Langmuir 1994,10, 2339-2343
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Aggregate Formation of Crocetindialdehyde in Langmuir-Blodgett Film: A Spectroscopic Study P. Pal, A. K. Dutta, A. J. Pal,?and T. N. Misra* Department of Spectroscopy and Department of Solid State Physics, Indian Association for the Cultivation of Science, Jadavpur, Calcutta 700 032, India Received January 3,1994. In Final Form: April 25, 1994@ The miscibility of crocetindialdehyde (CDA) and stearic acid (SA) in air-water subphase has been studied from the surfacepressure-area isotherms by using the surface phase rule and excess area criterion. Positive deviation from the additivityrule and positive values of Gibb's free energy indicate strong repulsive interaction between CDA and SAmolecules. This repulsiveinteraction may facilitate aggregateformation of CDA in air-water subphase. CDA has been incorporated into Langmuir-Blodgett (LB) films when mixed with SA. Absorption and emission spectra of CDA have been studied in solution,in LB films, and in an ethanol-water mixture. The resemblence between the absorption spectrum of CDA in a ethanolwater mixture and in LB film supports aggregate formation of CDA. Dual fluorescence of CDA has been observed in solution and in LB films and for the first time in an organized molecular layer of a carotenoid. A comparison of emission and absorption spectra of CDA in solution and in LB films confirms formation of aggregates in LB films. The aggregate formation of carotenoids has been discussed in the framework of possible self-organizationin biological membranes.
Introduction Carotenoids are one of the most important constituent molecules in biological systems. Particularly seen in photosynthetic unit,l in vertebrate retina macula,2s3and in cone oil droplets of some animals: they are linked to photoprotection and photosensitive pigment^.^^^ Because of their great ability to absorb light due to their polyenic chain, carotenoids can easily be studied by photomeric methods. Yet, their spectroscopic properties are far from clear. It has been observed that carotenoids undergo changes in their molecular state which are associated with changes in their spectroscopic proper tie^.^^^ The monolayer model (Langmuir-Blodgett film) is known to match the packing pattern of molecules in biological membranes of similar structures. Energetically low-lying triplet excitations from chlorophyll protect the organism from damage due to the formation of reactive singlet molecular oxygen.' The other role of the carotenoids is to absorb solar energy and transfer the excitations to the lower energy region where chlorophyll pigments absorb for use in p h o t o s y n t h e s i ~ . ~ It ~ ~is J~ believed that clusterdaggregates of carotenoids in lipid chlorophyllmatrix exist in biological membranes.11J2Such aggregates and their excited states greatly influence the energy and electron transfer efficiencies in photosynthesis. Hence, studies of aggregates of such carotenoids in LB Department of Solid State Physics. Abstract published in Advance ACS Abstracts, June 1,1994. (1)Cogdell,R.J.;Frank,H.A. Biochim. Biophys.Acta 1987,63,896. (2) Nishimura, M.; Takamatsu, K. Nature 1967, 180, 699. (3) Snodderly, D. M.; Brown, P. K.; Delory, F. C.; A m , J. D. Invest. t
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Ophthulmol. Vis. Sci. 1984,25,660. (4) Liebman, P. A. In Handbook ofsensory Physiology, Vol.WI, Part I , Photochemistry of Vision; Dartnall, H. J. A., Ed.; Springer-Verlag: . Berlin, 1972; pp-481. (5) Fujimuri, E.; Livingston, R. Nature 1967,180, 1036. (6) Claes, H.; Nakayama, T. 0. M. 2.Natul.forsch. 1969,14b, 746. (7) Yamamoto,H. T.; Bangham, A. D. Biochim. Biophys. Acta 1978,
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(8)Takagi, S.;Takeda, K.; Shiroishi,M.+. Biol. Chem. 1982,46, 2217. (9) Siefermann-Harms,D. H. Biochim.Biophys. Acta 1985,325,811. (10) Thrash, R. J.; Fang, L.-B.;Leroi, G. E. Photochem. Photobwl. 1979,29,1049. (11) Rebane, K. K. J. Phys. Chem. 1992,96,9683. (12) Alden, R. G.; Lin, S. H.; Blankenship,R. E. J . Lumin. 1992,51, 51.
films mimicking biological membranes may provide a better understanding of such fundamental process. In this paper we report our studies on the interaction of crocetindialdehyde (CDA) and stearic acid (SA),aggregate formation of CDA in LB film mixed with SA,and their relevance to photosynthesis.
Experimental Section Ultrapure crocetindialdehyde (CDA), whose molecular structure is shown in the inset of Figure 1,was obtained as a giR from Hoffmann La-Roche Co., Switzerland, and steric acid (SA) was purchased from Sigma Chemical Co., St. Louis, MO. Both compounds were used without further purification. Purity of the sampleswas checked uaingthin-layerchromatography(TLC) and absorption and IR spectra. A solution of CDA was prepared in spectroscopic grade chloroform and kept in dark bottles wrapped with aluminum foil in a refrigerator. For surface pressure measurements or lifting of films, freshly prepared sampleswere always used. All solventsusedwere of spectroscopic grade. A computer-controlled Langmuir-Blodgett alternate layer trough made of polytetrafluoroethylene (PTFE)(Model 4 Joyce-Laebl,U.K.) was used for the deposition ofthe monolayers. A filter paper Wilhelmy plate attached to a microbalance, which in turn was interfaced to a microcomputer,maintained constant pressure with an accuracy of f l mN/m over a very long time. Triply distilled water, further purified by a milli-Qplua water purification system was used as a subphase (pH GZ 6.2). The resistivity of water used was 18.2 MQ-cm. Fluorescence grade quartz slides were treated with chromic acid and boiling nitric acid to ensure that no traces of organic contaminants remained on the substrates. After acid treatments, the slides were repeatedly washed with deionized water and sonicated for about 10minin spectroscopicgrade chloroform. The slideswere finally dried in a hot air oven and stored in a dust-free chamber. Solutions of CDA, SA, and CDNSA mixture in Merent molar ratios were prepared in spectroscopicgrade chloroformand spread on the clean water surface of the Langmuir trough. After a wait of 20 min for the solvent to evaporate, the film was slowly compressed and isotherm data of surface pressure versus area per molecule were collected at room temperature (20 "C). By using the minimum dispersion of mean molecular area as a criterion for the optimum rate of compression, a rate of 5 cm2/ minwas chosen for these experiments. Depositionof mono- and Y-typemultilayers at a surfacepressure of 20 mN/m was achieved by allowing the substrate to dip with a speed of 5 d m i n with a drying time of approximately40 min. Absorption and emission spectra were recorded by a Shimadzu 2101 PC UV-vis spectro-
0743-7463l94I241Q-2339$04.5Ql0 0 1994 American Chemical Society
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figure represents the ideal concentration dependence of molecular area calculated by the equation
photometer (resolution = 0.05 nm) and a Perkin-Elmer MPF44A spectrofluorimeter (resolution = 0.05 nm), respectively.
Results and Discussion Monolayer Characterization. The pure CDA the at air-water interface was found to be very unstable as evidenced by a rapid drop in the surface pressure when the compression was stopped and also by a poor transfer ratio (0.2) on a quartz substrate. CDA was mixed with SA in order to increase the stability of the monolayer and to faciliate deposition on substrates. The film was transferred on the quartz substrate by Y-type deposition at 20 mN/m surface pressure using standard procedures.13 The transfer ratio of mixed monolayer was good enough (about 0.95 at 1:l mixture) to form multilayered LB film on the quartz substrate. Figure 1 shows the surface pressure (n)vs area per molecule (A) isotherms for pure and mixed CDNSA monolayers. Here, the mean area per molecule represents the total area occupiedhtal number of molecules. Each isotherm was obtained by averaging three to four runs. Our results for pure SA is in accord with published data.13 The specific molecular areas determined by “zeropressure” extrapolations of n-A isotherms are 0.23 f0.01 nm2 and 0.75 f 0.01 nm2 for pure SA and pure CDA, respectively, at 20 “C. This area per molecule for pure CDA is close to that of other polyenes reported e1~ewhere.l~ For mixed monolayer studies, we prepared a series of mixed CDN SA spreading solution ranging from 0 to 1mole fraction of CDA. n-A isotherms of mixed monolayers are shown in Figure 1at different concentrations of CDA. At higher concentration of CDA (>40%)a plateau is observed at around 25 mN/m surface pressure. From cyclic n-A isotherms (not shown in the figure), it was inferred that this plateau is indicative of collapse of the monolayer or formation of a multilayer. The mean area per molecule at extrapolated “zero-pressurenas a function of CDA mole fraction is shown in Figure 2. The straight line in the (13)Gaines, G. L., Jr. Insoluble Monolayers at Liquid-& Interfaces; Inter-science: New York, 1966. (14)N’soukpoe-Kossi,C . N.;Sielewiesiuk, J.; Leblanc, R. M.; Bone, R. A.; Landrum, J. T. Biochem. Biophys. Acta 1988,940, 255.
where N, and N , are the mole fractions and A, and A, are the area per molecule of pure CDA and SA, respectively. Figure 2 shows that the n-A isotherms do not follow the ideal additivity rule of eq 1. Not only have we observed a positive deviation from the additivity rule but also two maxima at 0.15 and 0.75 mole fractions of CDA. We have also calculated the Gibb’s free energy, a thermodynamic parameter of mixing from the isotherm data using the following equations.15-17
+ RTN, In N , + RTN, In N , + RTN, In N ,
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where AGE is the excess free energy of mixing, AGmis the free energy of mixing, AGf, is the free energy of mixing for an ideal system, A,,, A,, and A, are the areas of mixed and pure monolayers, n is the surface pressure, R is the universal gas constant, and Tis the absolute temperature. Figure 3 shows the plots of Gibb’s free energy of mixing (AG,) as a function of CDA mole fraction for different ranges of surface pressures. The positive values of AGm significantly depart from the ideal binary behavior with no intermolecular interaction.l* This positive value of AGm together with the positive deviation from the additivity rule suggest strong repulsive interaction between CDA and SA molecules. This strong repulsive interaction along with the dissimilar physical and chemical nature of CDA and SA may stimulate the formation of CDA microcrystals or crystallites. Our data moreover indicate that the dependences of AGm and area per molecule on mole fraction (Figures 2 and 3) are not monotonic but possess two maxima at (15)Vilallonga, F.Biochim. Biophys. Acta 1968,163, 290. (16)Ito,H.;Morton, T. H.;Vodyanoy,V. Thin Solid Films 1989,180, 180. (17)Vodyanoy, V.; Bluestone, G . L.; Longmuir, G. L. Bwchim. Biophys. Acta 1990,1047, 284. (18)Adamson, A. W. Physical Chemistry of Surfaces; WileyInterscience: New York, 1990.
Aggregate Formation of Crocetindialdehyde 20001
Langmuir, Vol. 10, No. 7, 1994 2341
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WAVELENGTH ( n m )
Figure 5. The absorption spectrum of CDA in chloroform solution(1x 10-6M)ona logarithmicscaleat mom temperature. Small bars on the spectxumindicatethelocations ofthe proposed one-photon &-SI transition. be observed in two-photon spectroscopy. While the solution absorption spectrum shows structured sharp bands with the origin at 477 nm, the LB film absorption spectrum shows a single broad band with its maximum at 390 nm. Such broadening and a blue-shift of the absorption band are the signature of H-type aggregates where several monomers are closely assembled in a “cardpack” array in LB filmsm to produce exciton-type of splitting with the higher (factor group) components being allowed in absorption. In addition to this broad band which can be assigned to the 1’4 llB, transition, a small shoulder appears at 528 nm which was unambiguously determinable from the second derivative spectrum. Indeed, weak but distinct humps are observable at 544 and 590 nm even in the solution absorption spectrum of CDA, when plotted on a logarithmic scale (Figure 5). To show that the weak humps are not due to instrumental error, we checked the absorption spectrum ofRhodamine6G in water (not shown in the figure) which showed the known exponential tail decreasing down to the noise level. The absorbances of the humps found in CDA are significantly higher than this noise level. Recently, Mimuro et aLZ1have used similar plots for other polyenes like neurosporene and spheroidene to observe the weak transition in the lowenergy region, which they have attributed to the onephoton SO S1transition. Therefore, one may tentatively assign this shoulder (at 528 nm) to n-n* transition from the ground (llAJ state to the first excited singlet state 2lA, (SI). In the LB film, this transition is blue-shifted and the relative intensity of the S1 compared to SZ transition increases compared to that in solution. Such a symmetry-breakingphenomenon is possible because the molecules are not strictly centrosymmetric in LB films where the molecules can be considered to be adsorbed onto the quartz substrate. This kind of distortion-induced intensity in an otherwise forbidden transition is often manifested in linear polyenes such as all-trans-1,3,5,7octatetraene and all-trans-2,4,6,8,10,12,14,16-octadecaoctaene in n-hexadecane at 4.2 K.223 Another elegant and established method for producing aggregates is the use of a binary mixture of hydrocarbon
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Figure 4. The absorption spectra of (a) CDA in chloroform solution (1x M); (b) 10 layers of CDNSA LB films with a molar ratio of 1:l; (c) 10 layers of CDNSA LB films with a molar ratio of 1:20; (d) redissolved LB film of CDA/SA in chloroform at mom temperature. around 0.15 and 0.75 mole fraction of CDA. This behavior is not readily explicable. The maximum at a mole fraction of 0.15 may correspond to the formation of miniclusters. Subsequent increase in CDA concentration may lead to agglomeration into a maxicluster or two-dimensional crystals as indicated by a peak corresponding to a mole fraction of 0.75. In other words, the size of CDA clusters and the responsible interaction varies with the concentration. The removal of SA-SA and CDA-CDA interactions as well as CDA-SA-forming interaction may dominate differently in the concentration range. W-Vis AbsorptionSpectroscopy. Figure 4 shows the electronic absorption spectra of CDA in chloroform solution, in an LB film, and of a redissolved LB film in chloroform a t room temperature. The absorption spectrum of the LB film is quite different from that of the solution. Redissolved LB film in chloroform solution reproduces the solution absorption spectrum. The absorption spectra of CDA, like other unsubstituted polyenes or carotenoids belonging to CU,symmetry group show the &(llAJ S2(11B,) transition, since the So(ll4) Sl(214) transition is symmetry forbiddenls and can only
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(19)Hudeon, B. 9.; Kohler, B. E.; Schulbn, K. In Excited States; Lim, E. C., Ed.; Academic Press: New York, 1982;Vol. 6, p 1.
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(20) West, W.; Caroll, B. J. Chem. Phys. 1951,19, 417. Naga~hima,U.; Nagaoka, S.;Takaichi, 5.;Yamaza(21)Mi”,M.; ki,I.; Nishimura, Y.; Katoh, T. Chem. Phys. Letts. 1993,204,101. (22)Kohler, B.E.; Spangler, C.; Westerfield, C. J.Chem.Phys. 1988, 89,5422. (23)Kohler, B.E.;Snow, J. B. J. Chem. Phys. 1988,79,2134.
2342 Langmuir, Vol. 10, No. 7, 1994
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WAVELENGTH ( n m ) Figure 6. The absorption spectra of (a)CDA in ethanol solution (1 x M) and (b) CDA in ethanol-water mixture keeping CDA concentration at 1 x M, volume fraction of water being 0.6 at room temperature. The absorption spectrum of 10 layers of CDNSA LB film with a molar ratio of 1:l (c) is also included for comparison.
and The hydrophobic interaction between solute molecules leads to strong molecular association and hence the formation of aggregates. Figure 6 shows the absorption spectra of CDA in pure ethanol and in the ethanol-water mixture (volume fraction of water 4 = 0.6) along with the absorption spectrum of LB films. When water is added to the ethanol solution, keeping the CDA concentration at 1 x M, the absorption spectrum shows very little change until the water volume fraction exceeds 0.40. Above this fraction, a significant change in the absorption spectrum occurs with a blue-shift of the maximum together with the broadening of the spectrum. The striking similarity between the absorption spectrum of CDA in the LB films and that in the ethanol-water mixture again confirms beyond doubt the formation of aggregates of CDA in LB films. Similar observation has been made in the case of Zeaxanthin in an ethanol-water mixture and has been assigned to card-pack array or H-aggregatmz6 The absorption spectrum of CDMSA LB films at very low concentrations (1:20) is shown in Figure 4 which is almost identical to that at the higher concentration. This suggests the formation of CDA aggregates even at very low concentrations. Similar behavior has also been observed for fatty acids,2' fullerenes,26 etc. Kajiyama et al.29 have recently reported a novel observation that aggregates dissipate into monomers with increasing temperature. Our temperature-dependence studies over the obtainable range (10-45 "C), however, do not show any change in the absorption profile of the mixed LB film. This indicates that aggregation dominates even at very low concentrations and at higher temperature, otherwise these spectra would have corresponded to that of solution absorption spectra. Emission Spectroscopy. Figure 7 shows the emission spectra of CDA in chloroform solution and in LB film. It is worth mentioning that a redissolved LB film of CDA in (24) Jiang, X. K. ACC.Chem. Res. 1988,21, 362. (25)Tung, C. H.; Xu, C. B. In Focus on Photochemistry and Photophysics; CRC Press: Boca Raton, FL, 1990; Vol. IV,p 242. (26) Gruszecki, W. I.; Zelent, B.; Leblanc, R. M. Chem. Phys. Lett. 1990, 171, 563. (27) Song, Y. P.; Petty, M. C.; Yanvood, J. Langmuir 1993, 9, 543. (28) Wang, Y.; Kamat, P. V.; Patterson, L. K. J.Phys. Chem. 1993, 97, 8793. (29) Kajiyama, T.; Oishi, Y.; Uchida, M.; Takashima, Y. Langmuir 1993, 9, 1978.
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550 6 50 750 WAVELENGTH ( n m ) Figure 7. (a)Emission spectra of CDA in chloroform solution (1 x M) at room temperature. (b) Emission spectra of 10 layers of CDNSA LB film with a molar ratio of 1:l. A thicker LB film with a molar ratio of 1:20 reproduces all the spectral features. chloroform reproduces the solution emission spectrum. Moreover, a change in molar concentration of CDA in SAmixed LB film has no effect on the features of the emission spectra. So far as the emission is concerned, shorter polyenes containing fewer than seven or eight double bonds generally fluoresce from the first singlet state S1(2lAJ, whereas longer carotenoids containing nine or more n bonds show an anti-Kasha emission from the SZ (llBu) ~tate.~Oal The number of n bonds where the conversion between SZ to S1 occurs is sensitive to substitution and other details of molecular structure.30 Emissions from both of the states, that is dual fluorescence, was observed fufor all-trans-tetradecaheptaene(seven n bond~),~O?~l coxanthin (nine n bonds),32&apo-€Y-carotenal (nine n bonds),33and some other modified carotenoids (nine and eleven n bond^).^^^^^ The carotenoids of the present study, CDA with nine n bonds, may thus emit dual fluorescence. Indeed that is specifically what we have observed in the emission spectrum of CDA in chloroform solution. The broad bands with maxima at 507 and 650 run may be assigned to the SZ SOand SI SOemissions, respectively. As can be expected from the fluorescence measurements of common ~ a r o t e n o i d s ,the 2 ~present ~ ~ ~ ~ compound ~~ shows a very small quantum yield. Quantum yields of 6 f 0.7 x for the high-energy emission and 1.2 f 0.3 x for the low-energy emission have been obtained by comparison with that of all-trans-/?-carotene (r#Jf = 2 x for S2)36and of rhodamine 6G (4f= 0.95). The emission spectrum of CDA in the LB films is similar to that observed in solution but is significantly red-shifted, supporting the thesis of aggregation of CDA in LB films. The bands in the 500-670 nm region is assigned to the Sz SOemission, while the band at lower energy (714 nm) is attributed to the SI-. SOemission. Biological Implications. Dual fluorescenceemissions in an organized carotene film have been observed for the
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(30) Cosgrove, 5.A.; Guite, M. A,; Burnell, T. B.; Christensen, R. L.
J.Phys. Chem. 1990,94, 8118. (31) Snyder, R.; Arvidson, E.; Foote, C.; Harrigan, L.; Christensen,
R. L. J . A m . Chem. SOC.1988,107,4117. (32) Shreve, A. P.; Trautman, J. K.; Owens, T. G.; Albrecht, A. C. Chem. Phys. 1991,154, 171. (33) Mimuro,M.;Nishimura,Y.; Yamazaki, I.; Katoh,T.;Nagashima, U. J. Lumin. 1992,51, 1. (34) Bettemann, H.; Bienioschek, M.; Ippendorf, H.; Martin, H. D. Angew. Chem. Int. Ed. Engl. 1992,31,1042. J . Lumin. 1993,55,63. (35) hdersson, P. 0.; Gillbro,T.;Asato, A. E.;Liu, R. S. H. J.Lumin. 1992, 51, 11.
Aggregate Formation of Crocetindialdehyde
first time; this is itself of interest because of similarity with the energy transfer mechanism in photosynthesis process where the carotenoid molecules are known to be organized.11J2 In LB films, the absorption is blue-shifted and emissions are red-shifted suggesting H-type "card pack" arrays. This blue-shifted absorption and red-shifted dual emissions in CDA organized in LB films may stimulate one to look for features of self-organization in photosynthesis. It is believed that excitations from the S1of carotenoids (donors) are transferred to the singlet state ofchlorophylls(acceptors). Thus a shift in the donor fluorescence due to self-organizationmay produce a larger overlap of donor emission and acceptor absorption and result in efficient energy transfer with higher quantum yield. Conclusion Surface pressure-area isotherms and Gibb's free energy data of mixed CDNSA monolayers indicate a strong
Langmuir, Vol. 10, No. 7, 1994 2343 repulsive interaction between CDA and SA molecules which may facilitate aggregate formation of CDA in an SAmatrix. We have observed dual fluorescence in solution and for the first time in an LB film of a carotenoid. The blue-shifted absorption and red-shifted emissions indicate H-aggregate formations of CDA. The resemblance between the absorption spectrum of CDA in an ethanolwater mixture and that in an LB film also supports the aggregate formation which may have an important role in photosynthesis.
Acknowledgment. The authors express their thanks to Dr. G. B. Talapatra of our institute for stimulating discussion and valuable suggestions. Thanks to H o h a n n La-Roche Co., Switzerland, for a generousgift of ultrapure CDA samples. Financial support from the Department of Science and Technology, Government of India, is gratefully acknowledged.
