to disaggregate the dimer in these solutions.6 It is therefore believed that the dimer present in the solutions for which the fluorescence spectra are presented in this work contains dimeric Chl a in which the Chl a-Chl a interaction occurs via a direct C=O. .Mg interaction, not involving water. There is however sufficient water (or adventitious nucleophile) in the solutions to give 19% monomeric Chl a, and it is not known whether under these conditions the dimer may possibly exist as an externally hydrated species [H,O.(Chl a-Chl a).HzO]. The main red absorption band of the Chl a monomer in solution is due to the Qy transition. The Q, transition lies to the blue, underneath vibronic components of the Qy band. If it is assumed that the bandwidths of the dimer transitions are similar to those of the monomer, the broad dimeric absorption band at 669 nm in hexane (Figure 1B) could be interpreted as containing contributions from the two Qytransitions of the dimer, split by 100 cm-’. On the other hand, if it is assumed that the absorption band at 669 nm arises from a single Qy transition of the dimer, which is red shifted relative to that of the monomer Qy transition, the band may be assigned to the transition terminating on the lowest-lying level of the singlet system of the dimer. In this case the second Qy transition of the dimer, which would be blue shifted relative to the monomer Qy transition, would be expected to lie underneath other transitions to shorter wavelength (5650 nm). This would imply that the splitting of the dimer bands corresponding to the Qy transition of the monomer is >450 cm-I. The absorption spectra of several covalently linked chlorophyll dimers have been published.I8 It has been shown that the optical properties of the dimers are strongly dependent on their structures. We note that the dimer spectrum of Figure 1B shows features similar to those of the absorption spectrum of the “folded” singly linked dimer. Both spectra are characterized by a broad red absorption band, which is shifted to longer wavelength compared to the narrower monomeric Qy absorption band. The broad red absorption band of the covalently linked “folded” dimer was assigned to the lower energy exciton transition, and the blue-shifted transition was predicted to have weaker intensity and to be shifted by -400 cm-I. The interpretation of the absorption spectra of the Chl a solutions is complicated firstly by the existence of several species of Chl a in equilibrium in solution and secondly by lack of unambiguous experimental data to support the assignment of the transitions of the Chl a dimer which are predicted from simple theoretical models. It is clear, however, that the main red and blue absorption bands of the dimer are red shifted with respect to those of the monomer. The differences in the absorption spectra of the monomer and the dimer suggest that interpretation of fluorescence data may be facilitated by wavelength-selective excitation of fluorescence from each of the Chl a species in solution.
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(26) R. J. Abraham and K. M. Smith, Tetrahedron Left., 24, 2681-4 (1983).
1080 The Journal of Physical Chemistry, Vol. 88, No. 6, 1984
de Wilton et al.
h C 80 0
750
700
650 nm
600
Figure 2. Fluorescence spectra of lo-, M Chl a in dry CCI4,selectively excited at wavelengths in resonance with the blue absorption bands of the Chl a monomer and dimer: (A)430.5,(B) 444.5,and (C) 450.5 nm. The bands at 627 and 685 nm are assigned to the dimer. Wavelength scale is in error: subtract 10 nm.
B. Selective Excitation and Time-Resolved Studies of Fluorescence. We have previously shown that fluorescence from M solution of Chl a in benzene may be excited with a a nanosecond pulsed tunable laser at wavelengths in resonance with either the red or the blue absorption bands of Chl a in solution.27 In addition to the strong fluorescence of the monomer other weaker emission bands were observed which were assigned to dimeric and more highly aggregated species of Chl a. In this work we present fluorescence spectra for Chl a in carbon tetrachloride (CC14) and hexane solutions which permit the assignment of specific fluorescence transitions to the Chl a dimer. The fluorescence spectra are shown in Figures 2-4. M solution of Chl a in dry CC14 is exposed to When a pulsed-laser radiation at a wavelength XI = 430.5 nm, which is in resonance with the peak of the Soret band, the spectrum recorded (Figure 2A) is predominantly that of the Chl a monomer. The monomeric fluorescence is characterized by a main band at 668 nm and a vibronic sideband at 720 nm. By tuning the laser to longer wavelength, away from the peak of the monomeric absorption into a region in which the dimeric absorption is greater than that of the monomeric species, we recorded a series of spectra (Figure 2A-C). Although the monomeric fluorescence dominates the spectra, additional emission bands at 627 and 685 nm are apparent. This is shown more clearly in the spectra obtained from a lo4 M solution of Chl a in dry hexane (Figure 3A-D). In this solution also, selective excitation in the spectral region corresponding to predominantly dimeric absorption results in strong fluorescence bands at 625 and 688 nm. Excitation at XI = 444.5 nm results in more intense emission at 625 nm, while excitation at XI = 450.5 nm enhances the intensity of the band at 688 nm, relative to the intensity of the monomeric emission at 668 nm. A weak broad emission band centered around 580 nm was also observed by excitation of Chl a in CC14, benzene, and hexane solutions at X = 460-470 nm. Addition of water to the dry hexane, benzene, or CCl, solutions of Chl a results in stronger monomeric emission and the bands at 625 and 685 nm diminish in intensity. Increasing the Chl a concentration of the dry solutions causes an increase in the intensity of the 625- and 685-nm bands relative to the monomeric band at 668 nm. The bands at 625 and 685 nm are observed only for solutions which are known to contain the Chl a dimer and are (27) A. C. de Wilton, L. V. Haley, and J. A. Koningstein, Chem. Phys. Lett., 97, 538-40 (1983).
0
L 800
750
70C
650
nm 600
Figure 3. Fluorescence spectra of 10" M Chl a in dry hexane, selectively excited at wavelengths in resonance with the blue absorption bands of the Chl a monomer and dimer: (A)430.5,(B) 438.5,(C) 444.5,and (D) 450.5 nm. The bands at 625 and 688 nm are assigned to the dimer.
800 1
750 I
700 I
I
I
Figure 4. Time-resolved fluorescence spectra of M Chl a in dry hexane. The times indicated are those recorded on the boxcar time scale, on which the maximum fluorescence intensity in time is recorded at t = 35 ns. The intensities of the three spectra are approximately normalized for comparison. The lifetime of the monomeric fluorescence band at 688 nm is - 5 ns. The intensities of the dimer band at 625 nm show approximately the same behavior in time as the monomeric emission. The dimer band at 688 nm decays significantly faster (A, = 444.5 nm).
not observed for Chl a in polar solvents, such as pyridine, in which Chl a is not believed to exist as the (Chl a)z dimer. The analysis of the absorption spectrum of the dry solutions of Chl a suggested the possibility of selective excitation of the monomer and the dimer at wavelengths in the range 430-450 nm, and indeed it is observed that selective excitation at wavelengths corresponding to the dimeric absorption band enhances the intensity of the 625- and 685-nm fluorescence bands. Thus the bands at 625 and 688 nm in the hexane solution of Chl a, and at 627 and 685 nm in benzene and CCll solutions of Chl a, may unambiguously be assigned to the Chl a dimer. Emission around 720 nm, which is additional to the monomeric vibronic sideband, may be assigned to (i) vibronic sidebands of
The Journal of Physical Chemistry, Vol. 88, No. 6, 1984 1081
Fluorescence Spectrum of Chlorophyll a Dimer
800
750
700
650 nm
600
Figure 5. Fluorescence spectra of 10-4 M Chl a in dry hexane, selectively excited at wavelengths in resonance with the red absorption bands of the Chl a monomer and dimer and oligomer: (A)614.0, (B)604.0, and (C) 120 nm.
the dimeric fluorescence at 688 nm and possibly to (ii) emission from oligomeric species of Chl a. Figure 4 is an example of time-resolved fluorescence spectra of the M solution of Chl a in hexane. Because (i) the fluorescence spectrum of this solution is composed of a number of overlapping bands and (ii) given the signal-to-noise ratio of the spectra and the 2-11s time resolution of the detection system, it was not possible to record fluorescence decay curves directly to obtain fluorescence lifetimes of the dimeric emission bands. However, the time-resolved fluorescence spectra shown in Figure 4 clearly show the differences in lifetimes of the fluorescence bands. That of the fluorescence of the monomer in solution (668 nm) is known to be -5 ns.14J7By recording the fluorescence spectra of the dimeric solution at time around t = 35 ns (boxcar time), the time at which the fluorescence intensity reaches a maximum, one can determine differences in lifetimes ( T ) between the monomeric and two dimeric fluorescence bands from the change in the relative intensities of the three bands. The behavior in time of the intensity of the emission band at 625 nm is similar to that of the monomeric emission band at 668 nm (T = 5 ns); the intensity of the dimeric fluorescence band a t 668 nm decays significantly faster than that of the monomeric band, and thus the fluorescence lifetime of this component is less than 5 ns. Wavelength-selective excitation of fluorescence has also been studied in the red spectral region. Typical spectra are shown in M Chl a in hexane. Excitation at XI = 614.0 Figure 5 for nm results in strong fluorescence of the monomer (Figure 5A) while excitation at XI = 604.0 nm (Figure 5B) results in a significant change in the relative intensities of the monomeric and dimeric emission bands. Since the fluorescence maximum of the dimer at 688 nm is shifted -410 cm-’ from the calculated
maximum of the corresponding absorption at 669 nm, it may be estimated that the wavelength of the absorption corresponding to dimeric emission at 625 nm should be at 609 nm. Both the spectra shown in Figure 5, A and B, are excited at wavelengths which are in resonance with the vibronic sidebands of the monomeric absorption band at 668 nm. However, the presence of an absorption band of the dimer at -609 nm is supported by the observation that excitation of fluorescence with XI = 604.0 nm results in strong fluorescence at 688 nm, with a weaker band at 625 nm, together with emission to longer wavelength (-720 nm) (Figure 5B) similar to the fluorescence spectrum observed with excitation in the blue absorption band at XI = 450.5 nm. Excitation at XI = 720 nm results in only the longer wavelength emission assigned to higher aggregates (Figure 5C). The results of selective excitation in the red indicate coupling between the two dimer exciton levels which absorb at 669 and -604 nm. However, the two dimeric fluorescence bands show a difference in their excitation profiles in the blue, and it would appear that energy relaxation to the two fluorescing levels following excitation with A, = 430-450 nm occurs via two different channels, and/or there is more than one electronic origin in this spectral region,z7 each of which is differently coupled with the two fluorescing levels of the dimer. It is known that the origins of the B, and the By transitions in the Soret band of the Chl a monomer in solution are approximately degenerate. The dimer absorption bands in the Soret region are therefore also expected to be due to transitions which would correspond to both By and B, monomer transitions. The difference in the excitation profiles of the two dimeric emission bands may reflect the symmetry of the states of the dimer from which the fluorescence transitions originate. C. Photoquenching of Fluorescence. The spectra displayed in Figures 2-5 were recorded with a photon flux density of -3 X 10I6photons cm-z s-l (laser pulses unfocused). The number of photons per laser pulse is approximately equal to the number of Chl a molecules in the irradiated volume for a M solution. Significant population of excited states with nanosecond lifetimes therefore does not occur. The photon flux density may be increased by a factor of lo4 by focusing the laser beam into the sample cell using a lens of focal length 80 mm. Under these conditions, excited states having lifetimes of -100 ps may be M Chl a. populated in a solution containing Selective quenching of the monomeric fluorescence of a solution of lo-’ M Chl a in benzene has been observedz8by excitation with focused laser pulses at wavelengths in resonance with the absorption maximum of the Soret band of the monomer. Fluorescence quenching was believed to be caused via population of the excited singlet level at -23300 cm-I, and subsequent absorption of a second photon which competes with relaxation to the first excited singlet state from which the red fluorescence originates. In that experiment selective quenching of fluorescence of the monomer permitted observation of weak fluorescence bands from dimeric and aggregated species of Chl a which have lifetimes comparable to that of the monomer, and which could not otherwise have been separated by the time resolution of our detection system (2 ns). The spectra shown in Figures 6 and 7 illustrate the effect of fluorescence quenching at high photon flux densities in solutions of Chl a in CC14 and in hexane, respectively. The M Chl a solution in CC14 was excited with XI = 450.5 nm, and a comparison of the spectrum in Figure 6B excited at low photon flux density with that of Figure 6A, excited at higher photon flux density, shows that more effective quenching of the dimeric fluorescence bands occurs compared to the quenching of the monomeric emission bands. A similar phenomenon is observed for excitation of the solution of Chl a in hexane with XI = 444.5 nm at different photon flux densities (Figure 7, A and B). Population of excited states of the dimer by excitation with wavelengths in resonance with the Soret absorption band has
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(28) A. C. de Wilton and J. A. Koningstein, J . Phys. Chem., 87, 185-8
(1983).
1082 The Journal of Physical Chemistry, Vol. 88, No. 6,1984
de Wilton et al. of the monomer and the dimer are therefore the absorption cross sections of the ground and excited states of the monomer and the dimer (which are wavelength dependent) and alternative pathways for deexcitation of the molecules besides fluorescence. Selective quenching of the fluorescence of the monomer or the dimer would be anticipated when the laser wavelength is tuned into resonance with the absorption maximum of the corresponding species of Chl a.
Whereas both the dimeric fluorescence bands are quenched to the same extent, relative to the monomeric emission band, by excitation with XI = 450.5 nm, quenching of the fluorescence band at 688 nm is more effective than for the 625-nm band with XI = 444.5 nm. This suggests the existence of more than one electronic origin in this wavelength region of the absorption band of the dimer, with specific channels for energy relaxation to the two exciton levels from which the fluorescence transitions originate. 800 I
750 1
650 nrn
700 I
~
1
600 I
Figure 6. Fluorescence spectra of lo4 M Chl a in CC14excited with XI = 450.5 nm: (B) laser beam unfocused, (A) laser beam focused with lens of focal length 80 mm, illustrating quenching of dimer fluorescence at 688 and 625 nm under irradiation with lo4 times higher photon flux
-
density.
IV. Discussion In this work we report pulsed-laser-induced fluorescence from the Chl a dimer in solution in hexane, benzene, and CC14. The dimeric fluorescence is characterized by two bands at 625 and 685 nm. Time-resolved fluorescence spectroscopy shows that the lifetime of the fluorescence at 625 nm is comparable to that of the Chl a monomer (- 5 ns) whereas that of the fluorescence at 685 nm is shorter than 5 ns. Analysis of the calculated absorption spectrum of the dimer together with the fluorescence results suggests several possible assignments for the splitting of the lower-lying singlet exciton levels of the Chl a dimer. The theoretical model which has been used previously to calculate the absorption spectrum of the Chl a dimer is based on a two-electron model in which one ground state and two excited singlet states are included.* This model predicts one allowed and one forbidden Qy transition and would be consistent with the assignment of the strong absorption band at 669 nm to the allowed Qy transition. The weaker absorption band which corresponds to the fluorescence band at 625 nm could then be assigned either to an allowed Q, transition or to a weakly allowed second Q, transition. The latter assignment would lead to an exciton splitting of 1400 cm-', whereas if the former assignment were correct the exciton splitting of the Qy transitions could not be determined from the absorption and fluorescence data. The full width at half-maximum (fwhm) of the fluorescence band of the dimer at 688 nm (550 & 50 cm-I) corresponds within experimental error to the fwhm of the red absorption band of the dimer at 669 nm (630 cm-I). If it is assumed that the bandwidths of the dimeric transitions are similar to those of the monomeric absorption band (460 cm-I), it is possible that the broad dimeric absorption band at 669 nm arises from two Qy exciton transitions of the dimer (with approximately equal intensities) which correspond to the Qy transition of the monomer at 659 nm. However, two bands of this width cannot be split by more than 100 cm-' to give a total bandwidth of -620 cm-'. The narrower fluorescence band at 625 nm may be assigned to a Q, transition of the dimer, although we have not detected fluorescence from the Q, level of the Chl a monomer. The activity of the Q, transition of the dimer would be an indication that the effects of symmetry considerations on the transition moments of the dimer are different from those on the transition moments of the monomer. A different theoretical model for the Chl a dimer was used for an analysis of the electronic Raman process between the two Q, exciton levels of the Chl a dimer which was reported29 recently. In this model for the dimer the splitting of both the highest occupied molecular orbitals and the lowest unoccupied molecular orbitals of the monomer are considered. In contrast to the twoelectron model,s this four-orbital model predicts two absorption bands of approximately equal intensity, separated in energy by the difference of the splittings of the two pairs of orbitals. This model would therefore be consistent with the assignment of the broad absorption band at 669 nm to the two Qy transitions of the dimer. However, to be consistent with the assignment of the observed fluorescence band at 625 nm (which would correspond
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800
700
750
650 nrn I
600
Figure 7. Fluorescence spectra of M Chl a in hexane excited with XI = 444.5 nm: (A) laser beam unfocused, (B) laser beam focused with lens of focal length 80 mm illustrating quenching of the dimer fluorescence at 688 and 625 nm, relative to that of the monomer at 668 nm, under irradiation with higher photon flux density.
recently been demonstrated by Raman scattering studies.29 It appears that for a Chl a concentration, 1 order of magnitude greater than that used when selective quenching of monomeric fluorescence was observed it is not possible to achieve effective population saturation of the 100-ps-lived excited state of the monomer under these conditions. It has been shown that the first excited singlet state of monomeric Chl a absorbs in the same region as the Soret absorption band,I5 and fluorescence quenching may be explained by excited-state absorption from this excited singlet state. A similar photoquenching of fluorescence would be expected for the dimer if the first excited singlet state of the dimer also absorbs in the blue. In the lo4 M solution of Chl a in hexane, the absorption data show that the molar concentration of the Chl a dimer is more than twice that of the monomer. However, the concentrations of both the monomer and the dimer are such that the number of molecules of each in the irradiated volume of solution is of the same order of magnitude as the number of photons per pulse. The important factors determining the relative photoquenching of fluorescence
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(29) L. V. Haley and J. A. Koningstein, Can. J. Chem., 61, 14-20 (1983).
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J. Phys. Chem. 1984,88, 1083-1086 to an absorption band at -609 nm) to a Q, transition, the model must be extended to include orbitals from which the monomer Q, transitions originate. Clearly this study shows that there are several possible alternative assignments for the dimeric absorption and fluorescence bands. Further experimental and theoretical work is required to
1083
determine unambiguously the origins of the fluorescence bands of the Chl a dimer.
Acknowledgment. This research was supported by the Natural Sciences and Engineering Research Council Of Canada. Registry No. (Chl u ) ~ 18025-08-6. ,
Chelation of Gas-Phase Anions. An Ion Cyclotron Resonance Study of Cooperative Binding to Fluoride and Chloride Ions J. W. Larson’ and T. B. McMahon* Department of Chemistry, University of New Brunswick, Fredericton, New Brunswick, Canada E3B 6E2 (Received: June 3, 1983; In Final Form: November 22, 1983)
The energetics of binding of the halide ions F and C1- to a number of compounds capable of binding anions at more than one site have been determined and compared to energetics of similar compounds with only one possible binding site. The data obtained show a definite increase in stability of adducts produced by anion attachment to (CHFz)20, (CHF2)2C0,and FCO(CF2)$OF compared to those of CHF20CF3,CHF2COCF3,and F(CF2),COF, respectively. These results thus indicate that chelation of halide ions in the former, multiple-binding-site, compounds is occurring. The chelation effect is observed to be more pronounced for Cl- than for F,a consequence of the largely electrostatic nature of C1- binding compared to covalent binding in F adducts.
Introduction Recent studies of the energetics of association of gas-phase ions with single neutral molecules have provided valuable insights into the nature of the solvation process and into the reasons for differences in relative stabilities and reactivities between the gas phase and s o l ~ t i o n . ~ -However, ~ one of the most interesting types of solvation interaction, that of multiple binding interactions within a single solvent molecule, has received little attention. In solution such reagents are well-knqwn and are exemplified most dramatically by crown ethers and cryptates which are valuable agents for solubilizing ionic salts in nonaqueous media and for producing highly reactive “naked” anions by preferential complexation of specific cations. Very recently Mautner’O has reported on the proton binding energies of both linear and cyclic (crown) polyethers and obtained intramolecular hydrogen bond energies. In both solution and the gas phase, however, while strong monodentate complexes of anions are known, evidence of compounds which exhibit multiple anion binding sites is almost nonexistent. Barcza and Pope1’J2 have reported that ethylene glycol and 1,2-dihydroxybenzene chelate ClO;, C1-, Br-, and I-, based on the increased stability of these adducts relative to their monofunctional analogues. While this conclusion may not be unreasonable, the increased adduct stability may also be due either (1) On sabbatical leave from Marshall University, Huntington, WV. (2) Kebarle, P. Annu. Reu. Phys. Chem. 1977, 28, 445. (3) French, M. A.; Cumming, J.; Kebarle, P. J. Am. Chem. SOC.1977,99, 6999. (4) French, M. A,; Ikuta, S.; Kebarle, P. Can. J . Chem. 1982, 60, 1907. (5) Mautner, M. J. Am. Chem. SOC.1979, 101, 2396. (6) Kessee, R. G.; Lee, N.; Castleman, A. W. J . Chem. Phys. 1980, 73, 2195. (7) Larson, J. W.; McMahon, T. B. J . Am. Chem. SOC.1982, 104, 6255. (8) Larson, J. W.; McMahon, T. B. J . Am. Chem. SOC.1983, 105,2944. (9) Larson, J. W.; McMahon, T. B. J. Am. Chem. SOC.,in press. (10) Mautner, M. Euchem Conference on Ion Chemistry: Gaseous vs. Solvated Ions, Lido di Ostia, Italy, 1982. (11) Barcza, L.; Pop, M. T. J . Phys. Chem. 1973, 77, 1795. (12) Barcza, L.; Pope, M. T. J . Phys. Chem. 1974, 78, 168.
0022-3654/84/2088-1083$01.50/0
wholly or in part to the increased acidity of the hydroxyl proton brought about by the electron-withdrawing ability of the neighboring O H group. An example of such an effect has been demonstrated by Huyskens and Lambeau13 who found that although citric acid binds C1- and Br- more strongly than does benzoic acid, correction for the increased acidity of citric acid eliminates the need to invoke chelation or multiple-site binding of the halide ions. While Lehn and ~ o - w o r k e r s have ~ ~ , ~shown ~ that macropolycyclic and related ammonium ions can indeed chelate anions, there is no firmly established evidence for chelation of anions by a neutral polyfunctional Bronsted acid. As part of a continuing program of investigation of anion binding energetics for both Bronsted and Lewis acids, we have encountered several cases where convincing arguments can be made for chelation of gas-phase halide ions. The results reported in the present work indicate that appreciable stabilization does occur for F and Cl- adducts due to multiple binding interactions. These results also suggest potentially useful directions for the design of anion-specific complexing agents. Experimental Section All experiments were conducted at ambient temperature with an ICR spectrometer of basic Varian V-5900 design extensively modified to permit routine operation in both conventional drift and trapped-ion mode^.'^^'^ Gas mixtures used in competitive halide transfer reactions were prepared with accurately known partial pressure ratios determined with a Validyne AP-10 absolute pressure gauge on a Monel vacuum line. Fluorinated dimethyl ethers, fluoroacetones, and perfluoroacyl fluorides were all obtained from PCR Inc. All other materials (13) Huyskens, P. L.; Lambeau, Y. 0. J . Phys. Chem. 1978, 82, 1886. (14) Dietrich, B.: Hosseini, M. W.; Lehn, J. M.; Sessions, R. B. J. Am. Chem. SOC.1981, 103, 1282. (15) Hosseini, M. W.; Lehn, J. M. J . Am. Chem. SOC.1982, 104, 3525 (16) McMahon, T. B.; Beauchamp, J. L. Reu. Sci. Instrum. 1973,43,509. (17) Lehman, T. A,; Bursey, M. M. “Ion Cyclotron Resonance Spectroscopy”; Wiley-Interscience: New York, 1976.
0 1984 American Chemical Society
