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Infrared spectra of Langmuir-Blodgett chlorophyll a films resolved into normal and tangential components
Infrared Spectra of Langmuir-Blodgett Chlorophyll a Films Resolved into Normal and Tangential Components JENNIFER A. BARDWELL~ AND MICHAEL J. DIGNAM Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A1 Received October 28, 1985; accepted June 19, 1986 The infrared spectra of Langmuir-Blodgett (LB) chlorophyll a films have been obtained by attenuated total internal reflection spectroscopy for both s- and p-polarized light (light polarized perpendicular to and parallel to the incident plane, respectively) using a Fourier transform (FT) spectrometer with KBr beam splitter. Through a Kramers-Kronig (KK) transformation of the reflectance data, followed by the necessary phase correction, the spectra have been separated into the absorption and dispersion spectra for directions both normal and tangent to the plane of the film. These display considerable anisotropy, and where band assignments have been made and straightforward interpretation is possible, the data are consistent with the Chapados and Leblanc model for the conformation of chlorophyll a in LB films. These results are the first direct spectroscopic evidence of optical anisotropy in LB chlorophyll a films and result from the first correct application of the KK transformation to resolving optical anisotropy in thin films. As the procedure is readily performed using the FT algorithm of the spectrometer, the techique shows considerable promise as a routine method for probing conformational structures in LB films and biological membranes. Even in those regions of the IR where band assignments are very difficult, resolved spectra can act as "fingerprints" for different conformational states. © 1987AcademicPress,Inc.
samples only the transverse component of the absorption, while the spectrum measured with The Langmuir-Blodgett technique is a light polarized in the p direction (parallel to simple and elegant method for fabricating orthe incident plane) samples the absorption in ganized molecular assemblies (1). These both the normal and transverse directions in structures are often assumed to be uniaxially the film. The four quantities Rs, Rp, 0s, and oriented; however, this assumption has been 0p allow one to calculate nt, kt, nn, a n d / ~ , spectroscopically verified in only a number of where n is the refractive index, k is the abcases. Some techniques which have been emsorption index, and the subscripts t and n refer ployed in the infrared region were reviewed in to the transverse and normal components, rea previous paper (2). In that paper we suggested spectively. For the calculation to be performed, the application of the Kramers-Kronig (KK) the refractive indices must be known or estitechnique to the resolution of the anisotropy mated at some frequency within the experiof these systems. The correct procedure was mental range where the film is essentially demonstrated theoretically and with the use nonabsorbing. This paper presents the first of synthetic data in model calculations. Briefly, correct application of the KK transformation reflectance spectra, R, are measured with poto the resolution of spectra of the optical conlarized light and transformed mathematically stants ofa uniaxial thin film and indeed to our to obtain phase spectra, 0. The spectrum meaknowledge the first such resolution performed sured with light polarized in the s direction correctly by any means. (polarized perpendicular to the incident plane) Chlorophyll a (Chl a) Langmuir-Blodgett films have been studied extensively, as they are thought to be good models for the phoPresent address: National Research Council of Canada, tosynthetic membrane (3). However, there has Metallic Corrosion on Oxidation Section, Montreal Road, Ottawa, Ontario K1A 0R6. been no effort to verify the proposed molecular INTRODUCTION
0021-9797/87 $3.00 Copyright© 1987by AcademicPress,Inc. Journal of Colloid and Interface Science, Vol. 116, No. 1, March 1987
All fights of reproduction in any form reserved.
2
BARDWELL AND DIGNAM
orientation in these layers by infrared spectroscopy, although they were found to exhibit optical anisotropy in the visible spectroscopy (4). In this paper the Kramers-Kronig technique is used to resolve the anisotropy in Chl a multilayers in the infrared. The transmission infrared spectra of Chl a mono- and multilayers have been thoroughly investigated in a series of papers by Chapados and Leblanc. Their careful work has included an investigation of the reorganization of multilayer arrays (5) and the effect of water on mono- and multilayer films (6). They conclude that dehydration of the multilayer films causes a reorientation from a more organized to a less organized film. EXPERIMENTAL
Spectra were collected on a Nicolet 8000 series FTIR spectrometer with a 7199 computer. A KBr beam splitter, Hg Cd Te (liquid nitrogen cooled) detector, and globar source were used. The multilayer Chl a film was supported on the top face of a ZnSe prism in a PLC-SIM liquid internal reflection cell (Harrick scientific) placed in the beam. The angle of incidence was 45 °. AgBr grid polarizers (Perkin-Elmer Model 186-0240) were placed before and after the sample cell. The polarizers could be rotated using stepping motors where one step corresponded to 0.015 ° of rotation. The light coming from the spectrometer's beam splitter is highly polarized, so it was necessary to align the polarizer's principal axes with care. The following procedure was used. First, a determination of the crossed position was made in the absence of the ceil. Because the efficiency of the polarizers varied with frequency, a minimum interferogram peak-topeak voltage did not determine the crossed position. Instead, it was necessary to collect single beam spectra and plot the intensity at one frequency as a function of the second polarizer position, as the first polarizer was held fixed. The minimum in this plot represents the crossed position. The principal axes of the sample cell can be aligned with the polarizer Journal of Colloid and Interface Science, Vol. 116, No. 1, March 1987
axes from a minimum in the intensity as the two polarizers are rotated together in a crossed position (7). Again, the polarizing properties of the spectrometer interfered with this alignment step. With the clean reflection accessory in place, the approximate location of this minimum was determined from the interferogram peak height. The cell was removed and single beam spectra were recorded in 20-step intervals in the region of the approximate minimum position. The cell was then replaced and the determination of the single beam spectra repeated at identical crossed polarizer positions. The spectra of the ratio of the signal for the ceil-in to that for the cell-out were computed for each angular setting of the crossed polarizers and the transmittance at one or more wave numbers versus crossed polarizer position plotted. The minimum represents a coincidence of the polarizer axes with the principal axes of the reflection cell. Making this plot at two frequencies is a check on the determination of the crossed position since it was found that these minima did not agree if the polarizers were not in fact in the crossed position. Such a discrepancy can also arise as a result of noncoincidence of the planes of incidence of the various mirrors and prism faces in the reflection accessory. In this particular experiment the discrepancy was about 1o and could not be eliminated by redetermining the crossed position, so it is likely due to a small internal misalignment of the accessory. This degree of misalignment is expected to have a negligible effect on the spectra (8). Chl a (from spinach) was purchased from Sigma and was handled in the clark or in dim white light. Although Chl a from this source is known to contain traces of contaminants, the reflectance spectra showed no trace of spurious bands, and thus we conclude that impurities were not present in a concentration sufficient to affect the result. The prism and its aluminum mount were cleaned by sonication first in benzene and then in isopropyl alcohol and blown dry. A Lauda Langmuir trough was used to prepare the monolayer films and to deposit them onto the prism. The
INFRARED SPECTRA OF CHLOROPHYLL a FILMS
3
subphase was water, doubly distilled from glass moved by a standard technique used in thin and buffered to pH 7.8 with 10-3 Mphosphate. film transmission work. By back-transforming The Chl a was spread from spectral grade ben- the spectra to interferograms it was easy to zene; deposition was made at a surface pres- identify a feature present in the p spectra but sure of 20 dyne/cm maintained automatically absent in the s spectra which was responsible by the feedback mechanism of the trough. for the interference fringes. By taking the difDue to the complicated shape of the prism ference between the s and p spectra and backand its mount, it was difficult to monitor ac- transforming, this feature was well isolated curately the deposition ratio. A rough estimate from those features giving rise to real differgave an average deposition ratio of 0.9 + 0.3. ences in the spectra, and it could be eliminated In addition, the prism showed the correct hy- by generating a straight line across it in the drophilic/hydrophobic behavior upon with- interferogram. This interferogram was then drawal/insertion, and thus it was concluded transformed and subtracted from the s specthat the deposition was satisfactory. A total of trum, yielding a p spectrum which did not 19 layers was deposited. The film was then exhibit the interference fringes. A close removed from the lower faces of the prism, comparison of the corrected and uncorrected through which the infrared beam is transmit- p-polarized reflectance spectra showed no ted, by carefully wiping them with benzene. distortion of the band shapes in fringe-free This procedure could not disturb the upper areas. The back-transform-transform selayer since the bottom of the prism is com- quence alone produced no detectable change pletely sealed from the top by the aluminum in the spectra. mount. The sample was then placed in the Application of the K K equations to the revacuum bench of the FTIR and evacuated to flectance data was made using the fast Fourier 800 mTorr. transform software of the F T I R as previously A background spectrum was collected prior described (9). To solve for the anisotropic opto deposition. The resolution was 2 cm -~ and tical constants using the thin film equations, 12,000 scans were averaged over a period of knowledge of the refractive index at, e.g., the 4 h. The spectral range was 4200-600 cm -1. upper integration limit (7899 cm-~), is reBecause of the long scan times required and quired (2). The refractive index ofChl a in the the length of time necessary to enable the near infrared does not appear to have been multilayer to reach its "dry" equilibrium state, measured. It was therefore estimated using the the spectrometer was subject to substantial molar refraction technique as described by baseline drift. This was corrected using the Levin (10). The value of the molar refraction "BLC" routine so that regions where the film for Mg was not included in these tables and did not absorb had 100% reflectance. At the was estimated at 8. The density of the monolow frequency end, the spectra in the s and p layer film was calculated using the known directions were baseline corrected to show thickness of 14 ,£, (11, 12) and the molecular identical percentage R in a region where they area at a surface pressure of 20 dyne/cm (3), had very similar shapes. Specifically, both were yielding p = 1.175 g/cm 3. The refractive index adjusted to 99.80% R at 663.2 cm -1. was thus estimated to be 1.60 at 7899 cm -1 Some of the p-polarized spectra showed an and was assumed to be isotropic. interference-type pattern in the frequency range 1350-600 cm -1. The exact origin of this RESULTS pattem is unknown, but appears related to a defect in the detector, as the interference The emphasis of this paper is to examine fringes were not present when the detector was the anisotropy of Chl a monolayers; therefore freshly filled with liquid nitrogen but grew in only the equilibrium spectra measured after with time. The interference fringes were re- long times under vacuum will be considered Journal of Colloid and Interface Science, Vol. 116, No. 1, March 1987
4
BARDWELL
AND DIGNAM
I00
O0 %R 99 %R )9
98
4000
98
3 oo WAVENUMBER
(crn-')
FIG. 1. Polarized reflectance spectra " d r y " Chl a rnultilayer, 4200-2400 c m -~.
here. These spectra show no band at 3275 c m -1 due to absorbed water. Figures 1 and 2 show the baseline-corrected spectra measured with light polarized in the s and p directions, respectively. Visual examination of these reveals little anisotropy. These spectra were KK transformed and the spectra of nt, kt, nn, and /q calculated as described in Ref. (2). Figure 3 shows the spectra of nt and kt in the region 4200-2400 cm -1. It exhibits the expected be-
havior of the dispersion spectrum (nt vs ~) in relation to the absorption spectrum. The spectra of kt and k~ in the range 2400600 cm -1 are compared in Fig. 4. At the present stage of development of this spectroscopic technique, definite error limits cannot be placed on kt and/q. Based on our experience with similar methods involving the KK transformation (8, 9, 13) a reasonable estimate is that they are in error by less than 10% at ab-
I00
I00
%R 99 %R 99
98
'
2~o
'
,6'00 ' WAVENUMBER
,~o (cm -I)
'
e~o
98
FIG. 2. Polarized reflectance spectra "dry" Chl a multilayer, 2400-600 c m -~. Journal of Colloid and InterfaceScience, Vol. 116, No. 1, March 1987
INFRARED SPECTRA OF CHLOROPHYLL a FILMS
rl t
5
;iii 209 1.5~ 3.06 1.52
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kt ).03
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'
3600
'
3200
'
2800'
'
WAVENUMBERS (cm 4)
1~o. 3. Spectraofnt and kt, "dry" Chl a multilayer,4200-2400 cm-~. sorption maxima (excluding random error or noise). Moreover, the sources of systematic error are such that they will affect the intensifies of bands that are close in frequency in a nearly equal manner, so that conclusions based on anisotropy in the relative intensities of such bands may be made with confidence. From Fig. 4 it is clear that the Chl a spectra display considerable anisotropy, particularly in the region below 1200 cm -~. Katz et al. (14) have assigned several of the Chl a bands and
-----~-
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-
1,3L.3i.t) ~-g~o
o - -
we now interpret the anisotropy displayed by these bands in accordance with their assignments and the structural model ofChl a Langmuir-Blodgett films proposed by Chapados and Leblanc (6). In their model, the porphyrin ring (Fig. 5) is oriented to maximize contact of the hydrophilic oxygen groups with the water surface. The aggregated ketone carbonyl stretch at 1657 cm -~ appears enhanced in the spectrum o f k , relative to neighboring bands. This
*t ~- ~ ~ - -"2 ~
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FIG. 4. Spectra o f k t a n d / q , " d r y " Chl a multilayer, 2 4 0 0 - 6 0 0 c m -~. Both spectra were s m o o t h e d to the
same extent. This level of smoothing did not distort the band shapes. Journal of Colloid and Interface Science, Vol. 116, No. 1, March 1987
6
BARDWELL AND DIGNAM
-'-'KA!--.
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FIG.5. Structureof Chl a, redmftedfromRef.(15), and orientedin accordancewiththe modelof Chapados and Leblanc(6). band is present when the ketone at carbon 28 attempt to draw structural conclusions from (Fig. 5) is coordinated to the central Mg of this anisotropy. another Chl a molecule. In the multilayer film The series of bands at 1200-1160 cm -1 have it is most likely coordinated to the Mg in the been assigned by Katz et al. to the ester antilayer directly below (5). Thus the Chapados symmetric carbon-oxygen stretching vibraand Leblanc model predicts a normally ori- tion, while those at 1160-1035 cm -1 to the ented dipole moment change for this band, in corresponding symmetric stretch. As illusagreement with the observed enhancement. trated in Fig. 5, these ester groups (positions A substantial enhancement of the 1377 33 and 35) are predicted by Chapados and cm-I band occurs in the spectrum o f / q . This Leblanc to be oriented to provide maximum band is assigned to the CH3 symmetric bend contact with the water surface, as they are the of the methyls on positions 51 and 55 (Fig. 5). principal hydrophilic groups in the molecule. The long hydrocarbon tail of the Chl a could If they are so oriented in the multilayer array, clearly exist in a number of conformations in the antisymmetric stretch will correspond to the monolayer film. As yet we have made no a dipole moment change primarily in the Journal of Colloid and InteoCaceScience, Vol. 116, No. 1, March 1987
INFRARED SPECTRA OF CHLOROPHYLL a FILMS
transverse direction, the symmetric stretch to one primarily in the normal direction. The spectra of Fig. 4 are in accordance with this, as the symmetric stretching band, almost completely absent in the transverse spectrum, has gained in intensity over the asymmetric streching band in the spectrum of ~ . The remainder of the fingerprint region displays strong anisotropy. As is generally the case in this region, these bands are extremely difficult to assign and in fact have not been assigned for Chl a. Thus specific conclusions concerning molecular orientation and/or conformation cannot be deduced from anisotropically resolved spectra covering only this region. Nevertheless, such resolved spectra could prove extremely valuable for "fingerprinting" various conformations of a given macromolecule brought about by, e.g., temperature changes, guest molecules (such as trapped solvent), and electric fields, etc. The wealth of the anisotropy displayed by the spectra in this region augers well for such applications. CONCLUSIONS
We have presented here the first anisotropically resolved spectra of a thin film, obtained by a method that is routine in nature when measurements are done on an FT spectrometer. As certain F-F instruments cover the range 45,000 to 10 cm -1, the method can be applied to the full UV visible and IR spectral regions. While the anisotropy associated with identifiable moieties can lead in a straightforward manner to direct information on orientation, conformation, and interactions in ordered molecular layers, the other spectral regions can be used to fingerprint different conformational states and assist in investigations involving the transformation between such states. In summary, the experimental technique, illustrated here for Chl a LB film, provides a very large amount of data that is related specifically to
•
7
the orientation and conformation of the molecules in the ordered layer, and this data can be obtained on a routine basis, involving little more effort than that required to obtain a conventional spectrum. In the present application of the technique, the model of Chl a Langmuir-Blodgett films proposed by Chapados and Leblanc has been confirmed. ACKNOWLEDGMENTS The authors thank the Natural Sciences and Engineering
ResearchCouncilofCanadaforsupportingthiswork.They are very grateful to Professor Michael Thompson and HectorWongforthe use oftheirLaudaLangmuirtrough. REFERENCES 1. Kuhn, H., in "Techniques of Physical Chemistry" (A. Weissberger and B. W. Rossiter, Eds.), Vol. 1, p. 579. Interscience, New York, 1972. 2. Bardwell, J. A., and Dignam, M. J., Langmuir 2, (1986), in press. 3. Costa, S. M. de B., Froines, J. R., Harris, J. M., Leblanc, R. M., Orger, B. H., and Porter, G., Proc. R. Soc. London A 326, 503 (1972). 4. Leblanc, R. M., Galinier, G., Tessier, A., and Lemieux, L., Canad. J. Chem. 52, 3723 (1974). 5. Chapados, C., Germain, D., and Leblanc, R. M., Biophys. Chem. 12, 189 (1980). 6. Chapados, C., and Leblanc, R. M., Biophys. Chem.
17, 211 (1983). 7. Dignam, M. J., and Moskovits, M., Appl. Opt. 9, 1868 (1970). 8. Bardwell, J. A., and Dignam, M. J., Anal. Chim. Acta. 181, 253 (1986). ' 9. Bardwell, J. A., and Dignam, M. J.,Anal. Chim. Acta. 172, 101 (1985). 10. Levin, S. W., in "Treatise on Analytical Chemistry" (I. M. Kolthoffand P. J. Elving, Eds.), Part I, Vol. 6, p. 3920. Interscience, New York, 1965. 11. Bellamy, W. D., Gaines, G. L., and Tweet, A. G., J. Chem. Phys. 39, 2528 (1963). 12. Jones, R., Tredgold, R. H., and O'Mullane, J. E., Photochem. Photobiol. 32, 223 (1980). 13. Bardwell, J. A., and Dignam, M. J., J. Chem. Phys. 83, 5468 (1985). 14. Katz, J. J., Dougherty, R. C., and Boucher, L. J., in "The Chlorophylls" (L. P. Vernon and G. R. Seely, Eds.), p. 188. Academic Press, New York, 1966. 15. Chapados, C., Biophys. Chem. 21, 227 (1985).
JournalofColloidandInterfaceScience,Vol.116,No. 1,March1987
samples only the transverse component of the absorption, while the spectrum measured with The Langmuir-Blodgett technique is a light polarized in the p direction (parallel to simple and elegant method for fabricating orthe incident plane) samples the absorption in ganized molecular assemblies (1). These both the normal and transverse directions in structures are often assumed to be uniaxially the film. The four quantities Rs, Rp, 0s, and oriented; however, this assumption has been 0p allow one to calculate nt, kt, nn, a n d / ~ , spectroscopically verified in only a number of where n is the refractive index, k is the abcases. Some techniques which have been emsorption index, and the subscripts t and n refer ployed in the infrared region were reviewed in to the transverse and normal components, rea previous paper (2). In that paper we suggested spectively. For the calculation to be performed, the application of the Kramers-Kronig (KK) the refractive indices must be known or estitechnique to the resolution of the anisotropy mated at some frequency within the experiof these systems. The correct procedure was mental range where the film is essentially demonstrated theoretically and with the use nonabsorbing. This paper presents the first of synthetic data in model calculations. Briefly, correct application of the KK transformation reflectance spectra, R, are measured with poto the resolution of spectra of the optical conlarized light and transformed mathematically stants ofa uniaxial thin film and indeed to our to obtain phase spectra, 0. The spectrum meaknowledge the first such resolution performed sured with light polarized in the s direction correctly by any means. (polarized perpendicular to the incident plane) Chlorophyll a (Chl a) Langmuir-Blodgett films have been studied extensively, as they are thought to be good models for the phoPresent address: National Research Council of Canada, tosynthetic membrane (3). However, there has Metallic Corrosion on Oxidation Section, Montreal Road, Ottawa, Ontario K1A 0R6. been no effort to verify the proposed molecular INTRODUCTION
0021-9797/87 $3.00 Copyright© 1987by AcademicPress,Inc. Journal of Colloid and Interface Science, Vol. 116, No. 1, March 1987
All fights of reproduction in any form reserved.
2
BARDWELL AND DIGNAM
orientation in these layers by infrared spectroscopy, although they were found to exhibit optical anisotropy in the visible spectroscopy (4). In this paper the Kramers-Kronig technique is used to resolve the anisotropy in Chl a multilayers in the infrared. The transmission infrared spectra of Chl a mono- and multilayers have been thoroughly investigated in a series of papers by Chapados and Leblanc. Their careful work has included an investigation of the reorganization of multilayer arrays (5) and the effect of water on mono- and multilayer films (6). They conclude that dehydration of the multilayer films causes a reorientation from a more organized to a less organized film. EXPERIMENTAL
Spectra were collected on a Nicolet 8000 series FTIR spectrometer with a 7199 computer. A KBr beam splitter, Hg Cd Te (liquid nitrogen cooled) detector, and globar source were used. The multilayer Chl a film was supported on the top face of a ZnSe prism in a PLC-SIM liquid internal reflection cell (Harrick scientific) placed in the beam. The angle of incidence was 45 °. AgBr grid polarizers (Perkin-Elmer Model 186-0240) were placed before and after the sample cell. The polarizers could be rotated using stepping motors where one step corresponded to 0.015 ° of rotation. The light coming from the spectrometer's beam splitter is highly polarized, so it was necessary to align the polarizer's principal axes with care. The following procedure was used. First, a determination of the crossed position was made in the absence of the ceil. Because the efficiency of the polarizers varied with frequency, a minimum interferogram peak-topeak voltage did not determine the crossed position. Instead, it was necessary to collect single beam spectra and plot the intensity at one frequency as a function of the second polarizer position, as the first polarizer was held fixed. The minimum in this plot represents the crossed position. The principal axes of the sample cell can be aligned with the polarizer Journal of Colloid and Interface Science, Vol. 116, No. 1, March 1987
axes from a minimum in the intensity as the two polarizers are rotated together in a crossed position (7). Again, the polarizing properties of the spectrometer interfered with this alignment step. With the clean reflection accessory in place, the approximate location of this minimum was determined from the interferogram peak height. The cell was removed and single beam spectra were recorded in 20-step intervals in the region of the approximate minimum position. The cell was then replaced and the determination of the single beam spectra repeated at identical crossed polarizer positions. The spectra of the ratio of the signal for the ceil-in to that for the cell-out were computed for each angular setting of the crossed polarizers and the transmittance at one or more wave numbers versus crossed polarizer position plotted. The minimum represents a coincidence of the polarizer axes with the principal axes of the reflection cell. Making this plot at two frequencies is a check on the determination of the crossed position since it was found that these minima did not agree if the polarizers were not in fact in the crossed position. Such a discrepancy can also arise as a result of noncoincidence of the planes of incidence of the various mirrors and prism faces in the reflection accessory. In this particular experiment the discrepancy was about 1o and could not be eliminated by redetermining the crossed position, so it is likely due to a small internal misalignment of the accessory. This degree of misalignment is expected to have a negligible effect on the spectra (8). Chl a (from spinach) was purchased from Sigma and was handled in the clark or in dim white light. Although Chl a from this source is known to contain traces of contaminants, the reflectance spectra showed no trace of spurious bands, and thus we conclude that impurities were not present in a concentration sufficient to affect the result. The prism and its aluminum mount were cleaned by sonication first in benzene and then in isopropyl alcohol and blown dry. A Lauda Langmuir trough was used to prepare the monolayer films and to deposit them onto the prism. The
INFRARED SPECTRA OF CHLOROPHYLL a FILMS
3
subphase was water, doubly distilled from glass moved by a standard technique used in thin and buffered to pH 7.8 with 10-3 Mphosphate. film transmission work. By back-transforming The Chl a was spread from spectral grade ben- the spectra to interferograms it was easy to zene; deposition was made at a surface pres- identify a feature present in the p spectra but sure of 20 dyne/cm maintained automatically absent in the s spectra which was responsible by the feedback mechanism of the trough. for the interference fringes. By taking the difDue to the complicated shape of the prism ference between the s and p spectra and backand its mount, it was difficult to monitor ac- transforming, this feature was well isolated curately the deposition ratio. A rough estimate from those features giving rise to real differgave an average deposition ratio of 0.9 + 0.3. ences in the spectra, and it could be eliminated In addition, the prism showed the correct hy- by generating a straight line across it in the drophilic/hydrophobic behavior upon with- interferogram. This interferogram was then drawal/insertion, and thus it was concluded transformed and subtracted from the s specthat the deposition was satisfactory. A total of trum, yielding a p spectrum which did not 19 layers was deposited. The film was then exhibit the interference fringes. A close removed from the lower faces of the prism, comparison of the corrected and uncorrected through which the infrared beam is transmit- p-polarized reflectance spectra showed no ted, by carefully wiping them with benzene. distortion of the band shapes in fringe-free This procedure could not disturb the upper areas. The back-transform-transform selayer since the bottom of the prism is com- quence alone produced no detectable change pletely sealed from the top by the aluminum in the spectra. mount. The sample was then placed in the Application of the K K equations to the revacuum bench of the FTIR and evacuated to flectance data was made using the fast Fourier 800 mTorr. transform software of the F T I R as previously A background spectrum was collected prior described (9). To solve for the anisotropic opto deposition. The resolution was 2 cm -~ and tical constants using the thin film equations, 12,000 scans were averaged over a period of knowledge of the refractive index at, e.g., the 4 h. The spectral range was 4200-600 cm -1. upper integration limit (7899 cm-~), is reBecause of the long scan times required and quired (2). The refractive index ofChl a in the the length of time necessary to enable the near infrared does not appear to have been multilayer to reach its "dry" equilibrium state, measured. It was therefore estimated using the the spectrometer was subject to substantial molar refraction technique as described by baseline drift. This was corrected using the Levin (10). The value of the molar refraction "BLC" routine so that regions where the film for Mg was not included in these tables and did not absorb had 100% reflectance. At the was estimated at 8. The density of the monolow frequency end, the spectra in the s and p layer film was calculated using the known directions were baseline corrected to show thickness of 14 ,£, (11, 12) and the molecular identical percentage R in a region where they area at a surface pressure of 20 dyne/cm (3), had very similar shapes. Specifically, both were yielding p = 1.175 g/cm 3. The refractive index adjusted to 99.80% R at 663.2 cm -1. was thus estimated to be 1.60 at 7899 cm -1 Some of the p-polarized spectra showed an and was assumed to be isotropic. interference-type pattern in the frequency range 1350-600 cm -1. The exact origin of this RESULTS pattem is unknown, but appears related to a defect in the detector, as the interference The emphasis of this paper is to examine fringes were not present when the detector was the anisotropy of Chl a monolayers; therefore freshly filled with liquid nitrogen but grew in only the equilibrium spectra measured after with time. The interference fringes were re- long times under vacuum will be considered Journal of Colloid and Interface Science, Vol. 116, No. 1, March 1987
4
BARDWELL
AND DIGNAM
I00
O0 %R 99 %R )9
98
4000
98
3 oo WAVENUMBER
(crn-')
FIG. 1. Polarized reflectance spectra " d r y " Chl a rnultilayer, 4200-2400 c m -~.
here. These spectra show no band at 3275 c m -1 due to absorbed water. Figures 1 and 2 show the baseline-corrected spectra measured with light polarized in the s and p directions, respectively. Visual examination of these reveals little anisotropy. These spectra were KK transformed and the spectra of nt, kt, nn, and /q calculated as described in Ref. (2). Figure 3 shows the spectra of nt and kt in the region 4200-2400 cm -1. It exhibits the expected be-
havior of the dispersion spectrum (nt vs ~) in relation to the absorption spectrum. The spectra of kt and k~ in the range 2400600 cm -1 are compared in Fig. 4. At the present stage of development of this spectroscopic technique, definite error limits cannot be placed on kt and/q. Based on our experience with similar methods involving the KK transformation (8, 9, 13) a reasonable estimate is that they are in error by less than 10% at ab-
I00
I00
%R 99 %R 99
98
'
2~o
'
,6'00 ' WAVENUMBER
,~o (cm -I)
'
e~o
98
FIG. 2. Polarized reflectance spectra "dry" Chl a multilayer, 2400-600 c m -~. Journal of Colloid and InterfaceScience, Vol. 116, No. 1, March 1987
INFRARED SPECTRA OF CHLOROPHYLL a FILMS
rl t
5
;iii 209 1.5~ 3.06 1.52
co
kt ).03
40~
'
3600
'
3200
'
2800'
'
WAVENUMBERS (cm 4)
1~o. 3. Spectraofnt and kt, "dry" Chl a multilayer,4200-2400 cm-~. sorption maxima (excluding random error or noise). Moreover, the sources of systematic error are such that they will affect the intensifies of bands that are close in frequency in a nearly equal manner, so that conclusions based on anisotropy in the relative intensities of such bands may be made with confidence. From Fig. 4 it is clear that the Chl a spectra display considerable anisotropy, particularly in the region below 1200 cm -~. Katz et al. (14) have assigned several of the Chl a bands and
-----~-
o.t
-
1,3L.3i.t) ~-g~o
o - -
we now interpret the anisotropy displayed by these bands in accordance with their assignments and the structural model ofChl a Langmuir-Blodgett films proposed by Chapados and Leblanc (6). In their model, the porphyrin ring (Fig. 5) is oriented to maximize contact of the hydrophilic oxygen groups with the water surface. The aggregated ketone carbonyl stretch at 1657 cm -~ appears enhanced in the spectrum o f k , relative to neighboring bands. This
*t ~- ~ ~ - -"2 ~
,,o to ~^ ~
-~
~-~ ~
~0
•
..3 ~
~
co {.o
~
~-
2doo
'
,6'0o
,zbo
'
WAVENUMBER
0.2
o~
-
'
~
~
'
{
o~o
~"
0.1 kn
8~o
(crn -i )
FIG. 4. Spectra o f k t a n d / q , " d r y " Chl a multilayer, 2 4 0 0 - 6 0 0 c m -~. Both spectra were s m o o t h e d to the
same extent. This level of smoothing did not distort the band shapes. Journal of Colloid and Interface Science, Vol. 116, No. 1, March 1987
6
BARDWELL AND DIGNAM
-'-'KA!--.
.--_~C. \
\I.
!
l
)c
.~'~." N
./.
/
17
/I
_ /
36C
./'\
3,~. ~
34
\
")\
"'" ,oL~ , ,J "~'~ 33
\
26C~---"
/
"
x\
C.
3o~,~t
3,~"
%_c/
II
~ N~ ,. ../2 :.C
X C
2,ic-...,
.
.__ / / C2o
410' ' ' ~
"/
!
~
t2
,~.c /
~-"
C29 "----C2,
.;
'o.iC-~o^ %
FIG.5. Structureof Chl a, redmftedfromRef.(15), and orientedin accordancewiththe modelof Chapados and Leblanc(6). band is present when the ketone at carbon 28 attempt to draw structural conclusions from (Fig. 5) is coordinated to the central Mg of this anisotropy. another Chl a molecule. In the multilayer film The series of bands at 1200-1160 cm -1 have it is most likely coordinated to the Mg in the been assigned by Katz et al. to the ester antilayer directly below (5). Thus the Chapados symmetric carbon-oxygen stretching vibraand Leblanc model predicts a normally ori- tion, while those at 1160-1035 cm -1 to the ented dipole moment change for this band, in corresponding symmetric stretch. As illusagreement with the observed enhancement. trated in Fig. 5, these ester groups (positions A substantial enhancement of the 1377 33 and 35) are predicted by Chapados and cm-I band occurs in the spectrum o f / q . This Leblanc to be oriented to provide maximum band is assigned to the CH3 symmetric bend contact with the water surface, as they are the of the methyls on positions 51 and 55 (Fig. 5). principal hydrophilic groups in the molecule. The long hydrocarbon tail of the Chl a could If they are so oriented in the multilayer array, clearly exist in a number of conformations in the antisymmetric stretch will correspond to the monolayer film. As yet we have made no a dipole moment change primarily in the Journal of Colloid and InteoCaceScience, Vol. 116, No. 1, March 1987
INFRARED SPECTRA OF CHLOROPHYLL a FILMS
transverse direction, the symmetric stretch to one primarily in the normal direction. The spectra of Fig. 4 are in accordance with this, as the symmetric stretching band, almost completely absent in the transverse spectrum, has gained in intensity over the asymmetric streching band in the spectrum of ~ . The remainder of the fingerprint region displays strong anisotropy. As is generally the case in this region, these bands are extremely difficult to assign and in fact have not been assigned for Chl a. Thus specific conclusions concerning molecular orientation and/or conformation cannot be deduced from anisotropically resolved spectra covering only this region. Nevertheless, such resolved spectra could prove extremely valuable for "fingerprinting" various conformations of a given macromolecule brought about by, e.g., temperature changes, guest molecules (such as trapped solvent), and electric fields, etc. The wealth of the anisotropy displayed by the spectra in this region augers well for such applications. CONCLUSIONS
We have presented here the first anisotropically resolved spectra of a thin film, obtained by a method that is routine in nature when measurements are done on an FT spectrometer. As certain F-F instruments cover the range 45,000 to 10 cm -1, the method can be applied to the full UV visible and IR spectral regions. While the anisotropy associated with identifiable moieties can lead in a straightforward manner to direct information on orientation, conformation, and interactions in ordered molecular layers, the other spectral regions can be used to fingerprint different conformational states and assist in investigations involving the transformation between such states. In summary, the experimental technique, illustrated here for Chl a LB film, provides a very large amount of data that is related specifically to
•
7
the orientation and conformation of the molecules in the ordered layer, and this data can be obtained on a routine basis, involving little more effort than that required to obtain a conventional spectrum. In the present application of the technique, the model of Chl a Langmuir-Blodgett films proposed by Chapados and Leblanc has been confirmed. ACKNOWLEDGMENTS The authors thank the Natural Sciences and Engineering
ResearchCouncilofCanadaforsupportingthiswork.They are very grateful to Professor Michael Thompson and HectorWongforthe use oftheirLaudaLangmuirtrough. REFERENCES 1. Kuhn, H., in "Techniques of Physical Chemistry" (A. Weissberger and B. W. Rossiter, Eds.), Vol. 1, p. 579. Interscience, New York, 1972. 2. Bardwell, J. A., and Dignam, M. J., Langmuir 2, (1986), in press. 3. Costa, S. M. de B., Froines, J. R., Harris, J. M., Leblanc, R. M., Orger, B. H., and Porter, G., Proc. R. Soc. London A 326, 503 (1972). 4. Leblanc, R. M., Galinier, G., Tessier, A., and Lemieux, L., Canad. J. Chem. 52, 3723 (1974). 5. Chapados, C., Germain, D., and Leblanc, R. M., Biophys. Chem. 12, 189 (1980). 6. Chapados, C., and Leblanc, R. M., Biophys. Chem.
17, 211 (1983). 7. Dignam, M. J., and Moskovits, M., Appl. Opt. 9, 1868 (1970). 8. Bardwell, J. A., and Dignam, M. J., Anal. Chim. Acta. 181, 253 (1986). ' 9. Bardwell, J. A., and Dignam, M. J.,Anal. Chim. Acta. 172, 101 (1985). 10. Levin, S. W., in "Treatise on Analytical Chemistry" (I. M. Kolthoffand P. J. Elving, Eds.), Part I, Vol. 6, p. 3920. Interscience, New York, 1965. 11. Bellamy, W. D., Gaines, G. L., and Tweet, A. G., J. Chem. Phys. 39, 2528 (1963). 12. Jones, R., Tredgold, R. H., and O'Mullane, J. E., Photochem. Photobiol. 32, 223 (1980). 13. Bardwell, J. A., and Dignam, M. J., J. Chem. Phys. 83, 5468 (1985). 14. Katz, J. J., Dougherty, R. C., and Boucher, L. J., in "The Chlorophylls" (L. P. Vernon and G. R. Seely, Eds.), p. 188. Academic Press, New York, 1966. 15. Chapados, C., Biophys. Chem. 21, 227 (1985).
JournalofColloidandInterfaceScience,Vol.116,No. 1,March1987