Langmuir 1991, 7, 1483-1490
1483
Tetraarylporphyrins in Mixed Langmuir-Blodgett Films: Steady-State and Time-Resolved Fluorescence Studies Devens Gust,' Thomas A. Moore,* Ana L. Moore, David K. Luttrull, Janice M. DeGraziano, and Nancy J. Boldt Department of Chemistry and Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, Arizona 85287
Mark Van der Auweraer and Frans C. De Schryver Afdeling Organische Scheikunde, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium Received August 13,1990. In Final Form: January 23, 1991 The absorption, fluorescenceemission,and fluorescenceexcitationspectra of mixed Langmuir-Blodgett (LB) films of 5-(4-acetamidophenyl)-10,15,20-tri-p-tolylporphyrin and each of 10 different lipid diluents have been determined in order to investigate those factors that affect porphyrin association. LB films on glass of this porphyrin mixed with dioleoyl-, dielaidoyl-, or oleoylstearoylphosphatidylcholineyield absorption and fluorescencespectra similarto those of dilute solutions of the porphyrin in organic solvents, whereas other lipids give varying amounts of porphyrin aggregation. On fused silica plates, LB films of this porphyrin and the same three phosphatidylcholine diluents in molar ratios of 1:20 or 1:50 yield fluorescence decays at 655 nm, which may be analyzed as single exponentials to yield lifetimes of ca. 9.8 ns, which are similar to the lifetimes of monomeric porphyrins in organic solvents. Three other tetraarylporphyrins bearing acidic, basic, or hydrophobic substituents in place of the acetamidogroup showed evidence of aggregation when deposited as mixed films with dioleoylphosphatidylcholine.
Introduction Recent studies of dyads,lI2triads,lv3and more complex molecular devices113consisting of porphyrins covalently linked to electron acceptor and/or donor moieties have shown that such species are able to mimic certain aspects of photosynthetic energy conversion. In particular, the triads and more complex molecules can undergo photoinitiated electron transfer from the porphyrin first excited singlet state to yield energetic charge separated states in high quantum yield and with long life times'^^ (more than 300 ps in some cases'). A next step in the evolution of these artificial photosynthetic systems is to devise ways by which to gain access to the energy stored in the charge separated states in order to carry out other chemical or physical processes. In natural photosynthetic reaction centers, this is accomplished by performing the charge separation in a protein which spans a lipid bilayer. This process compartmentalizes the oxidizing and reducing equivalents, and mechanisms are provided for extracting useful work from them. The incorporation of artificial systems into lipid bilayers or monolayers has therefore been a promising area for investigation. LangmuirBlodgett (LB) films are particularly attractive candidates for study which can be formed on conducting or semiconducting substrates and therefore in principle constitute parts of electronic devices.6 To be most useful, the porphyrin-based species must usually be present in the monolayer in monomeric form (1) Gust, D.; Moore, T. A. Science 1989,244,35-41. (2) Connolly, J. S.; Bolton, J. R. In Photoinduced Electron Transfer, Part A; Fox, M. A., Channon, M., Eds.; Elsevier: Amsterdam, 1988, Chapter 6.2. (3) Gust, D.; Moore, T. A. Top. Curr. Chem., in press. (4) Gust, D.; Moore, T. A.; Moore, A. L.; Lee, S.-J.;Bitteremann, E.; Rehms, A.; Belford, R. E.; Luttrull, D. K.;DeGraziano, J. M.; Ma, X. C.; Gao, F.; Trier, T. T. Science 1990,248, 199-201.
in essentially dilute solution. There are two reasons for this. Molecular association may alter the photophysical and photochemical properties of the molecule to the point where it will no longer function in the desired way. Porphyrin aggregates, for example, usually have very short singlet excited state lifetimes (see below). In addition, singlet energy transfer, although it can lead to potentially useful antenna effects, often results in the draining of singlet excitation energy into traps and a consequent reduction in quantum yield of desirable photochemical processes. Cyclic tetrapyrroles bearing hydrophilic groups have long been known to form monolayers a t the air-water interface, which can often be deposited on glass or other substrates. Chlorophyll a monolayers were reported by Langmuir and Schaefer in 19376and later investigated by Gaines and many As might be expected, pure chlorophyll monolayers have spectroscopic properties indicative of strong interactions among the chromophores. Some porphyrins bearing hydrophilic substituents and sometimes long hydrophobic side chains form monolay(5) See, for example, (a) Kuhn, H. Thin Solid F i l m 1983,99,1-16. (b) MBbius, D. Mol. Cryst. Liq. Cryst. 1979, 52, 236-238. (c) Kuhn, M.; MBbiue, D. Angew. Chem.,Znt.Ed. Engl. 1971,10,620-637. (d) Sugi, M. Thin Solid F i l m 1987,152,306-326. (e) Fujihira, M.; Nishiyama, K.; Yamada, H. Thin Solid F i l m 1986, 132,77-82. (f) Fujihiia, M. Mol. Cryst. Liq. Cryst. 1990,183,59-69. (g) Metzger, R. M.; Schumaker, R. R.; Cava, M. P.;Laidlaw, R. K.; Panetta, C. A.; Torree,E.Langmuir 1988, 4,298-304. (6) Langmuir, I.; Schaefer, V. J. J. Am. Chem. SOC. 1937,59, 20762076. (7) Gaines, G. L., Jr. Insoluble Monolayers at Liquid-Gae Interfaces: Interscience: New York, 1966, and references cited therein. (S! Picard, G.; Munger, G.; Leblanc, R. M.; Le Sage, R.; Sharma, D.; Siemiarczuk, A.; Bolton, J. R. Chem. Phys. Lett. 1986, 129,4147, and references cited therein.
0743-7463/91/2407-1483$02.50/0 0 1991 American Chemical Society
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ers,gJO but the tetrapyrroles tend to aggregate or interact strongly. Pigment-pigment interactions can be reduced by dilution with lipids to form mixed monolayers. For example, Gaines reported that dilution of chlorophyll a with oleyl (but not stearyl) alcohol reduced the amount of aggregation." Dilution of methyl chlorophyllide with triolein12 or chlorophyll a with lecithins13 also reduced aggregate formation. In a pioneering study, Patterson and cow o r k e r ~ 'found ~ that ca. 1251 mixtures of dioleoylphosphatidylcholine and chlorophyll a give monolayers at the gas-water interface which exhibit truly monomeric properties as detected by absorption and fluorescence emission and excitation spectra. Time-resolved fluorescence emission experiments, which are sensitive probes for association, yielded a single exponential decay with a lifetime similar to that of dilute chlorophylla in solution. However, studies of similar monolayers of chlorophyll a diluted with egg phosphatidylcholine and deposited on fused silica plates gave fluorescence decays that could not be fit as single exponentials and were characteristic of mixtures of monomeric and aggregated chlorophyll.* Even so, lecithins seem to be particularly promising as diluents for reducing chlorophyll aggregation in LB films. Patterson and coworkers'& and Bolton, Leblanc, and c o - w o r k e r ~have ~~ recently extended this work to porphyrins.16 Bolton, Leblanc and co-workers reported that 5-(4-carboxyphenyl)10,15,20-tri-p-tolylporphyrindiluted 50-fold with dioleoylphosphatidylcholine yielded monolayers that, when deposited on fused silica plates, gave absorption and steady-state fluorescence excitation and emission spectra similar to those of the dilute porphyrin in organicsolvents. In addition, the fluorescence decay was analyzed as two exponentials with lifetimes of 10.7 and 2.2 ns. The amplitudes were 93 % and 7%, respectively. This encouraging result demonstrates that under some conditions, it has been possible to observe nearly monomeric behavior for tetraarylporphyrins in mixed monolayers. But which structural features of the lipid diluent, and of the porphyrin itself, are necessary for monomeric behavior? In order to investigate this question, we have used absorption, steady-state fluorescence excitation and emission, and time-resolved fluorescence emission spectroscopies to examine the aggregation of a tetraarylporphyrin bearing a neutral but hydrophilic side chain, 544acetamidophenyl)-l0,15,20-tri-p-tolylporphyrin (TTPNHAc), when present in mixed LB films with 10 different lipid diluents. In addition, we have studied porphyrins bearing a hydrophilic but basic substituent (544-aminophenyl)-10,15,20-tetra-p-tolylporphyrin, TTPNHd, a hydrophilic b u t acidic substituent (5-(4-carboxyphenyl)- 10,15,20tri-p-tolylporphyrin, TTPCOOH) , and a neutral, hydrophobic substituent (tetra-p-tolylporphyrin, TTP) as mixed LB films with one of the lipid diluents. (9) Ringuet, M.; Gynon, J.; Leblanc, R. M. Langmuir 1986,2, 700-
704, and references cited therein.
(IO)Bardwell, J.; Bolton, J. R. Photochem. Photobiol. 1984,39,735-
746, and references cited therein.
(11)Gaines, G.L., Jr.; Bellamy, W. D.; Tweet, A.
G.J. Chem. Phys.
1964,41, 538-542. (12) Gaines, G.L., Jr.; Bellamy, W. D.; Tweet, A. G. J. Chem. Phys. 1964,41, 2572-2573. (13) DBsormeaux,A.; Leblanc, R. M. Thin Solid Films 1986,132,91aa (14) (a) Agrawal, M. L.; Chauvet, J.-P.; Patterson, L. K. J.Phys. Chem. 1985,89,297%2982. (b) Boulu, L. G.;Patterson, L. K.; Chauvet, J. P.; Kozak, J. J. J. Chem. Phys. 1987,86,503-507. (c) See also Bohorquez, M.; Patterson, L. K.; Brault, D. Photochem. Photobiol. 1987,46,953-957. (15) Dick, H. A.; Bolton, J. R.; Picard, G.;Munger, G.;Leblanc, R. M. Langmuir 1988,4, 133-136. I".
al.
,. TTPCOOH : R = O -!H
TTP:R=
-CH3
Experimental Section Porphyrins "PNHAc,'~ "PNH2," "PCOOH," and TTp,l* were prepared by using methods described in the literature. Lecithinsdioleoyl-L-a-phosphatidylcholine(DOPC),distearoylL-a-phosphatidylcholine (DSPC), dielaidoyl-L-a-phosphatidylcholine (DEPC),and j3-oleoyl-ystearoyl-L-a-phosphatidylcholine (OSPC) as well as stearic, arachidic, oleic, elaidic, linoleic, and brassidic acids were obtained from Sigma Chemical Co. and used as received. Monolayers were prepared on a circular Teflon trough of the Fromherz type. Solutions of the lipids (ca. 1 X lo4 M in chloroform) containingthe appropriate porphyrin were applied dropwise to the clean subphasesurfaceby syringe and the monolayer was then compressed by the moveable Teflon barrier at a rate of 2.4 X 1Oam2s-l. The subphase was pure (MilliporeMilli-Q system),unbuffered distilled water,pH ca. 5.3, or 5 X l(r CdCl2, pH ca. 5.3, as noted, at 21 f 1O C . The glass or fused silica elides for deposition of the monolayers were cleaned by washing with dichloromethane, heating at 110 O C for 2 h in concentrated chromic acid, rinsing repeatedly in Milli-Q purified water, and soaking in M NaOH in Milli-Q purified water for 24 h. The slides were then rinsed 8 times with Milli-Q purified water and kept in the final rinse until used. For deposition of LB films, the slide waa immersed in the subphase and the monolayer was formed and compressed to 25 mN m-l, unless otherwise noted. The slide was then withdrawn at a rate of 1.5 X lo-' ms-'. This procedure resulted in a single monolayer on each side of the slide. For time-resolved fluorescence experimentscarried out with a microchannel plate photomultiplier based spectrometer (see below), slides were cleaned employing a slightly different pro~edure.~e~~ The slides were boiled for 15 min each in chloroform and isopropyl alcohol, respectively. After rinsing well in pure Milli-Q water, the slides were immersed for 3 min in a stirred solution of H2SOd:H202 (30 7% so1ution):HaO (4:l:l volume ratio), rinsed 10 times in Milli-Q water, and subsequentlyplaced for 1min in a stirred solution of 1:20 (volume ratio) H202(30% solution):H20 containing 2 g of NaOH per 100 mL of solution. The slides were then rinsed 10times in and stored under Milli-Q water untilused. Monolayer deposition on theseslideswas similar to that described in the foregoing paragraph but resulted in a (16)Gust, D.; Moore, T. A.; Liddell, P. A,; Nemeth, G.A,; Makings, L. R.; Moore, A. L.; Barrett, D.; Pessiki, P. J.; B e n w o n , R. V.;Rougb, M.; Chachaty, C.; De Schryver, F. C.; Van der Auweraer, M.;H o h a r t h , A. R.; Connolly, J. S. J. Am. Chem. SOC.1987,109,846-866. (17) Little, R. G.;Anton, J. A.; Loach, P. A.; Ibers, J. A. J.Heterocycl. Chem. 1975,12,343-349. (18)Fuhrhop, J.-H. In Porphyn'ne and Metalloporphyrine, Smith, K. M., Ed.; Elsevier: Amsterdam, 1975; pp 769-770. (19) Tredgold, R. H.; Smith,G.W.Thin Solid Film 1983,99,215220. (20) Jones, R.; Tredgold, R. H.; Hoorfar, A.; Hodge, P. Thin Solid Films 1984, 113, 115-128.
Langmuir, Vol. 7, No. 7, 1991 1485
Tetraarylporphyrins in Mixed LB Films monolayer being deposited on only one side of the slide (double dipping mechanism). Absorption spectra were obtained on an SLM Aminco DW2000 UV-VIS spectrophotometerusing a single slide,a reference slide bearing no porphyrin pigment, and a band-pass of 3 nm. Steady-statefluorescence spectra were obtained for single slides on a Spex Fluorolog using the geometry described by Whitten and co-workers.*l For excitation spectra, the excitation bandpass was 4.5 nm and the emission band-pass was 9 nm. For the corrected emission spectra, the excitation band-pass was 9 nm and the emission band-pass 4.5 nm. Fluorescence decays, with excitationat 590 nm, were obtained by using the time-correlated single photon method. The single decays were measured on an instrumentnwhose excitation source was a mode-locked argon ion laser coupled to a synchronously pumped, cavity dumped dye laser,which featureddetectionusing a conventional Philips XP2020 photomultiplier. The slide was placed at an angle of ca. 20° with respect to the excitation beam, so that reflected light was directed away from the monochromator. Detection was at 655 nm, as selected either by the monochromatoror by a 655-nm band-passinterferencefilter. In some cases (noted in the text), decays and decay associated spectra were determined by using a second instrumentBwith excitation via a frequency doubled, mode-locked Nd-YAG laser coupled to a synchronously pumped,cavity dumped dye laser and detection by a microchannel plate photomultiplier (Hamamatau R2809U01). The instrument response time was ca. 35 ps, and no interference filters were used. Results Monolayers. The porphyrin with the neutral, hydrophilic side chain, TTPNHAc, was studied as amixed monolayer with each of the 10 lipid diluents. The subphase was 5X M CdCl2 a t 21 f 1 OC. The pH was ca. 5.3 in all cases, although exact determination and maintenance of pH was difficult in the essentially unbuffered solutions. A lipid diluent to porphyrin molar ratio of 50 to 1 was used in all cases. Of course, this ratio would not lead to identical average interporphyrin spacings even when the pigment molecules were randomly dispersed, as the lipids have different areas per molecule a t any given surface pressure. The resulting surface pressure-area isotherms are shown in Figure 1. High accuracy in the area-per-molecule determinations cannot be claimed due to the small amounts of materials available. However, the shapes of these isotherms and the areas per molecule at 25 mN m-1 are in general not very different from those reported in the literature for pure DOPC,24-26OSPC,26-27DSPC,2k27 the saturated fatty acids: and oleic and elaidic acids.28 The conditions of pH, subphase, and temperature in the literature studies differed somewhat from those reported here. At the 50 to 1 dilution ratio, the presence of the porphyrin would be expected to have only a small effect upon the area per molecule in the monolayer, relative to the pure lipid. Inspection of Figure 1 shows that films with three types of isotherms are represented. These will be called class (21) (a) Mooney, W.; Brown, P.; Russell, J.; Coeta, S.; Pedersen, L.; Whitten, D. J. Am. Chem. SOC.1984,106,5659-5667. (b) Mooney, W.; Whitten, D. J. Am. Chem. SOC.1986,108,5712-5719. (22) Boens, N.; Van den Zegel, M.; De Schryver, F. C. Chem. Phys. Lett. 1984,111, 340-346. (23) Gust, D.; Moore, T. A.; Luttrull, D. K.; Seely, G. R.; Bittersmann, E.; Bensasson, R. V.; RougBe, M.; Land, E. J.; De Schryver, F. C.; Van der Auweraer M. Photochem. Photobiol. 1990, 51, 419-426. (24) Ducharme, D.; Salesee,C.;Leblanc, R. M. Thin SolidFilms 1985, 132,83-90. (25) Demel, R. A.;van Deenen, L. L. M.; Pethica, B. A. Biophys. Biochim. Acta 1967,135, 11. (26) Eibl, H.; Demel. R. A.; van Deenen. L. L. M. J. Colloid Interface Sci. 1969, 29, 381-387. (27) Van Deenen, L. L. M.; Hontemuller, U. M. T.; De Haas, G. H.; Mulder, E. J. Pharm. Pharmacol. 1962, 14,429. (28) Feher, A. I.; Collins, F. D.; Healy, T. W. A w t . J. Chem. 1977,30, 511-519.
50 I
-10 I
0
20 40 60 80 100 Area per Molecule (Square Angstroms)
I 120
Figure 1. Surface pressure-area isotherms for mixed monolayers of 501 molar ratios of diluent lipid to TTPNHAc. The diluents are (a)stearic acid, (b) brassidicacid, (c)arachidicacid, (d) elaidic acid, (e) linoleic acid, ( f ) oleic acid, (g) DSPC, (h) OSPC, (i) DEPC, and (i) DOPC. The subphase is 5 X lo-' M CdClz in water, pH ca. 5.3, at 21 f 1 O C .
I, class 11, and class I11 for convenience. Class 1 films comprise the mixtures of TTPNHAc with stearic, arachidic, and brassidic acids, and with DSPC. These mixtures all give condensed-filmtype isotherms with steep pressure area plots. Oleic, elaidic, and linoleic acids give class I1 behavior, which is characterized by curved, liquidexpanded isotherms with areas per molecule of 25-30 A2 at 25 mN m-l. Class I11 films from DOPC, OSPC, and DEPC feature very expanded liquid-type isotherms with areas per molecule greater than 50 A2 at 25 mN m-l. SpectroscopicProperties of LB Films. As described in the Experimental Section, LB films from the mixed monolayers of TTPNHAc and the 10 lipid diluents were deposited on glass slides. For this phase of the study, the subphase was 5 X lo4 M CdC12,pH ca. 5.3, and the surface pressure was maintained a t 25 mN m-l during deposition. For elaidic acid, the deposition was at 15 mN m-l because the monolayer was not very stable at the higher pressure. Only slides displaying a deposition ratio of 1:l within experimental error were used for spectroscopic studies. Pure TTPNHAc, when spread on the same subphase, gave a very rigid monolayer which tended to collapse in the region of the moving barrier as the surface pressure was increased. However, the monolayer far from the barrier appeared normal to the eye, and a film of pure TTPNHAc from this region was deposited on a glass slide at 15 mN m-l. The LB films appeared to be stable to ambient light and air for many days. Similar stabilities were noted by Bolton, Leblanc, and co-workers.15 Absorption Spectra. Absorption spectra of the single LB films on the glass slides could be obtained with reasonable signal to noise in the Soret region. Figure 2 shows some typical spectra. The spectra have been normalized at the maxima, and the negative absorptions around 380 nm, which are seen in some cases, are due to a slight mismatch between the slide with the LB film and a reference slide with no porphyrin pigment. Figure 2 illustrates that in dichloromethane solution, TTPNHAc has a Soret band with a maximum a t 420 nm and a width a t half maximum of 730 cm-1. The LB film of pure TTPNHAc on glass has a broad Soret at 435 nm with a halfwidth of ca. 1800 cm-', which is characteristic of aggregated porphyrins.15 The spectrum of the LB film of 5 0 1 DOPC: TTPNHAc resembles that of the solution, with a Soret maximum of 421 nm and a width at half maximum of 790 cm-l. The Soret bands of the films of 50:l DSPC:TTPNHAc and arachidic acidTTPNHAc have maxima at 432
1486 Langmuir, Vol. 7, No. 7, 1991
Gust et al.
1.100,
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0.700 0.500 C 1-
.u
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380
400
420
440
460
480
600
500
620
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Figure2. Normalized absorption spectraof TTPNHAc in dichloromethane (a),as a pure LB film on a glass substrate (e),and in mixed LB films on glass consisting of 501 molar ratios of DOPC (b),DSPC (c),and arachidicacid (d)to porphyrin. The negative absorptionsin the 380-nmregion are due to a poor match between the sample and a reference film in this region.
and 434 nm, respectively, and are broad, as was observed for the pure porphyrin film. All of the class I11 films had Soret spectra similar to that of the D0PC:TTPNHAc mixed film (maximum at 421 nm). All of the class I films, on the other hand, had spectra that resembled those for DSPC and arachidic acid mixed films in Figure 2 (maximum at 433 nm for the stearic and brassidic acid mixed monolayers). The spectra of class I1 films (oleic, elaidic, and linoleic acid diluents) all featured a major band a t 421 nm with a shape similar to that of the DOPC mixture in Figure 2, but with a minor shoulder in the 440-nm region. The relative intensity of this shoulder varied from slide to slide, and sometimes it could not be distinguished from noise. The four porphyrin Q bands were observable in most of the films a t 5 0 1 dilution, but the signal-to-noise ratio was very poor. However, their observation does indicate that the porphyrin was still present in the free base form. In this connection, it was found that stirring TTPNHAc with a large excess of CdC12, water, chloroform, and arachidic acid for 24 h did not result in metalation of the porphyrin. In summary, the visible absorption results suggest that under the conditions of the experiment, class I films and pure TTPNHAc films feature essentially aggregated porphyrin, class I11 films contain monomeric unassociated porphyrin, and class I1 films feature both associated and monomeric porphyrin. The 50:l D0PC:TTPNHAc mixture was used to investigate the effect of substrate, subphase, and dilution ratio upon the absorption spectra. Changing the subphase from 5 X 10"' M CdC12 to unbuffered water at pH ca. 5.3 gave identical absorption spectra within experimental error ( f l nm), as did changing the substrate for deposition to fused silica. Deposition onto two monolayers of pure arachidic acid on glass resulted in a ca. 1-nm red shift of the Soret band but did not affect the bandwidth significantly. Changing the D0PC:TTPNHAc ratio to 2 0 1 did not lead to any observable change in the position or shape of the Soret band. However, at 1O:l the maximum shifted to 422 nm and the band broadened slightly to 890 cm-1. A t 51 the Soret appeared at 423.5 nm with a width a t half maximum of 106Ocm-l. At 5:l the Q bands were observable at 515,553,595, and 649 nm. In dilute solution in dichloromethane the corresponding values are 516,552,592, and 647 nm. Steady-State Fluorescence Spectra. The fluorescence emission and excitation spectra of the LB films on
660
680
700
720
740
Wavelength (nm)
Wavelength (nm)
Figure 3. Corrected fluorescence emission spectra of TTPNHACwith excitation at 420 nm. The spectra are from (a) pure TTPNHAc in dichloromethane, (b)an LB film on glass consisting of 50:l DOPC:TTPNHAc, (c) an LB film on glass consisting of 501 DSPC:n'PNHAc, and (d) an LB film on glass Consisting of 501arachidicacid:l'TF"HAc. The intensitiesof the emissions from the LB films are the relative intensities for single plates, all with the same geometry in the fluorometer and measured within a few minutes of one another. The solution spectrumhas been normalized to the DOPC curve at 655 nm.
0
600
620
640
660
680
700
720
740
Wavelength (nm)
Figure 4. Corrected fluorescence emission spectra of LB films on glass consisting of (a) 501 linoleic acid/TTPNHAc, (b)501 oleic acid/TTPNHAc, and (c) 501 DOPC/TTPNHAc. The excitation wavelength was 420 nm, and the spectraare normalized at ca. 655 nm.
glass prepared from the 1:50 dilutions of TTPNHAc with the 10 lipids were also studied. Some typical corrected emission spectra with excitation a t 420 nm appear in Figure 3. It may be seen from the figure that a dichloromethane solution of TTPNHAc and the 50:l LB film with DOPC as diluent yield identical emission spectra, within experimental error. Two maxima are observed a t 655 and 720 nm in a ratio of intensities of ca. 1.6:l. This was also observed for the other class I11 films. The class I films (represented by DSPC and arachidic acid in Figure 3) all have very weak emission. This was also observed for the LB film of the pure porphyrin. The two maxima are still present, but the signal-to-noise ratio is verylow. The class I1films feature emission spectra in which the 720-nm band is roughly equal to the 654-nm band in intensity (e.g. Figure 4). The fluorescence excitation spectrum of an LB film on glass with a 5 0 1 molar ratio of D0PC:TTPNHAc (emission at 655nm) is identical with the absorption spectrum, within experimental error, with a maximum a t 421 nm (Figure 5). Scanning the excitation monochromator from 430 to 620 nm revealed maxima corresponding to three Q bands at 515, 553, and 593 nm. The other class I11 films gave similar results. The class I films have very weak, broadened excitation spectra with apparent maxima in the 428-435-
Tetraarylporphyrim in Mixed LB Films
Langmuir, Vol. 7, No. 7, 1991 1487 1 1
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Figure 5. Corrected,normalized fluorescenceexcitation (a) and absorption (b) spectra of an LB film on glass consisting of 501 DOPC/TTPNHAc. The fluorescence emission was detected at 655 nm. nm region. The class I1 films (oleic, linoleic and elaidic acids) yielded excitation spectra with maxima at 421 nm which resembled those of the class I11 films. Thus, in steady-state fluorescence emission and excitation, as well as absorption, the class I11 films resemble monomeric porphyrin in solution, the class I films appear to contain mostly aggregated porphyrin, as does the pure porphyrin film, and the class I1 films demonstrate both types of behavior simultaneously. The choice of subphase and substrate has little effect upon the fluorescence properties of the DOPC films. The 50:l D0PC:TTPNHAc LB films deposited from 5 X 10" M CdC12 solution and from pure water have identical emission and excitation spectra, within experimental error, and films on glass and fused silica yield essentially identical emission and excitation spectra. Films deposited on a glass slide covered with two monolayers of pure arachidic acid have emission spectra identical with those of films deposited directly on glass but excitation spectra that are red shifted by ca. 1 nm. The steady-state emission spectra of the LB films on glass slides with 50:1, 20:1, l O : l , and 5:l molar ratios of DOPC to porphyrin were essentially identical in shape, with maxima at 655 and 720 nm. (At the lower lipid-pigment ratios, singlet-singlet energy transfer effects might be expected to influence the fluorescence decay times.) Excitation spectra for the 501 and 20:l dilutions had maxima a t 421 nm, but those for the 1O:l and 5:l dilutions were slightly red shifted to 422 and 424 nm, respectively. Similar behavior was noted in the absorption spectra (see above). Time-Resolved Fluorescence Emission Spectra. Time-resolved fluorescence spectroscopy is a valuable tool for the study of LB films because it allows detection of associated or aggregated species with emission spectra very similar to those of the monomeric species but having different lifetimes and of short-lived states that do not contribute much intensity to the steady-state spectra. These associated species manifest themselves through changes in the rate constants for exponential decay components or the appearance of multiexponential or nonexponential decays. Time-resolved emission studies were therefore undertaken with some of the mixed monolayer systems discussed above. In order to reduce any potential interference from foreign species, the LB films for the time-resolved work were prepared from monolayers formed on pure water and deposited as single layers directly onto fused silica slides. As discussed above, the presence or absence of cadmium ions and the substitution of fused silica for glass had no significant effects on the absorption
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Figure 6. Decay of fluorescence from an LB film on fused silica consisting of 201 DOPC/TTPNHAc,the associated instrument response function, and the best fit to the data using a single exponential decay function. The spectrometer featured conventional photomultiplier detection. Excitation was at 590 nm and detection was at 655nm using a band-passinterferencefilter. A total of loo0 counts were collected in the highest channel, and each channel corresponds to 0.120 ns. The goodness of fit is shown by the plots of residuals, the autocorrelation function, and the heterosedacity as well as by the x2 value of 0.97. and steady-state emission and excitation spectra of DOPC/ TTPNHAc mixed LB films. The initial studies were undertaken with 20:l mixtures of DOPC and TTPNHAc using the conventional photomultiplier based apparatus described in the Experimental Section. Excitation was at 590 nm into a porphyrin Q band, and emissiondecays were measured at 655 nm. Decay times were determined by fitting the experimental decay to a convolution of an instrument response function including a correction for scattered light and a sum of exponential^.^^ The instrument response function was determined by scattering light off the slide at the excitation wavelength. The combination of the very low emission intensity from the two single monolayers (one on each side of the slide) and the necessarily high intensity of the exciting laser light made the elimination of artifacts at early times in the decay due to scattered light difficult. These artifacts include not only scattered light itself striking the detector but also weak emission from components of the spectrometer excited by the scattered light. However, with very smooth, scratch-free slides and careful reduction of the amount of scattered light, good results were obtained. For 20:l D0PC:TTPNHAc films, the decays could be satisfactorily analyzed as single exponentials. Figure 6 shows a typical example. The reduced x2goodness-of-fit parameter was 0.97 for this experiment. Analysis of 14 decay curve-instrument response function pairs yielded an average fluorescence lifetime of 9.8 f 0.4 ns with an average x2of 1.13. In nondeoxygenated dichloromethane solution, TTPNHAc has a lifetime of 8.0 ns. Although in most cases it was possible to slightly improve the x2 value by analysis as a two-exponential decay (for the 14 experiments mentioned above, analysis as two exponentials gave an average x2 of LOB), the amount and lifetime of the minor component varied substantially from experiment to experiment, and there is no evidence for (29) Van den &gel, M.; Boens,N.;Daems, D.;De Schryver, F. C. Chem. Phys. 1986,101,311-335.
Gust et al.
1488 Langmuir, Vol. 7, No. 7,1991 0.25
Ri I
0.20
I
0.15 ~
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I
=0
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-J 0
0.05 1.0
0.00
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Figure 7. Decay of fluorescence from an LB film on fused silica consisting of 201 DOPC/TTPNHAc,the associated instrument response function, and the best fit to the data using a single exponential decay function (9.4 ns). The spectrometer in this case featured microchannel plate detection. Excitation was at 590 nm, and detection was at 655 nm as selected by a monochromator. Each channel correspondsto0.0103 ns. Thegoodneas of fit is shown by the plot of the residuals and the ~2value of 1.09. the presence of a physically significant second exponential component in the porphyrin decay. Time-resolved experiments were also performed with LB films containing 50:l ratios of D0PC:TTPNHAc on fused silica. With these slides, emission intensities were lower,and deviations from exponentiality at the beginning of the decay due to the effects of scattered light were more difficult to eliminate. However, with care, reasonable results could be obtained. For example, four experiments with DOPC as the diluent yielded single exponential fits to the data with an average lifetime of 9.6 f 0.1 ns and an average x2of 1.22. In this case, two-exponential analyses gave an improvement in the average x2 ( L l l ) , but the lifetime of the shorter component varied from 0.60 to 2.96 ns. The effect of two-exponential analysis is mainly to help correct for scatter artifacts, and there is no good evidence for the presence of a second component in the porphyrin fluorescence decay. Similar time-resolved experiments were carried out with the microchannel plate based fluorescence spectrometer, which has a considerably shorter instrument response function. Excitation was at 590 nm. In this case, decay times were determined by fitting the experimental decay to a convolution of an instrument response function and a sum of exponentials, with no correction for scattered light. The emission decay a t 655 nm from fused silica slides bearing a single LB film containing a 20:l ratio of D0PC:TTPNHAc could be satisfactorily analyzed as a single exponential with a lifetime of 9.4 ns (x2 = 1.09, Figure 7). Analysis as two exponentials did not significantly improve the fit. Continued irradiation of the slide with the relatively intense light from this laser system resulted in the appearance of a second component in the decay, which had a lifetime of about 0.5 ns. This component increased in amplitude with continued irradiation and, evidently, represents light- or heat-induced disruption of the monolayer and/or porphyrin pigment. The decays for these LB films were also measured a t seven different wavelengths in the 645-735 nm region using the microchannel plate based instrument, and fit to two exponentials using a global analysis technique.30 The results were scaled to the steady-state emission spectrum to yield the decay-associated spectrum. A typical result is shown in Figure 8. The global x 2 value was 1.10. The (30)Wendler, J.; Holzwarth, A. R.Biophys. J. 1987,52, 717.
’
-0.05
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655
675
695
715
735
Wavelength (nm) Figure 8. Decay associated spectra resulting from excitation at 590 nm of an LB film on fused silica consisting of a 20:l mixture of DOPC/TTPNHAc. Decays were measured at seven wavelengths,and the data were fit by using a global analysis technique. The global x2 value was 1.10. major portion of the decay has maxima corresponding to emission from the porphyrin and a lifetime of 9.0 ns. The minor component, with a lifetime of 0.47 ns, is an artifact related to the laser-induced disruption of the sample mentioned above (thedecay a t 655 nm, for example, cannot be fit by a single exponential). The relative amplitude of this component at each wavelength increases with the irradiation time, and with long or repeated irradiation it can become a significant part of the decay. In a global analysis of results from a similar LB film on fused silica containing a 50:l ratio of DOPC:TTPNHAc, the second component ( T = 0.38 ns) made up a substantially larger fraction of the decay. Fused silica slides bearing LB films of 201 0SPC:TTPNHAc were studied by using the conventional photomultiplier based instrumentation and also gave decays at 655 nm, which could be satisfactorily fit as single exponentials. Six sample decay-instrument response function pairs gave an average lifetime of 9.6 f 0.1 119 with an average x2 of 1.07. As in the case of DOPC, only slightly better x2 values (average x2 = 1.02) could be obtained by twoexponential fits, which retain the 9.6-11s component but include from 10 to 21 % by initial amplitude of a second component with a lifetime ranging from 0.7 to 1.3 ns. The LB films containing 501 ratios of DEPC and OSPC to TTPNHAc gave similar results, with single exponential decay lifetimes of 10.2 ns (average x2 of 1.12 for three experiments) and 9.6 ns (average x2 of 1.21 for three experiments), respectively. Two exponential analyses again gave slightly better fits, but the lifetimes for the shorter components varied from 0.5 to 1.9 ns and their amplitudes from 12% to 44%. Time-resolved studies of fused silica plates bearing LB films containing 1OO:l molar ratios of oleic or linoleic acid to TTPNHAc gave quite different results. In these two cases, the films were considerably less emissive than those of class 111, and only about 300 counts were collected in the highest channel. Analysis as single exponential decays proved completely unsuitable. Analysis as two exponentials gave reasonable fits with no systematic errors. For 1OO:l oleic acid:TTPNHAc, lifetimes of 8.8 and 1.7 ns with amplitudes of 40% and 60%,respectively, were measured with a x2 of 1.14. In the case of 1OO:l linoleic acidTTPNHAc, the lifetimes were 8.8 and 1.3 ns, with amplitudes of 24% and 76% and a x2 of 1.16. Because of the low signal-to-noise ratio, these lifetimes are relatively impre-
Tetraarylporphyrins in Mixed LB Films cise, and it is possible that the decays could be fit by functions other than sums of two exponentials. Porphyrin Structure. Given the results for TTPNHACdiscussed above, it was of interest to investigate the behavior of other porphyrins in the mixed LB f i i . Monolayers of 5 0 1 DOPC and TTPNH2, TTPCOOH, or TTP, were prepared on pure water and deposited on fused silica slides as described in the Experimental Section. The deposited films were studied by using absorption and fluorescence spectroscopies. In dichloromethane solution, TTPNH2 hasa Soret band with a maximum at 421 nm and a width at half maximum of 840 cm-l. The same molecule in an LB film as a 1 5 0 mixture with DOPC has a Soret maximum at 421 nm and a width of ca. 900 cm-l. With emission a t 655 nm, the fluorescence excitation spectra in solution and the monolayer are identical, within experimentalerror, with maxima a t 423 nm. The corrected emission spectra differ slightly, however. Both have two emission maxima at 659 and 723 nm, but the ratio of these maxima is 1.7:l in dichloromethane solution and only 1 5 1 in the LB film. Timeresolved studies of a fused silica slide bearing an LB film containing a 20:l ratio of D0PC:TTPNHz were performed by using the microchannel plate based instrument. Excitation at 590 nm yielded decays at 655 nm which could not be satisfactorily fit as single exponentials. Twoexponential fits gave components of 6.7 ns (73 %) and 0.46 ns (27%) with a x2 value of 1.01. In dichloromethane solution, TTPNH2 yields a single exponential decay with a lifetime of 7.7 ns. Dichloromethane and chloroform15 solutions of TTPCOOH have Soret bands with maxima at 420 nm and narrow widths similar to those reported for TTPNHAc and TTPNH2. In 5 0 1 mixed films with DOPC as the major component (deposited on fused silica at 20mNm-9, Bolton, Leblanc and co-workersnote15that the absorption and fluorescence excitation spectra are nearly identical with those in solution. Emission spectra in solution and the film are very similar, but in the film, a slight decrease in emission intensity, relative to the maxima, in the 660710 nm region was reported. We have observed the same behavior in our laboratories. Bolton, Leblanc and coworkers report that a careful series of measurements on similar films yielded two-exponential decays with lifetimes of 10.7 ns (93%)and 2.2 ns (7% of the decay). LB films could also be prepared from monolayers with 50:l molar ratios of DOPC and TTP itself. In dichloromethane solution, TTP spectra feature a narrow Soret band at 419 nm similar to those of the other porphyrins. In the films, however, a broader band with a maximum at 440 nm was observed, which has a shoulder with about 40% of the intensity of the main band at ca. 422 nm. The fluorescence emission spectrum in solution with excitation a t 420 nm has the usual two maxima a t 655 and 720 nm in a ratio of 1.65:l. In the film, the maxima occur at 653 and 720 nm in a ratio of 2.3:l. Excitation spectra with detection at 655 nm resemble the absorption spectra. In solution, a narrow intensity profile with a maximum at 420 nm is observed, while the LB film gives a broader profile with a peak at 438 nm and a shoulder at ca. 420 nm. Time-resolved emission studies were very difficult due to the low emission intensity of the sample, but most of the intensity decayed within 1 ns, and the emission was seen to consist of a t least two and very likely more components.
Discussion With respect to the effect of diluent lipid on TTPNHACassociation in LB films, it is clear that the three groups
Langmuir, Vol. 7, No. 7, 1991 1489 of lipids, classified on the basis of the surface area-pressure isotherms, behave differently from one another. The class I LB films from stearic, arachidic, and brassidic acids and DSPC, under the conditions of this study, give absorption spectra which differ considerably from that of the porphyrin in dichloromethane solution and resemble that of aggregated porphyrin in LB films of the pure porphyrin. The fluorescence emission and excitation spectra are also characteristic of aggregation, and emission is very weak. Class I11 films from DOPC, OSPC, and DEPC, on the other hand, have absorption and steady-state fluorescence emission and excitation spectra that are very similar to those of monomeric porphyrins in solution. In addition, these films give fluorescence decays that are satisfactorily fit as single exponentials at the porphyrin emission maximum. The lifetimes measured are about the same as those of porphyrin monomers in solution. The class I1 films from oleic, linoleic, and elaidic acids have absorption and steady-state and time-resolved fluorescenceproperties that seem to be superpositions of the two types of behavior mentioned above. The absorption and fluorescence excitation, emission, and decay data have features that resemble monomeric porphyrins, but the red-shifted shoulders usually observed in the absorption spectra of the Soret region, the altered ratio of intensities of the two emission bands, and the impossibility of fitting the fluorescence decays as single exponentials are reminiscent of the behavior of aggregated porphyrins. Taken together, this evidence suggests that in the class I11films the porphyrin is essentially all in monomeric form with no significant association of the pigments. Class I films contain porphyrin which is nearly all aggregated in domains of unknown size. Class I1films appear to contain a mixture of monomeric and aggregated porphyrin molecules. The monomeric behavior observed in the class I11 films is essentially insensitive to the presence of cadmium ions in the subphase and the nature of the substrate (glass, fused silica, arachidic acid). It is interesting to speculate upon the structural features of the lipid diluent which appear to be necessary to ensure monomer-type behavior in the LB films. It is perhaps easiest to say what is not sufficient. Neither cis nor trans double bonds in the hydrophobic chains guarantee monomeric behavior, as demonstrated by the results with brassidic acid and the three class I1 acids. Also, none of the lipids with single hydrophobic chains gave completely monomeric behavior. The phosphatidylcholine moiety was necessary for monomeric behavior within the bounds of this study, but not sufficient, as illustrated by the DSPC films. Within the group of lipids demonstrating class I11 behavior, all had double bonds in the fatty acid chains, but it did not seem to matter whether there were one or two double bonds or whether they were cis or trans. Indeed, the best predictor of film formation with monomeric porphyrin appears to be the isotherm itself, rather than any particular lipid structural features. Lipids giving steep, condensed-type isotherms invariably led to aggregated porphyrin regardless of lipid structure. The lipids of class I1 with more liquid-expanded isotherms evidently gave some monomeric porphyrin, but also featured aggregated regions. The phosphatidylcholines which gave strongly curved isotherms characteristic of strongly liquidexpanded behavior were the only diluents to produce monomeric porphyrin. These lipids, unlike the saturated fatty acids and other class I diluents,do not readily pack together to form stable, ordered arrays in the monolayer, and therefore they evidently do not tend to "squeeze out" the porphyrins. In addition, the behavior of the class I1films
1490 Langmuir, Vol. 7, No. 7, 1991 shows that monomeric and associated porphyrin can evidently coexist in the deposited monolayer. Thus, it is possible that the class I11phosphatidylcholinesalso act to prevent formation of porphyrin aggregates. Of course, the spectroscopicdata directlyrelate only to the deposited LB films, whose structures may differ from those of the monolayers at the air-water interface. With respect to porphyrin structure, it seems probable that at least in the absenceof long, hydrophobic side chains, the tetraarylporphyrins must have hydrophilic groups in order to display monomeric behavior in these mixed monolayers. TTP itself was strongly aggregated in DOPC mixtures, whereas the other porphyrins were not. It is also interesting to note that of the three porphyrinsbearing hydrophilic groups, only the one with the neutral acetamido group, which cannot demonstrate acidic or basic behavior in the pH region of interest, gave acceptable single exponential fluorescence decays a t 655 nm. It is evident that more experiments with a wider variety of porphyrin structures will be necessary before one may predict with certainty which porphyrins will tend to yield single exponential behavior and which will not.
Gust et al.
Clearly, many unanswered questions concerning the behavior of porphyrins in LB films remain. However, the monomeric behavior of TTPNHAc in the class I11 films and the encouraging results for TTPCOOH reported by Bolton, Leblanc, and c o - w ~ r k e rsuggest s ~ ~ that meaningful electron and energy transfer studies of porphyrin-based molecular species may be performed and that the results may be related to solution behavior.
Acknowledgment. This work was supported by the National Science Foundation (CHE8903216, INT-8701663; D.G. and T.A.M.). The time-resolved fluorescence apparatus a t Arizona State University was constructed with a grant from the US. Department of Energy University Research Instrumentation Program (DE-FG05-87ER75361). This is publication No. 65 from the Arizona State University Center for the Study of Early Events in Photosynthesis. The center is funded by U.S. Department of Energy Grant No. DE-FG02-88ER13969as part of the USDA/DOE/NSF Plant Science Center program. D.G. thanks F.C.D.S. for his hospitality and support during a sabbatical stay at KUL.
