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Langmuir 1997, 13, 4676-4682
Control of Molecular Arrangement in Langmuir-Blodgett Films of Chlorophyll a Prepared in Various Gas Phases Studied by Ultraviolet-Visible and Infrared Spectroscopies and Atomic Force Microscopy†,‡ Hidetoshi Sato§ and Yukihiro Ozaki* Department of Chemistry, School of Science, Kwansei-Gakuin University, Uegahara, Nishinomiya 662, Japan
Yushi Oishi, Miyuki Kuramori, and Kazuaki Suehiro Department of Applied Chemistry, Faculty of Science and Engineering, Saga University, Honjo-machi, Saga 840, Japan
Kenichi Nakashima Department of Chemistry, Faculty of Science and Engineering, Saga University, Honjo-machi, Saga 840, Japan
Kaku Uehara Research Institute for Advanced Science and Technology, Osaka Prefecture University, Gakuen-cho, Sakai 593, Japan
Keiji Iriyama Institute of DNA Medicine, The Jikei University School of Medicine, Nishi-shinbashi, Minato-ku, Tokyo 105, Japan Received February 4, 1997. In Final Form: May 23, 1997X Attenuated total reflection (ATR)/Fourier-transform infrared (FT-IR) spectra were measured for onemonolayer Langmuir-Blodgett (LB) films of chlorophyll a (Chl-a) fabricated in an argon (Ar) atmosphere. The frequencies of CdO stretching bands and marker bands for the coordination number of the central Mg atom suggest that Chl-a takes a five-coordinated dimer in the films prepared in the Ar atmosphere. Ultraviolet-visible (UV-vis) as well as IR spectra were obtained for multilayer LB films of Chl-a prepared in air, Ar, nitrogen (N2), and oxygen (O2) atmospheres. In the cases of the multilayer LB films, spectral features in the CdO stretching band region suggest that Chl-a exists as a monomer in the film prepared in air while it assumes a dimer in the films prepared in the Ar, N2, and O2 atmospheres. The marker bands for the coordination number of the Mg atom in the IR spectra indicate that Chl-a is in a five-coordinated state. These results imply that one can control the molecular arrangement in the LB films of Chl-a by changing the atmosphere in which they are prepared. Both the UV-vis and IR spectra of the LB films fabricated in the Ar, N2, and O2 atmospheres are almost identical to each other. This means that O2 does not affect the stability and structure of Chl-a in the monolayer on the aqueous subphase. On the basis of the postulate that CO2, which exists only in air, caused a change in pH of the aqueous subphase, we investigated pH-dependent IR spectral changes for the LB films. The results indicate that the dimer of Chl-a changes into pheophytine a (Phe-a) below pH 6.0 and that the monomer species do not exist through pH 6.0-4.0; therefore, it is unlikely that CO2 changes the pH. Probably CO2 coordinates to the central Mg atom as a fifth ligand in the LB films prepared in air, preventing the keto carbonyl group of another Chl-a molecule from coordinating to it.
1. Introduction Since the middle of the 1950’s, researchers of chlorophylls (Chls), encouraged by electron micrographs showing a lamellar structure in the chloroplast,1 made much effort to investigate the function and structure of packed Chls * To whom correspondence should be addressed. FAX: +81798-51-0914. E-mail:
[email protected]. † This work was supported by a Grant-in-Aid to H.S. (4533) from the Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. ‡ Abbreviations: AFM, atomic force microscopy; ATR, attenuated total reflection; Chl-a, chlorophyll a; Chls, chlorophylls; FT, Fouriertransform; HPLC, high-performance liquid chromatography; IR, infrared; LB, Langmuir-Blodgett; Phe-a, pheophytine a; TEM, transmission electron microscopy; Tris, tris(hydroxymethyl)aminomethane hydrochloride; UV-vis, ultraviolet-visible; π-A, surface pressure-surface area. § Present address: Laboratory for Photo-biology, Photodynamics Research Center, RIKEN, Nagamachi, Aoba-ku, Sendai 980, Japan. X Abstract published in Advance ACS Abstracts, August 1, 1997.
S0743-7463(97)00113-3 CCC: $14.00
at the interface between hydrophilic and hydrophobic phases.2,3 The studies of monolayer films of Chls reported before 1966 were reviewed by Ke in The Chlorophylls.4 In 1977, Chapados and Leblanc,5,6 in their pioneering studies on the structure of Langmuir-Blodgett (LB) films of Chla, carried out an infrared (IR) study of molecular arrangement and orientation in LB films of Chl-a. According to their reports,5-8 Chl-a takes the form of a monomer in a one-monolayer LB film, but assumes a five-coordinated oligomer and dimer in the multilayer LB films. Zelent et (1) Thomas, J. B.; Minnaert, K.; Elbers, P. F. Acta Bot. Neerl. 1956, 5, 315. (2) Rodrigo, F. A. Biochim. Biophys. Acta 1953, 10, 342. (3) Jacobs, E. E.; Holt, A. S.; Rabinowitch, E. J. Chem. Phys. 1954, 22, 142. (4) Ke, B. In The Chlorophylls; Vernon, L. P., Seely, G. R., Eds.; Academic Press: New York, 1966; p 253. (5) Chapados, C.; Leblanc, R. M. Chem. Phys. Lett. 1977, 49, 180. (6) Leblanc, R. M.; Chapados, C. Biophys. Chem. 1977, 6, 77. (7) Chapados, C.; German, D.; Leblanc, R. M. Biophys. Chem. 1980, 12, 189. (8) Chapados, C.; Leblanc, R. M. Biophys. Chem. 1983, 17, 211.
© 1997 American Chemical Society
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al.,9 in a report about LB films of hydrated oligomers of Chl-a, suggested that wet Chl-a forms self-organized molecular assemblies consisting of nonhydrated or hydrated monomers and/or oligomers depending on conditions for preparation of the LB films of Chl-a. They also described the oligomer of Chl-a in freshly prepared multilayer LB films as consisting of six Chl-a molecules and some bound water molecules.9 Investigations on LB films of Chl-a may be classified into four categories: studies on their structure, stability, morphology, and function. Iriyama10,11 examined the stability of Chl-a in multilayer LB films and suggested preferable conditions for fabricating high-quality LB films of Chl-a. Iriyama and Yoshiura12 explored morphological properties of LB films of Chl-a by electron microscopy combined with a plasma replica method. The resultant micrographs revealed the existence of irregular structure due to uric acid that they employed as an antioxidant in the LB films. A number of studies on the functions of Chl-a LB films have been carried out extensively, aiming at developing new molecular devices such as photoelectric cells based upon the Chl-a assemblies.13-21 Most of the studies suggested that Chl-a is not a suitable organic semiconductor and a sufficient photoelectric conversion effect has not been observed. Some research groups investigated energy transfer from/to Chl-a in LB films containing another molecule as well as Chl-a. For example, Diarra et al.22 studied photoelectric properties in Chl-a LB films containing canthaxanthin carotenoid and suggested the existence of energy transfer from the carotenoid to Chl-a in the films. Es-Sounni and Leblanc23 investigated energy transfer between Chl-a and plastoquinone 9 in LB films and indicated the absence of any interaction between the two components. Deisenhofer et al.24 determined the three-dimensional structure of a photosynthetic reaction center of Rhodopseudomonas viridis, in which bacteriochlorophylls are organized in a suitable manner for electron separation along the pigment array. This study reaffirmed the importance of investigations on the molecular arrangement and orientation of Chls. We have been investigating LB films of Chl-a.25,26 Our final goal is to propose a new molecular device based upon a supramolecular structure of Chls. In order to reach the goal, we have explored the possibility of controlling the (9) Zelent, B.; Gallant, J.; Volkov, A. G.; Gugeshashvili, M. I.; Munger, G.; Tajmir-Riahi, H.-A.; Leblanc, R. M. J. Mol. Struct. 1993, 297, 1. (10) Iriyama, K. Photochem. Photobiol. 1979, 29, 633. (11) Iriyama, K. J. Membr. Biol. 1980, 52, 115. (12) Iriyama, K.; Yoshiura, M. Chem. Lett. 1989, 1635. (13) Simpson W. H.; Reucroft, P. J. Thin Solid Films 1970, 6, 167. (14) Reucroft, P. J.; Simpson, W. H. Discuss. Faraday Soc. 1971, 51, 202, (15) Janzen, A. F.; Bolton, J. R. J. Am. Chem. Soc. 1979, 101, 6342. (16) Lawrence, M. F.; Dodelet, J. P.; Ringuet, M. Photochem. Photobiol. 1981, 34, 393. (17) Tang, C. W.; Albrecht, A. C. J. Chem. Phys. 1975, 62, 2139. (18) Miyasaka, T.; Watanabe, T.; Fujishima, A.; Honda, K. J. Am. Chem. Soc. 1978, 100, 6657. (19) Miyasaka, T.; Honda, K. Surf. Sci. 1980, 101, 541. (20) Lawrence, M. F.; Dodelet, J. P.; Dao, L. H. J. Phys. Chem. 1984, 88, 950. (21) Watanabe, T.; Machida, K.; Suzuki, H.; Kobayashi, M.; Honda, K. Coord. Chem. Rev. 1985, 64, 207. (22) Diarra, A.; Hotchandani, S.; Max, J.-J.; Leblanc, R. M. J. Chem. Soc., Faraday Trans. 1986, 82, 2217. (23) Es-Sounni, A.; Leblanc, R. M. Langmuir 1992, 8, 1578. (24) Deisenhofer, J.; Epp, O.; Miki, K.; Huber, R.; Michel, H. Nature 1985, 318, 618. (25) Sato, H.; Ozaki, Y.; Uehara, K.; Araki, T.; Iriyama, K. Appl. Spectrosc. 1993, 47, 1509. (26) Sato, H.; Oishi, Y.; Kuramori, M.; Suehiro, K.; Kobayashi, M.; Uehara, K.; Araki, T.; Iriyama, K.; Ozaki, Y. J. Chem. Soc., Faraday Trans. 1997, 93, 621.
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molecular arrangement in assemblies of Chls. As the first step of our studies, we carried out an ATR/FT-IR investigation of a one-monolayer LB film of Chl-a. The IR spectra of the one-monolayer LB films could be obtained with a good signal-to-noise ratio, leading us to conclude that Chl-a exists as a five-coordinated monomer in the films.25 We also showed that the films have a flat and homogenized surface by use of transmission electron microscopy (TEM). Of note in our first study was that we prepared the LB films neither in argon (Ar) nor in nitrogen (N2) but in an air atmosphere.25 Next, we undertook a comprehensive investigation on the stability, morphology, and structure of multilayer LB films of Chl-a prepared in the air atmosphere by use of high-performance liquid chromatography (HPLC), TEM, atomic force microscopy (AFM), and IR and UV-vis spectroscopies.26 The chromatogram of the sample retrieved from the LB films suggested that Chl-a is stable during the LB film fabrication even in the air atmosphere. It was revealed by the microscopic observations that not only the one-monolayer but also multilayer films have a very smooth surface, suggesting that Chl-a is packed very tightly and welloriented in the films. The resultant IR spectra indicated that Chl-a exists in a five-coordinated monomer in the multilayer LB films as well as in the one-monolayer films. Our conclusion on the molecular arrangement of Chl-a in the LB films did not always agree with that reached by Chapados and Leblanc.5,8 The purpose of the present study is to give answer to the conflict about the molecular arrangement of Chl-a between our conclusion and their conclusion. We inferred that the structural difference is brought about by the difference in an atmosphere where the LB films are prepared; therefore, we fabricated LB films of Chl-a in Ar, N2, and O2 atmospheres. Another purpose of the present study is to explore if one can control the structure of Chl-a in the LB films by changing the atmosphere. 2. Experimental Section 2.1. Sample Preparation. Chl-a was isolated from fresh spinach leaves and purified according to the method of Iriyama et al.27-29 Sepharose CL-6B (Pharmacia, Sweden) was employed for gel chromatography. The sample was more than 99% pure as estimated by high-performance liquid chromatographic (HPLC) equipped with a UV detector. HPLC and thin-layer chromatography analyses showed that the sample did not include carotenoids or lipids.27-29 The LB films of Chl-a were made by using a USI model 110 Langmuir trough with a Wilhelmy balance (USI System Co. Fukuoka). The whole trough was put in a small plastic container which was filled with gas or air, and the container was placed in a larger housing to keep the trough away from vibration made by human activity. During the LB film fabrication, Ar, N2, or O2 gas was flowed at a constant rate of about 5 L/min, which was gentle enough to keep the surface of the aqueous subphase flat. All of the procedures for the preparation of the LB films were carried out at 293 K under dim green light. Several drops of Chl-a in a chloroform solution (6 × 10-4 M) were placed onto an aqueous subphase of tris(hydroxymethyl)aminomethane hydrochloride (Tris) buffer solution (0.1 mM). The buffer was controlled at pH 8.1 ( 0.1 for the usual film fabrications while it was adjusted to 9.2, 6.0, 5.0, and 4.0 for examinations of pH-dependent structural changes in the LB films. In order to prepare water for the buffer, water was passed through activated charcoal and reverse osmosis filters and then distilled. Finally, it was purified by a Ultrapure Water System model CPW101 (Advantec, Tokyo). The resistance of the finally prepared water was larger than 18.1 MΩ. (27) Iriyama, K.; Ogura, N.; Takamiya, A. J. Biochem. 1974, 76, 901. (28) Iriyama, K.; Shiraki, M.; Yoshiura, M. Chem. Lett. 1977, 787. (29) Iriyama, K.; Yoshiura, M.; Shiraki, M. J. Chromatogr. 1978, 154, 302.
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Figure 1. (A) AFM image of a one-monolayer LB film of Chl-a deposited on mica in an Ar atmosphere (800 × 800 nm). (B) Cross section of the same AFM image. After evaporation of the solvent, the monolayers were compressed at a constant rate of 30 cm2/min (whole trough area was 855 cm2) up to the surface pressure of 16.5 mN m-1. The surface pressure-surface area (π-A) isotherm showed that the monolayers were solid condensed films at this surface pressure. They were transferred by the vertical dipping method onto CaF2 plates covered with a one-monolayer LB film of barium arachidate (for UV-vis, transmission IR, and fluorescence measurements of multilayer films), ZnSe plates (for ATR/IR measurements of the one-monolayer films), and mica (for AFM measurements). The moving speed of a lift, onto which the substrates had been attached, was controlled at 5.0 mm/min. The substrates were dried in the atmosphere for 5 min before each downstroke for the fabrication of multilayer LB films. The transferred LB films of Chl-a were always Y-type. The ZnSe and CaF2 windows were cleaned by a homemade UV-ozone cleaner. To prepare the one-monolayer film of barium arachidate, a chloroform solution of arachidic acid (210-3 M) was spread onto the surface of a barium chloride solution (3 × 10-5 M) and the monolayer film was transferred onto the CaF2 windows at a surface pressure of 20 mN m-1. 2.2. Microscopy. AFM images of the monolayers were obtained with a SFA300 (Seiko Instruments) in air at 293 K. A 0.8 µm × 0.8 µm scan head and a silicon nitride tip on a cantilever with a spring constant of 0.022 N m-1 were used. The images were recorded in the “constant-force” mode (utilizing feedback to keep the cantilever at constant deflection and measuring sample motion). The applied force during the scanning was about 10-9 N. 2.3. Spectroscopy. UV-vis spectra of the LB films were measured with a Shimadzu UV-vis 3101 PC spectrophotometer. Their FT-IR spectra were obtained at a 4 cm-1 resolution with a Nicolet Magna-IR spectrometer 550 with an MCT detector. To generate the spectra with a high signal-to-noise ratio, 256 interferograms were added. The ATR/IR spectra were measured by using a JEOL ATR-100 internal reflection attachment with a incidence angle of 45°.
Fluorescence spectra of the LB films were measured on a Hitachi F-4000 spectrofluorometer with a cell-holder in the frontface configuration. A sharp cut-off filter (Toshiba UV-35) was placed between a sample and the excitation monochromator in order to remove the second-order component of excitation light (435 nm). The LB films were deaerated by nitrogen-gas purging during the fluorescence measurements. The obtained spectra were corrected by the use of a standard tungsten lamp with a known color temperature.
3. Results 3.1. Structure and Morphology of the One-Monolayer LB Films Prepared in an Ar Atmosphere. Figure 1 depicts the AFM image of a one-monolayer LB film of Chl-a prepared in an Ar atmosphere (A) and its cross section (B). The image reveals a very flat and condensed surface of the LB film without collapsed or island structure. The surface roughness estimated by the cross-sectional profile (Figure 1B) is less than 0.28 nm, which is much smaller than the size of the molecule (diameter of the chlorin ring is about 0.8 nm). It is suggested from the image that Chl-a molecules are packed tightly enough (no holes) but not too much (no island) to keep the smooth surface of the LB films. In our previous study,26 we showed by AFM that the surface of a onemonolayer LB film of Chl-a prepared in an air atmosphere was very much homogenized and smooth. The quality of the LB film arranged in the Ar atmosphere seems to be as high as that of the films fabricated in air. Figure 2a shows the ATR/FT-IR spectrum of a onemonolayer LB film of Chl-a deposited on a ZnSe prism in an Ar atmosphere. Intense bands at 1693 and 1660 cm-1 are assignable to CdO stretching modes of the 131-keto carbonyl groups that are free and coordinating to the
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Figure 2. (a) ATR/IR spectrum of a one-monolayer LB film of Chl-a prepared on a ZnSe prism in an Ar atmosphere. (b) Transmission IR spectrum of a six-monolayer LB film of Chl-a fabricated on a CaF2 substrate with a one-monolayer film of Ba arachidate in an Ar atmosphere.
central Mg atom of another Chl-a, respectively. It is noted that the frequency of the latter band is higher by 5 cm-1 than that of the corresponding band observed at 1655 cm-1 for dimers in solutions and cast films. Since these two bands have similar intensities, it is likely that Chl-a exists as a dimer in the film.7,30 Features at 1608, 1551, 1533, and 1492 cm-1 are assigned to IR1, IR4, IR5, and IR6 modes of the chlorin ring of Chl-a, respectively.31,32 These frequencies lead us to conclude that the central Mg atom of dimeric species is five-coordinated.31,32 Previously, we measured ATR/FT-IR spectra of one-monolayer LB films of Chl-a prepared in an air atmosphere.25 They are clearly different in the CdO stretching band region from that in Figure 2a; the former does not have a band near 1660 cm-1 while the latter does have it. It can be concluded, therefore, that there are structural differences between the LB films fabricated in air and Ar atmospheres. Figure 2b depicts the transmission IR spectrum of a six-monolayer LB film of Chl-a deposited on a CaF2 substrate with a one-monolayer LB film of Ba arachidate in an Ar atmosphere. The two spectra in Figure 2 are very similar to each other, indicating that the structure of the LB films of Chl-a changes little with the number of monolayers. 3.2. Multilayer Films Prepared in Air, N2, O2, and Ar Atmospheres. Figure 3 exhibits UV-vis spectra of multilayer LB films prepared in (a) air, (b) N2, (c) O2, and (d) Ar atmospheres. The spectral features in Figure 3b-d bear close resemblance to each other. It is known that the use of Ar or N2 gas as an atmosphere prevents Chl-a from degrading. On the other hand, Chl-a has been thought to be decomposed by O2 in the monolayer on an aqueous subphase because Chl-a produces singlet O2 by light irradiation.33 The spectrum shown in Figure 3c, however, suggests that significant decomposition does not occur during the LB film fabrication even in the O2 atmosphere under dim green light. (30) Ballschmiter, K.; Katz, J. J. J. Am. Chem. Soc. 1969, 91, 2661. (31) Tasumi, M.; Fujiwara, M. In Advances in Spectroscopy; Clark, R. J. H., Hester, R. E., Eds.; John Wiley & Sons: Chichester, 1987; Vol. 14, p 407. (32) Fujiwara, M.; Tasumi, M. J. Phys. Chem. 1986, 90, 250. (33) Hynninnen, P. H. In Chlorophylls; Scheer, H., Ed.; CRC Press: Boca Raton, 1991; p 145.
Figure 3. UV-vis absorption spectra of four-monolayer LB films of Chl-a prepared in (a) air, (b) N2, and (c) O2 atmospheres and that of (d) a six-monolayer LB film of Chl-a fabricated in an Ar atmosphere.
Another notable point in Figure 3 is that absorption bands in the spectrum in Figure 3a show a blue shift by several nanometers compared with those in the other spectra. The shifts are not large but reproducible. These shifts may be caused by carbon dioxide (CO2) in air, because N2, O2, and Ar in air must be excluded from the suspects. The blue shift of the Qy band at 673 nm may be ascribed to a change in molecular orientation in the film; however, UV-vis spectroscopy cannot offer a solid explanation of these spectral changes. Figure 4 compares the IR transmission spectra of fourmonolayer LB films of Chl-a prepared in (a) air, (b) N2, and (c) O2 atmospheres. The spectrum of the multilayer film fabricated in the Ar atmosphere is presented in Figure 2b. The film prepared in air gives a spectrum largely different from the others. Bands at 1736 and 1699 cm-1 in Figure 4a are assigned to CdO stretching modes of the ester carbonyl groups and the 131-keto carbonyl group, respectively,25,26 and their frequencies suggest that all of the carbonyl groups are free from any interaction. In our previous papers,25,26 we concluded from the IR measurements of LB films of Chl-a prepared in an air atmosphere that Chl-a exists as a five-coordinated monomer in the films. The IR spectra in Figures 4b,c, and 2b resemble each other closely as in the case of their corresponding UV-vis spectra. An additional peak at 1655 cm-1 in Figure 4b,c arises from a CdO stretching mode of the 131-keto carbonyl group coordinating to the Mg atom of another molecule.7,8,30 Bands due to IR1, IR4, IR5, and IR6 modes of the chlorin ring are identified at 1608, 1551, 1533, and 1492 cm-1, respectively. The existence of the additional CdO band, together with the frequencies of IR1, IR4, IR5, and IR6 bands, indicates that Chl-a assumes a five-coordinated dimer in the LB films prepared in the N2, O2, and Ar atmospheres. Of note is that the frequencies of the IR1, IR4, IR5, and IR6 bands are almost unchanged between the LB films prepared in air and those in the other
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Figure 4. Transmission IR spectra of four-monolayer LB films of Chl-a prepared in (a) air, (b) N2, and (c) O2 atmospheres. Table 1. The Formation and Coordination Statesa of Chl-a in LB Films Prepared in Various Gas Phases gas phase
air
Ar
N2
O2
formation
monomer
dimer
dimer
dimer
a
Figure 5. Transmission IR spectra of four-monolayer LB films of Chl-a prepared in an Ar atmosphere on an aqueous subphase at pH (a) 6.0, (b) 5.0, and (c) 4.0.
The coordination no. ) 5 in each case.
atmospheres. It seems, therefore, that there is no significant difference in the structure of the chlorin ring and its environment between those LB films. This conclusion is consistent with the fact that their UV-vis spectra are similar to each other (Figure 3b-d). Table 1 summarizes the structure of Chl-a in the LB films prepared in various atmospheres. Table 1 suggests the possibility of controlling the aggregation states of Chl-a in LB films by changing the atmosphere. We inferred that the structural difference between the LB film prepared in air and those in other gases was brought about by CO2 gas because CO2 is contained only in air. The interesting question is how CO2 affects the structure of LB films. We assumed that CO2 decreases the pH value of the aqueous subphase. The considerable reduction of the pH value in the whole buffer solution is very unlikely; however, it may be possible for CO2 to reduce the pH value in a limited area of the interface between the subphase and air. Therefore, we investigated the dependency of the structure of LB films of Chl-a on the pH value of the aqueous subphase. 3.3. Structural Changes and Stability of Chl-a in LB Films. Their Dependences on pH of Subphase for LB Film Fabrications. IR spectra of four-monolayer LB films of Chl-a prepared at pH 6.0, 5.0, and 4.0 in an Ar atmosphere are presented in Figure 5, panels a, b, and c, respectively. The spectrum in Figure 5a is similar to that shown in Figure 2b, while the spectrum in Figure 5c is close to that of a LB film of pure Phe-a which we reported in our previous paper.26 Therefore, it may be concluded that little decomposition of Chl-a takes place during the preparation of the LB film at pH 6.0, but that it is pheophytinized easily on the subphase at pH 4.0. The
Figure 6. Difference spectrum between Figure 5b and Figure 5a.
band due to IR1 shows a clear upward shift by 8 cm-1 from 1609 to 1617 cm-1 upon the pheophytinization. This band is useful as a marker band for Phe-a.26 Figure 5b resembles Figure 5a but shows some characteristics of the spectrum of Phe-a. Thus, a difference spectrum between Figure 5a and Figure 5b was calculated as shown in Figure 6. It shows a band at 1617 cm-1 assignable to the IR1 mode of Phe-a. Bands at 1220 and 1162 cm-1 seem to be also characteristic for Phe-a.26 These observations suggest that both Chl-a dimer and Phe-a coexist in the LB film prepared in the Ar atmosphere at pH 5.0. We also prepared a four-monolayer LB film of Chl-a transferred from an aqueous subphase of pH 9.2 to examine the possibility of dimerization caused by a basic subphase. We assumed that it is possible to keep the acidity of the water surface greater than pH 6.0 by using this subphase in air during the LB film fabrication. An IR spectrum of the LB film fabricated at pH 9.2 is shown in Figure 7. The spectral features in Figure 7 resemble
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Figure 7. IR spectrum of a four-monolayer LB film of Chl-a prepared in an air atmosphere on an aqueous subphase at pH 9.2.
those in Figure 4a. Lack of a band near 1655 cm-1, together with the frequencies of bands due to IR1, IR4, IR5, and IR6 modes, indicates that Chl-a exists as a fivecoordinated monomer in the LB film. An intensity of the band at 1700 cm-1 due to the CdO stretching mode of the 131-keto carbonyl group decreases compared with that in the spectrum in Figure 4a, suggesting the existence of allomeric species. The allomerization is another process of decomposition of Chl-a that is inhibited by acids.33 If the pH value of the subphase is kept high (above pH 6.0) through the preparation, the pheophytinization, that is protonation, should not occur. The acidic subphase did not cause the monomerization of Chl-a in the Ar atmosphere, while the basic subphase did not bring about the dimerization in air. These experimental facts lead us to conclude that CO2 directly plays a key role in producing the monomeric species of Chl-a in the LB films. It seems likely that a CO2 molecule coordinates to the central Mg atom of Chl-a as a fifth ligand, preventing the 131-keto carbonyl of another Chl-a molecule from coordinating to it. In order to confirm this, we attempted to prepare LB films of Chl-a in pure CO2 and Ar containing 10% CO2, but the pheophytinization occurred as the major reaction in these experiments. We have not succeeded in making LB films of monomeric Chl-a under controlled conditions, such as Ar containing CO2, yet. Probably, the content of CO2 must be as small as that in air. 3.4. Fluorescence Spectroscopy. Figure 8 compares fluorescence spectra between (a) 12-monolayer LB and (b) cast films of Chl-a prepared in the Ar atmosphere. In our previous paper,25 we presented an IR spectrum of cast film of Chl-a made from a diethyl ether solution, in which Chl-a exists in a five-coordinated dimer. The IR spectrum of the cast film resembles closely those of the LB films in Figures 2 and 4b,c; however, the present results of fluorescence spectroscopy reveal clear differences in structure between these films. A band at 735 nm appearing in Figure 8a becomes weak and shifts to 760 nm in Figure 8b. This spectral change indicates that Chl-a takes nonhomogeneous orientation of dimer species in the cast film, while it assumes homogeneous arrangement in the LB films prepared in the Ar atmosphere. Detectable fluorescence emission was not observed for the LB films prepared in air, indicating that Chl-a is well oriented and takes a face-to-face configuration in the films prepared in the air atmosphere. 4. Discussion 6
Leblanc et al. and Chapados et al.7,8 suggested that Chl-a exists as a monomer in one-monolayer LB films
Figure 8. Fluorescence spectra of (a) a 12-monolayer LB film of Chl-a prepared in an Ar atmosphere and (b) a cast film of Chl-a.
prepared in Ar and N2 atmospheres. On the other hand, it was indicated that freshly prepared multilayer LB films consist of a polymer of Chl-a molecules linked through keto CdO‚‚‚Mg coordinate bonds and that a water molecule coordinates to the Mg atom as a sixth ligand of the polymeric species. According to them,6-8 the multilayer array collapses and the original polymeric state of the Chl-a molecule is replaced by dimers of Chl-a when the water molecules leave. Since Chl-a exists as a fivecoordinated dimer in cast films which have irregular molecular order, their hypothesis seems reasonable. We did not observe any time-dependent structural change in the multilayer LB films prepared in the Ar atmosphere, however. The AFM observation revealed that the dimeric species make smooth, homogenized and rigid one-monolayer films in Ar, as the monomeric species do in air. We also did not observe any morphological irregularity on the films. The fluorescence study revealed the difference in the molecular arrangements between the LB and cast films. Our conclusion is that Chl-a is stabilized as a five-coordinated dimer from the first in the one-monolayer LB films as well as in the multilayer films fabricated in an Ar atmosphere and there is little interaction between each monolayer in the multilayer array. We also concluded that the dimerization accompanied with collapsing of molecular array does not take place in the LB films and molecular order in the LB films is different from that in the cast films. Those structural differences between the LB films prepared by Chapados et al.7,8 and by us might arise from the difference in types of deposition for fabricating the multilayer LB films of Chl-a; the multilayer LB films made by Chapados et al. can be classified into Z-type, but our multilayer LB films have a Y-type structure. It seems reasonable to consider that molecular reorganization occurs in the LB films of Z-type in which monolayers are deposited in tail-to-head configuration. After the collapsing of multilayer array, that Chapados et al. pointed out, molecular order in the LB films may resemble that in the cast films in which Chl-a takes the form of a fivecoordinated dimer. It is rather difficult to depict the structure of the fivecoordinated dimer because the molecular aggrangements in the LB films are not clear. However, judging from the similarity in the infrared spectra, the structure of the
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five-coordinated dimer of Chl-a in the LB films may be similar to that of the dimer proposed by Ballschmiter and Katz.34 5. Conclusions The present study has demonstrated the possibility of controlling the molecular arrangement in LB films of Chla. The LB films of Chl-a were prepared in air, Ar, N2, and O2 atmospheres. The IR studies revealed that Chl-a exists as a five-coordinated monomer in the LB films prepared in the air atmosphere while it takes the form of a fivecoordinated dimer in those prepared in the Ar, N2, and O2 atmospheres. It was also suggested that Chl-a exists as a five-coordinate dimer in a one-monolayer LB film prepared in the Ar atmosphere. In order to elucidate the cause which brings about the change in the molecular arrangement, the pH dependence of the structure of the LB films of Chl-a was investigated. (34) Ballschmiter, K.; Katz, J. J. J. Am. Chem. Soc. 1969, 91, 2661.
Sato et al.
The study elucidated two important facts. First, the dimer species of Chl-a in the LB films prepared in an Ar atmosphere are decomposed directly into Phe-a below pH 6.0. Second, both the acidic and basic subphases do not cause the monomerization of Chl-a in the films; it turned out that the pH change is not a major cause for changing the molecular arrangement. These results led us to reach the conclusion that CO2 directly controls the structure of Chl-a in the LB films. CO2 may be the fifth ligand of Chl-a in the LB films prepared in air. Probably, it prevents the 131-keto carbonyl group of another Chl-a from coordinating to the Mg atom. The present study has provided important knowledge about the stability of LB films of Chl-a; Chl-a is stable during LB film fabrication even under an O2 atmosphere. Acknowledgment. The authors thank Prof. D. Borchman (Department of Ophthalmology, University of Louisville) for correcting English. LA9701135
