J. Phys. Chem. 1986, 90,6611-6616
6611
ARTICLES Vibrationally Resolved Optical Spectra of Chlorophyll Derivatives in Dlfferent Solid Media Indrek Renge,* Koit Mauring, Priit Sam, and Rein Avarmaa Institute of Physics, Estonian S S R Academy of Science, Riia 142, 202400 Tartu, Estonian S S R , USSR (Received: January 23, 1986; In Final Form: July 8, 1986)
The site-selection vibronic fluorescence and excitation spectra of chlorophyll a (Chl a), protochlorophyll (PChl), and some of their derivatives in a number of organic solvent matrices are recorded at 5 K. The strength of electron-phonon coupling, estimated as a relative line-to-background intensity in the excitation spectra, was found to be dependent mainly on the state (microcrystallic or glassy) of the matrix. The resolution is better in vitreous samples than in snowy ones. For PChl and 4-vinyl-PChl marked differences were found in both fluorescence and excitation spectra. Chl a, Chl a', and pyro-Chl a could be distinguished on the basis of some characteristic &-state vibrational frequencies, while the spectra of Chl a and ethylchlorophyllide a (where the phytyl ester group is replaced by ethyl) are practically identical. The coordination number of the Chl a and PChl central magnesium atom (with one or two axial extra ligands) is found to influencespecifically the vibrational modes and the S1-Szsplitting in the excitation spectra. It has been established that a bisligated Chl a species prevails in sterically uncrowded alcohols, e.g., 1-butanol.
Introduction Recently the possibility of obtaining high-resolution optical spectra for large organic molecules by cooling them in supersonic expansions has been demonstrated.' Internally fine-structured vibronic spectra of isolated molecules are essentially modified by the incorporation of the latter into a solid m a t r i ~ . ~First, ? ~ the coupling with intermolecular lattice modes gives rise to phonon sidebands near zero-phonon vibronic lines. Second, the intermolecular fields induce dispersed shifts of the frequency of a purely electronic 0-0 transition, which are characterized by an inhomogeneous distribution function. The latter has discrete, comparatively narrow (1-5 cm-I) peaks in the case of simple porphyrins as impurity centers in crystalline n-alkane matrices (the Shpolskii system^).^,^ The derivatives of a more complicated structure, e.g., chlorophylls, have broad-band low-temperature absorption and luminescence spectra at white-light excitation in all conceivable solvent matrices. This corresponds to the wide smooth inhomogeneous distribution of centers (fwhm up to 300 cm-'). The application of the siteselection effect6 as well as the spectral hole-burning method' at liquid helium temperature has enabled one to remove partially the inhomogeneous broadening and to reveal some homogeneous characteristics of the spectra. A number of chlorophyll derivatives exhibit sharp-line fluoroescence and excitation spectra under monochromatic excitation (detection) in the region of the first vibronic transition. Zerophonon lines have been observed at temperatures, less than 40 K (mostly at 5 K) in different hosts with a comparatively weak electron-phonon coupling (EPC), including frozen organic solutions,8-'0 solidified vesicular systems," and even etiolated and
greening plants.12J3 The analogues of Chl a, such as protochlorophyll (PChl), Chl b, and the corresponding pheophytins, possess quite distinguishable sets of ground- and excited-state vibrational frequer~cies.~J~ In order to test more exhaustively the selectivity of the method, we investigated the subtle substituent effects in the spectra of PChl and 4-vinyl-PChl as well as Chl a analogues Chl a', pyro-Chl a, and ethylchlorophyllide a (EtChlide a ) (Figure 1). The latter have slight differences in the peripheral region not conjugated with the *-electronic system of the phorbine macrocycle. Further, it will be demonstrated that the number of axial extra ligands at the central Mg atom manifests itself in the vibrational frequencies and the SI-S2 splitting in selectively recorded fluorescence excitation spectra. The line-to-background intensity ratio of the spectrum depends on the coupling strength of the vibronic transition with the matrix modes and so-called pseudolocal modes, which correspond to the low-frequency intermolecular vibrations as well as to librational and torsional motions of the guest molecule. In order to correlate the electron-phonon coupling strength with the physical and chemical properties of the matrix, the Chl a excitation spectra are recorded in a number of frozen solvent hosts. The possibilities of the low-temperature laser spectroscopy as a tool for studying chlorophylls will be discussed and compared with IR and N M R spectroscopy.
(1) Even, U.; Magen, J.; Jortner, J.; Friedman, J.; Levanon, H. J . Chem. P h p . 1982, 77, 4391-4399. (2) Rebane, K. K. Impurity Spectra of Solids; Plenum: New York, 1970. (3) Rebane, K. K. Zh. Prikl. Spektrosk. 1982, 37, 906-922. (4) Gurinovich, G. P.; Sevchenko, A. N.; Solov'ev, K. N. Spectroscopy of Chlorophyll and Related Compounds; Nauka i Tekhnika: Minsk, 1968. (5) Canters, G. W.; van der Waals, J. H. In The Porphyrins; Dolphin, D., Ed.; Academic: New York, 1978; Vol. 111, Part A, pp 531-582. (6) Personov, R. I. Spectrochim. Acta, Part B 1983, 388, 1533-1544. (7) Rebane, L. A.; Gorokhovskii, A. A.; Kikas, J. V. Appl. Phys. B 1982, 829, 235-250.
(8) Bykovskaya, L. A,; Litvin, F. F.; Personov, R. E.; Romanovskii, Yu. B. Biojizika 1980, 25, 13-20. (9) Rebane, K. K.; Avarmaa, R. A. Chem. Phys. 1982, 68, 191-200. (10) Avarmaa, R.; Rebane, K.Spectrochim. Acta, Part A 1985, 41A,
0022-3654/86/2090-6611$01.50/0
Experimental Section PChl and 4-vinyl-PChl were extracted from pumpkin seeds following Houssier and Sauer.14 Chl a was isolated from barley
1365-1 380. (1 1) Flinfschilling, J.; Walz, D. Photochem. Photobiol. 1983, 38, 389-393. (12) Avarmaa, R.; Renge, I.; Mauring, K. FEES Lett. 1984,167, 186-190. (13) Renge, I.; Mauring, K.; Avarmaa, R. Biochim. Biophys. Acta 1984, 766, 501-504. (14) Houssier, C.; Sauer, K. Biochim. Biophys. Acta 1969,172,476-491, 492-502.
0 1986 American Chemical Society
6612 The Journal of Physical Chemistry, Vol. 90, No. 25, 1986
Renge et al.
TABLE I: Line-to-Background Intensity Ratios in the Excitation Spectra of Chl B for the Vibronic Mode we = 346 f 2 state of solvent matrix LBIR, % Amax! nm if,nm F, D matrix 96 668.8 669 1.40 0.8 glass TEA 1-butanol 17 679 678.9 1.40 1.65 glass methanol-ethanold 75 677 675 1.34 1.6 glass diethyl ether 33 669.5 670 1.35 1.2 ?
greening barley' cyclohexane + 1% TEA y-collidine
33 32 30 22 20 14 10 0
DMF
THF pyridine 2-methylpropanol
dioxane
680 672 674 67 1 612 68 1 677.3 676
614 672 674 667.8 672 68 1 676 676
1.43 1.50 1.43 1.41 1.51 1.39 1.44
0 1.93 3.8 1.87 2.2 1.66 0.4
at 5 K"
prevailing solvate typec Ll L2H L2H Ll
?
Ll
snow
Ll Ll Lz
snow snow snow snow snow
L2
LIH L2
"The recording wavelengfth is set at A[; n20Dis the refractive index of the liquid solvent; p is the dipole moment of the solvent molecule (ref 32). bFluorescenceband maximum at A, = 514.5 nm. 'Reference 21. "Reference 28. 'Reference 12. absorption intensity of selected centers, because the laser output energy was maintained constant during the scanning. Narrowband detection (0.05-0.1 nm) at the wavelength Af in the region of the inhomogeneously broadened 0-0 SI So fluorescence band was used. Vibrationally resolved fluorescence spectra of PChl and 4-vinyl-PChl were obtained by setting the laser wavelength A, (bandwidth -0.01 nm, excitation density, -0.2 mW within the 0-0 S1 So band of the pigment. The slit width of the spectrometer did not exceed 0.05-0.1 nm. Nonselective fluorescence spectra were run under excitation with Ar (457.9 or 514.5 nm) or Cd laser lines (441.6 nm). These showed no vibrational structure, since higher electronic states are excited by these lines, while fluorescence is emitted after relaxation to the SI state. Some fluorescence spectra were measured at 77 K in liquid nitrogen. The visible absorption spectra of the liquid solutions were recorded on a Beckman Acta MVII spectrophotometer.
-
-
R1 R2 R3 ________
R
PCHL 4-VINYL-PCHL
bH5 - CH
CH2
CHL
A
CHL
A'
ETCHLIDE A PYROCWLA
Ci~Hjg C20H3q C2H5 Czo%g
U
COOCH3
COOCH3
H
H
COC' 3
H
H
Figure 1. Structure of protochlorophyll and chlorophyll a derivatives.
shoots according to S ~ e c . 'EtChilde ~ a was prepared after Chow et a1.I6with the exception that Ailanthus was replaced by another plant rich in chlorophyllase, Heracleum sp. Chl a'and pyro-Chl a were prepared following Pennington et al." The SI and Soret band absorption maxima and the Soret-to-SI band intensity ratios of the pigments in dry ether were in good accordance with published values."-19 The pigments were stored as ether solutions at -4 OC. The solvents diethyl ether, dioxane, terahydrofuran (THF), and triethylamine (TEA) were stored and then distilled over K-Na alloy. Pyridine and 2,4,6-trimethylpyridine (y-collidine) were allowed to stand on BaO and then distilled. The reagent-grade alcohols, cyclohexane, and dimethylformamide (DMF) were used as received. Immediately before the run of a spectrum the pigment solution in ether was vacuum-evaporated in a test tube and the desired solvent added. The solution (2 X 10"-2 X M) in a l-mmthick quartz cuvette was plunged into liquid N 2 and then transferred to a liquid H e cryostat, where the temperature was maintained at 5 K. The experimental setup, consisting of a Coherent CR-490 dye laser (with rhodamine 6G and DCM dye, tuning range of 570-700 nm) pumped by a CR-3 argon laser and a DFS-24 spectrometer supplied with a photon-counting detection system, was mainly the same described by Rebane and A ~ a r m a a . ~It, ' was ~ supplemented with an automatic Ne optogalvanic marker-line unit in conjuction with computer processing.20 The excitation spectra represent the (15) Svec, W. A. In The Porphyrins; Dolphin, D., Ed.; Academic: New York, 1978; Vol. V, Part C, pp 342-399. (16) Chow, H.-C.; Serlin, R.; Strouse, C. E. J . Am. Chem. SOC.1975, 97,
7230-7242. (17) Pennington, F. C.; Strain, H . H.; Svec, W. A.; Katz, J. J. J. Am. Chem. SOC.1964, 86, 1418-1426. (18) Seely, G. R.; Jensen, R. G. Spectrochim. Acta 1965, ? I , 1835-1845. (19) Hynninen, P. H.; Wasielewski, M. R., Katz, J. J. Acta Chim. Scand., Ser. B 1979, 833, 637-6423. (20) Avarmaa, R.; Suisalu, A. Proc. Acad. Sci. Estonian SSR, Phys. Math. 1984, 33, 333-3323,
Results and Discussion Electron-Phonon Coupling (EPC) of Chl a SI So Transition in Different Hosts. The quality of site-selection spectra of inhomogeneously broadened systems depends mainly on the coupling of a given vibronic transition with low-frequency lattice modes (phonons). A quantitative measure of the EPC, the Debye-Waller factor, is defined for homogeneous spectra as an integral line to line plus wing intensity ratio. Site-selection spectra observable for inhomogeneous systems are always of poorer quality in the sense of line-to-background intensity ratio (LBIR). For practical purposes, we have characterized the EPC as the LBIR in fluorescence excitation spectra when fluorescence is recorded at the spectral maximum. We preferred the excitation spectra, since the effect of optical holeburning leading to a cancellation of lines is negligible in this case. The LBIR values (in percent) for a prominent frequency (346 i 2 cm-l) in a number of solvents are listed in Table I. The nonspecific solvation parameters, the dominating forms of Chl a-solvent complexes,21and the state of the low-temperature matrix-transparent (glassy) or snowy-are also indicated in Table I. In some cases (cyclohexane, diethyl ether) it is hard to decide whether a badly cracked glass or microcrystalline sample is formed. An inspection of Table I shows that spectra are particularly good with LBIR values as high as 75-96% in vitreous hosts: TEA, 1-butanol, and methanol-ethanol (1 / 1) mixture. Remarkably low (
