J . Phys. Chem. 1986, 90, 4938-4941
4938 For IWl/u >> 1, r l ( w ) peaks at z l In this case
= w I / w o , and (zl - 11 >> c.
At low temperatures such that k T
> kT, we obtain eq 66 of the text.
ARTICLES Determination of in Vitro Chlorophyll a Using Wavelength-Selective and Time-Resolved Fluorescence N. E. Binnie, L. V. Haley, T. A. Mattioli, D. L. Thibodeau, W. Wang, and J. A. Koningstein* The Ottawa- Carleton Chemistry Institute, Department of Chemistry, Carleton University, Ottawa, Ontario, Canada K l S 5B6 (Received: September 18, 1985: In Final Form: May 5, 1986) From an analysis of the wavelength-selectivefluorescence spectra of chlorophyll a in dry and wet hexane, spectral assignments of monomeric and aggregate species are made. The monomeric species are penta- or hexacoordinated water complexes; the aggregate species are (1) a donor/acceptor type of complex with the chlorophyll macrocycles linked via the ring V keto group of one with the magnesium atom of the other, and (2) other higher aggregates with predicted electronic origins in the 690-722-nm spectral region
Introduction and other laboratorIn earlier published work from ies4-6*'w1 the wavelength-selective excitation of the fluorescence from solutions containing chlorophyll a (Chl a ) and bacteriochlorophyll a (BChl a ) have been discussed. The emission spectra of these solutions reveal the presence of multiple species which may not be totally resolved by conventional absorption spectroscopic methods since this technique is sensitive mainly toward the bulk constituents of the solution. The aim of this article is to propose possible assignments for several species of Chl a in various hexane solutions and to characterize each of these species by their fluorescence properties. Unlike absorption spectroscopy, the emission spectra of the various Chl a species present in solution can be time-resolved and selectively enhanced by inducing fluorescence with lasers tuned to regions where the absorption cross section for a particular species is larger than for other species. In addition, the species present in solution depends on the respective equilibria which will vary with the total concentration of Chl a and the solvent. Experimental Section Chl a was extracted from fresh spinach according to the method of Omata and Murata7 and purity established by standard ~~
* Killam Fellow 0022-3654 I86 12090-493830 1.50lO , 8
,
The pure Chl a was stored in the dark in a nitrogen-purged drybox. This Chl a did not contain contamination from protochlorophyll as seen by the absence of fluorescence in the 625-630-nm region for Chl a in ether. Chl a was dried by codistillation sequentially with dry CCll (three times) and dry hexane (two times) prior to preparation of a concentrated stock solution. All solvents were dried by passing them through a column of neutral aluminum oxide (Woelm Activity 1) and then degassed. Hexane (Caledon Labs Inc., spectral grade) and CC1, (Baker Photrex) were used. Fluorescence samples were used in air-tight quartz cells with Teflon stopcocks. The dilute 10-7-106 M Chl a hexane solutions were prepared by dilution of the stock solution with dried hexane, (1) de Wilton, A. C.; Haley, L. V.; Koningstein, J. A. J. Phys. Chem. 1984, 88, 1077.
(2) de Wilton, A. C.; Koningstein, J. A. J . A m . Chem. Soc. 1984, 106, 5088. (3) de Wilton, A. C.; Koningstein, J. A. Chem. Phys. Lett. 1984, 114, 161. (4) Hunt, J. E.; Katz, J. J.; Svirmickas, A,; Hindman, J. C. Chem. Phys. 1983, 82, 413. ( 5 ) Baugher, J.; Hindman, J. C.; Katz, J . J. Chem. Phys. Lett. 1979, 63, 15Q
( 6 ) Yuen, M. J.; Shipman, L. L.; Katz, J. J.; Hindman, J. C. Photochem. Photobioi. 1982, 36, 21 1. ( 7 ) Omata, T.; Murata, N. Photochem. Photobiol. 1980, 31, 183. (8) Svec, W . A. In The Porphyrins, Vol. V , Dolphin, D., Ed.; Academic: New York, 1978; pp 341-399.
0 1986 American Chemical Society
The Journal of Physical Chemistry, Vol. 90, No. 21, 1986 4939
Chl a Fluorescence
1
0.065
1
426
w
2 0.050 a m
8 0.045 m
a
0.040
t
0.035
300
400
600
500
300
700
400
Figure 1. Absorption spectrum of a 2.7
X lo-’
M Chl a in dry hexane.
undried hexane (used as received), or water-saturated hexane. The latter two samples were equilibrated at longer times before use. The lo4 M samples were prepared with dried hexane. Absorption spectra were recorded with a DMS-90 spectrophotometer and a H P 8450A UV/vis spectrophotometer using 1-, 2-, or 10-mm quartz cells fitted with Teflon stopcocks. Fluorescence spectra in the absence of excited-state population saturation effects (Figure 4, A and B) were obtained by using excitation from a Coherent Radiation CR-4 supergraphite ion laser chopped at 250 Hz (square wave), and detected by a RCA 31034 water-cooled photomultiplier tube and a Jobin-Yvon Ramanor HG2 spectrometer (1-m double pass, resolution of 2 cm-I maximum). Signal processing was carried out with a Stanford Research SR250 gated integrating boxcar averager (15-ps gate). A front face reflection technique (55O geometry) was used to minimize signal reabsorption effects in the fluorescence measurements. The pulsed laser experiments of were performed9 with a tunable dye laser pumped with a nitrogen laser (Lambda Physik F12000 and M2000, respectively), repition rate 25 Hz, pulse fwhm 4 ns, and a Princeton Applied Research (PAR) boxcar integrator. Lifetime determinations for fluorescence decays were made with the same N, laser system with the SR250 boxcar integrator (2-11s gate). Lifetimes were calculated with a modified phase-plane method by computer.
Results and Discussion Fluorescence Spectra of Dilute Chl a Solutions. It is wellknownI0 that solutions of Chl a in a polar, electron-donating solvents such as ether, ethanol, or pyridine are dominated by monomeric species or low n-number aggregates at relatively high concentrations. Also, dry nonpolar solvents such as hexane, carbon tetrachloride, and benzene tend to forrm higher aggregates at even relatively low concentrations in order to fulfill the coordination requirements of the magnesium atom. The extent of aggregation depends strongly upon the following solvent behavior: ( 1 ) labile coordination with magnesium (poor Lewis basicity) and (2) solvation of the porphyrin ring moiety. Low temperatures have also been used to effect aggregation.” In contrast, trace amounts of impurity nucleophiles are known to terminate aggregation chains. Fong et aLI1 have recorded the room temperature fluorescence spectra of Chl a in deoxygenated, water-saturated 1:l methyl(9) de Wilton, A. C. Ph.D. Thesis, Carleton University, Ottawa, Canada, 1984. (1 0) Schaafsma, T. J. In Triplet State ODMR Spectroscopy: Techniques and Applications to Biophysical Systems, Clarke, R. H., Ed.; Wiley-Interscience: New York, 1982; p 316 and references therein. (1 1) Fong, F. K.; Kusunoki, M.; Galloway, L.; Matthews, T. G.; Lytle, F. E.: Hoff, A. J.; Brinkman, F. A. J . Am. Chem. SOC.1982, 104, 2579. Alfano, A. J.: Fong, F. K. J . Chem. Phys. 1985, 82, 758. (12) Svec, W. A. In The Porphyrins, Vol. V, Dolphin, D., Ed.; Academic: New York, 1978; p 381.
500
600
700
WAVELENGTH (nrn)
WAVELENGTH ( n m )
Figure 2. Absorption spectrum of a 1.6 X lo-’ M Chl a in water-saturated hexane converted to pheophytin. nm 769
714
14000
666
625
I5000
16000
em-’
Figure 3. Fluorescence spectra of a lO-’-lO” M Chl a in hexane of various solvent dryness: (A) dried, (B) undried, and (C) water saturated. Intensities normalized. Pulsed laser excitation at 436 nm.
cyclohexaneln-pentane solution ( 10-5-10-4 M) and found an emission band at 672 nm which was assigned to the Q,fluorescence from a hydrated Chl a monomer (Chl.H,O). Also, a weaker band at 726 nm was attributed to the monohydrate Chl a dimer (Chl-H,0)2. In our work, to avoid the formation of higher aggregates, solutions of approximately 10-7-104 M Chl a in hexane were used to observe the monomeric species. In Figures 1 and 2 we show the absorption spectra of lo-’ M chlorophyll in hexane solution. Both the Soret and the Q band regions for the wet hexane solution (Figure 2) show shifts in the maxima due to pheophytinization of the chlorophyll molecule.I2 The fluorescence spectra of these very dilute solutions were observed to change with water content in the solvent (Figure 3, A-C) and clearly indicate the presence of two distinct emission maxima at 664, 666, and 673.4 nm. We associate these bands with two different monomeric species having different degrees of hydration while the third band is due to pheophytin.20 The 664-nm fluorescence from our driest preparation (Figure 3A) is assigned to a pentacoordinated hydrated Chl a monomer since it is unlikely’O that our solvent and sample drying procedure can remove the strongly axial-bound water ligand. It has been observed (Figure 3B) that a small increase in the water content of the solvent and sample equilibration produces a slight broadening and red shifting of the 664-nm band to 666 nm for the same excitation wavelength, whereas the use of water-saturated hexane (Figure 3C) gives a new band at 673.4 nm free of interference from the 664-nm emission. The 673.4-nm fluorescence (pheophytin) has a measurably longer fluorescence lifetime than that of the 664-nm fluorescence (Table I). With a radiative lifetime
4940
The Journal of Physical Chemistry, Vol. 90, No. 21, 1986
TABLE I: Fluorescence Band Maxima and Lifetimes Assigned to Chlorophyll a species in Hexane Chl a fluorescence
concn
max Q, region, nm 10-6 664 10” 666 10-5-10-4 -668
(total), M
-688
species assignmentb
lifetime,” ns
6.2 5.3 -4.2 -4.1
Chl O H ~ O Chl 0 2 H 2 0 “acceptor” “donor”
QxlQy region -4.7
625
unknown chlorophyll species
With 436-ns excitation. magnesium bound water is indicatWater-saturated hexane. e Donor-acceptor ed. e Dry hexane. M solution. fluoiescence is most intense in the nm 169
666
625
1
Figure 4. Fluorescence spectra of (A) and (B) IO4 M Chl a in dry hexane. Intensities not normalized. Chopped continuous wave laser excitation at 472.7 and 476.5 nm, respectively.
of 5.3 ns the 666-nm band represents a new species. We exclude the possibility of a solvent effect on the pentacoordinated species because such an effect was not observed for pheophytin in dry and wet hexane solutions (Le. there was no observable red shift” in absorption and fluorescence bands). This new species is most likely the hexacoordinated species Chl a.2H20. Connoly et aLi3 have found (1) that the fluorescence lifetime of BChl a is longer for the hexacoordinated species than for the pentacoordinated species, and (2) that the fluorescence lifetime is shorter when the macrocycle is hydrogen bonded than when it is not. Fluorescence and Absorption Spectra of More Concentrated, Dry Solutions. Figure 4A shows a fluorescence spectrum of a M Chl a sample prepared from a concentrated dry stock solution diluted with dried hexane. Emission from the pentacoordinated monohydrate in this solution appears as a shoulder on the high-energy side of a new band maximized at -668 nm. This 668-nm emission cannot be assigned to the Q, band of the hexacoordinated Chl @ 2 H 2 0because (1) conditions are not favorable to produce this species (requires a wet but not watersaturated hexane and equilibration), (2) the emission decay is 4.2 ns instead of the 5.3 ns obtained for the Q, band of the hexacoordinated species, and (3) the concentration is not high enough to induce concentration quenching. The second major spectral feature in the fluorescence spectrum (Figure 4A) is an emission band at 688 nm and is characterized by a decay time of -4.1 ns. Extensive evidence exists in the literature (NMR,I4 IR,I5 optical CD,I6 and f l ~ o r e s c e n c edata) ~ ~ indicating that Chl a aggregates can exist where the keto group of ring V of one chlo(13) Connolly, C. S.; Samuel, E. B.; Janzen, A. F. Photochem. Photobiol.
Binnie et al. rophyll molecule (donor) is coordinated to the magnesium atom of another chlorophyll molecule (acceptor). The constituent “monomers” of the donor/acceptor complex should have absorption and fluorescence spectra differing from the free monomers. Based upon the above and other published we assign the Q, emission at 668 nm to an acceptor molecule. This is consistent with our observation that the Q, emission of hexacoordinated Chl a is red-shifted compared to a pentacoordinated Chl a monomer. The donor molecule, fluorescing at -688 nm, is expected to be pentacoordinated but cannot be directly compared to the pentacoordinated monohydrate monomer because the keto (and/or acetyl) group of the donor is linked to the magnesium atom of the acceptor molecule. Analysis of the fluorescence excitation spectra for the various bands leads us to conclude that the emissions at 668 nm and 688 nm are correlated and represent a single species. The emission spectra of a M Chl a sample in dry hexane is shown in Figure 4B. The solution yields a strong emission band positioned at 690 nm and can be tentatively assigned to fluorescence from aggregates. In these solutions, the fluorescence at 690 nm is difficult to resolve because of contributions from another fluorescence band positioned at -688 nm. The emissions at 690 and 688 nm are seen to be different from fluorescence measurements where a lod M solution is excited at 476.5 nm and where a M solution is excited at 472.7 nm (Figure 4). A weak emission band at 625 nm and a broad band extending from 704 to 770 nm are also observed if the emission is induced with the laser tuned to 436 nm.’ As well as the donor/acceptor complex, other aggregated species may be present in hexane solution. For cofacial-type Chl a dimers where the ring V keto group of one Chl a monomer is bonded to the magnesium atom of a second monomer, and vice-versa, or where the intermolecular bonds are made via water molecules between the ring V keto groups and magnesium atoms, we can place an upper limit of 726 nm to the fluorescence. Fong has assigned a 726-nm fluorescencel*~’* to a Chl a dimer, linked by water molecules directly between the macrocycles via the carbomethoxy group of C10. Interactions between the constituent “monomers” of cofacial dimers should be equal and reciprocal and can be expected to cause a red shift of the singlet energy levels as compared to a donor/acceptor complex and the monomeric species. If such “cofacial” dimers exist in hexane solution, we predict that the center of gravity of the donor’s Q, emission should be observed to the red of the 688-nm emission (for small exciton splitting of the first set of excited states). Hence we predict that the 690-722-nm spectral region could contain the Q, emissions of (1) the dry and wet “cofacial” dimers, and (2) higher aggregates that do not contain water molecules between the macrocycles. The absorption spectrum‘ of a M Chl a solution in dry hexane (not shown here) shows maxima at in the Q region at 664 and 672 nm. Titration of this solution with pyridine (Le., a nucleophile) dissociates the dimers and aggregates to form a monomeric species (Chl @pyridine). The Q, emission of this species] is at 668 nm with an absorption maximum at 659 nm. If the contribution of the pyridinated monomeric species is subtracted from the overall absorption spectrum, then the spectra of the donor molecule and the aggregated species are left over. The main absorptions in the subtracted spectrum are found at 432 and 669 nm. For the donor molecule, with a fluorescence at 688 nm and an estimated absorption at 669 nm, we project the origin of the SI (Q,) So band system at 678.5 nm. By a similar method, another donor electronic origin is projected at 447 nm (S, So). Nonlinear resonance Raman studies of lod M solutions of Chl a in hexane indicated the presence of two electronic origins about 80 cm-’ apart’ as revealed by sharp interferences in the Raman
-
-
1982, 36, 565.
(14) (a) Katz, J. J.; Norris, J. R. Curr. Top. Bioenerg. 1973, 5, 41. (b) Kooyman, R. P. H.; Schaafsma, T.J. J . Am. Chem. Sor. 1982, 106, 551. (15) Ballschmiter, K.; Katz, J . J. J . Am. Chem. SOC.1969, 91, 2661. (16) Houssier, C.; Sauer, K. J . Am. Chem. SOC.1970, 92, 779. (17) Keller, W. W. A.; Albers, B. A.; Straws, E.; Maier-Schwartz, K. J . Lumin. 1984, 31/32, 892.
(18) Fong, F. K.; Koester, V. J.; Galloway, L. J . Am. Chem. SOC.1977, 99, 2372. (19) Clarke, R. H.; Hotchandani, S . ; Jagannathan, S. P.; Leblanc, R. M. Chem. Phys. Lett. 1982, 89, 37. (20) Avarmaa, R.; Sovik, T.; Tamkivi, R.; Tonissoo, V . Stud. Biophys. 1977, 65, 213.
J. Phys. Chem. 1986,90, 4941-4945 excitation profile in the Soret region (433-436 nm). These origins are consistent with the electronic structure of a species having at least three close-lying electronic levels. A more substantial assignment of the species and chemical bonding discussed in this work awaits the outcome of the resonance Raman studies of the intensity of normal modes in the ground state, specifically of the carbonyl vibrational modes.
Concluding Remarks In this article, it has been shown that wavelength-dependent and time-resolved fluorescence spectroscopy of Chl a in hexane can be used to study the presence of various complexes including aggregated species. These results indicate the extreme usefulness of fluorescence techniques in areas where absorption spectroscopy techniques fail to discriminate between the properties of various species. The fluorescence spectra reveal mono- and dihydrated
4941
monomeric Chl a species in highly dilute hexane solutions while the monohydrated complex is also present in solutions of higher concentration. At higher concentrations evidence from fluorescence data has indicated the presence of a donor/acceptor type dimer as well as other aggregated species. The exact structure of these latter species is not known, but this species has interesting properties as observed from the resonance Raman excitation profile.
Acknowledgment. The authors thank the Natural Science and Engineering Research Council for partial funding of this research. We thank T. L. Collier for assistance in the deconvolution of the lifetimes. Also, L.V.H. thanks Dr. J. D. Cooney of the National Research Council (Ottawa) for the use of the HP8450A spectrophotometer. Registry No. Chlorophyll a , 479-61-8.
Hydrogen-Bonding Properties of a Room-Temperature Phosphorescence Cellulose Substrate Georg W. Suter,* Alan J. Kallir, Urs P. Wild, Physical Chemistry Laboratory, Swiss Federal Institute of Technology, ETH- Zentrum. CH-8092 Zurich, Switzerland
and Tuan Vo-Dinh Advanced Monitoring Development Group, Health and Safety Research Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 (Received: November 12, 1985; In Final Form: April 8, 1986)
The luminescence of heterocyclic compounds on Whatman 4 filter paper, a substrate frequently used in room-temperature phosphorimetry, is investigated with the view to characterizing the moleculesubstrate interaction. Three molecules having known spectroscopicproperties, benzo[a]phenazine, 1,4-diazatriphenyIene,and “Michlers ketone”, adsorbed on the substrate and dissolved in rigid solvents, are used as luminescent probes to investigate the nature of adsorbate-substrate interaction. The results indicate that the paper substrate provides a very polar environment with high hydrogen-bonding activity for the adsorbed species.
Introduction The phosphorescence of organic molecules at room temperature is normally difficult to observe in liquid solutions where quenching processes are many orders of magnitude more efficient than the spin-forbidden radiative transition between the lowest excited triplet state and the ground state. One of the most effective quenchers is dissolved oxygen, which is present in nondegassed M.’ In order to aliphatic solutions in concentrations up to observe phosphorescence, this quenching process and molecular deactivation mechanisms have to be eliminated either by thoroughly degassing the solution or by working in rigid solvents, where the diffusion of oxygen and other quenchers is effectively inhibited. For routine analytical applications these techniques are rather tedious and complicated, and therefore a series of simpler methods have been developed to observe phosphorescence at room temperature.2 One simple and successful procedure involves the adsorption of chromophores on solid substrates, such as filter paper or silica gel: small amounts (2-5 wL) of the sample solution are spotted on a solid substrate and thoroughly dried. Samples prepared in this manner are largely protected against deactivation of the triplet states by collisional quenching, and phosphorescence may easily be observed a t room temperature. This room-temperature phosphorescence (RTP) is usually enhanced by heavy (1) Birks, J. B. Photophysics of Aromatic Molecules; Wiley: New York, 1970. (2) Vo-Dinh, T. Room Temperature Phosphorimetry for Chemical Analysis; Wiley: New York, 1984.
atoms, either added to the solution or adsorbed onto the substrate prior to spotting. Based on this very simple and inexpensive sample preparation, RTP has become a most useful analytical technique.2 In contrast to the well-known analytical applications of RTP, little is known about the photophysical aspects related to the interactions of molecules with the substrate. Experimental observations for these interactions are diverse. Various filter paper substrates have been compared, and differences in the RTP signal of about 1 order of magnitude were found.3 Even more pronounced effects have been reported when different heavy-atom enhancers were a ~ p l i e d . ~The solvent used to spot the sample may also have an influence on the RTP signal. So far, no general rules have been established with respect to these effects. In a recent paper the electron-phonon coupling of polyaromatic molecules adsorbed on paper substrate at 4 K was s t ~ d i e d . It ~ has been reported that H-bond formation plays an important role in the immobilization of the chromophore^.^^^ The aim of our study is to investigate the particular aspect of the chromophore-substrate interaction, which is related to the formation of H bonds between the adsorbed chromophores and the substrate. The phosphorescence spectra of adsorbed probe molecules are used (3) Vo-Dinh, T.; Walden, G. L.; Winefordner, .I.D. Anal. Chem. 1977, 49, 1126. (4) Vo-Dinh, T.; Hooyman, J. R. Anal. Chem. 1979, 51, 1915. (5) Vo-Dinh, T.; Suter, G. W.; Kallir, A. J.; Wild, U. P.J . Phys. Chem. 1985, 89, 3026. (6) Schulman, E. M.; Parker, R. T. J . Phys. Chem. 1977, 81, 1932 (7) Ford, C. D.; Hurtubise, R. J. Anal. Chem. 1978, 50, 610.
0022-3654/86/2090-4941$01.50/0 0 1986 American Chemical Society
