Characteristics of Long-Chain Fatty Acid Monolayers Studied by

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Characteristics of Long-Chain Fatty Acid Monolayers Studied by Infrared External-Reflection Spectroscopy Takeshi Hasegawa* and Jujiro Nishijo Kobe Pharmaceutical University, Motoyama-kita, Higashinada-ku, Kobe 658-8558, Japan

Motoko Watanabe Tokyo College of Pharmacy, Horinouchi, Hachioji, Tokyo 192-0392, Japan

Junzo Umemura Institute for Chemical Research, Kyoto University, Gokasho Uji, Kyoto-fu 611-0011, Japan

Yuqiu Ma, Guodong Sui, Qun Huo, and Roger M. Leblanc* Department of Chemistry, University of Miami, P.O. Box 249118, Coral Gables, Florida 33124-0431 Received December 5, 2001. In Final Form: April 8, 2002 Unique monolayer properties of a long-chain fatty acid, hexacosanoic acid (C26), have been studied by surface pressure-area (π-A) isotherms, Brewster angle microscopy (BAM), and a new analytical technique using infrared external-reflection (IR-ER) spectroscopy, in comparison to other shorter long-chain fatty acids (C23-C25). The shorter fatty acids gave an identical limiting molecular area, although the shape of the isotherms was different from one another. Nevertheless, C26 yielded a significantly larger limiting molecular area than the shorter ones, with a relatively simple isotherm in shape. The IR-ER spectra of the C26 monolayer deposited on GaAs suggested that the molecules were ordered well in the film, which is not consistent with the large limiting molecular area. The dielectric dispersion analysis of the spectra, which has recently been developed, supported that the C26 monolayer had a highly ordered molecular conformation. The key to understanding the inconsistency was found in the degree of lateral molecular aggregation. The extraordinarily strong aggregation property has been suggested via the real part of the complex refractive-index analysis using the IR-ER spectra and confirmed by comparison of the C26 monolayer to the cadmium salt of the C26 monolayer using BAM.

Introduction Mycolic acid is a major chemical component in the cell envelope of mycobacteria represented by Mycobacterium tuberculosis and Mycobacterium leprae.1-5 It has been suggested that the virulence of mycobacteria is strongly correlated to the chemical structures or conformations of mycolic acids, but the relationship has never been clarified.5 The general chemical structure of mycolic acid (Chart 1) consists of two characteristic parts: a simple saturated carboxylic acid part and a very long (∼C48) fatty alcohol part that is called the mero group. The longer mero group greatly varies in structure, and some unsaturated bonds or functional groups are substituted.5 On the other hand, the saturated carboxylic acid part is known to have only two kinds of fatty acids: C24 and C26 (k ) 21 and 23 in Chart 1, respectively).1 Therefore, it is considered that * To whom correspondence should be addressed. Takeshi Hasegawa: fax, +81 78 435 2080; e-mail, [email protected]. Roger M. Leblanc: fax, +1 305 284 4571; e-mail, [email protected]. (1) Minnikin, D. E. Lipids: Complex Lipids, Their Chemistry, Biosynthesis and Roles. In The Biology of the Mycobacteria; Ratledge, C., Stanford, J., Eds.; Academic Press: London, 1982. (2) Minnikin, D. E.; Goodfellow, M. Chemical Methods in Bacterial Systematics; Academic Press: London, 1985. (3) Besra, G. S.; Chatterjee, D. Lipids and Carbohydrates of Mycobacterium tuberculosis. In Tuberculosis: Pathogenesis, Protection, and Control; Bloom, B. R., Ed.; ASM Press: Washington, DC, 1994. (4) Draper, P. Front. Biosci. 1998, 3, d1253. (5) Brennan, P. J. Chem. Eng. News 1999, 77 (20), 56.

Chart 1. General Structure of Mycolic Acida

a X and Y are replaceable by various groups, represented by the cyclopropyl and the CdC double bond groups. The numbers of CH2 units, k, l, m and n, depend on the species of mycolic acids, but only k is 21 or 23.

the characteristics of mycobacteria can be discussed in terms of the structural variations of the mero group and the shorter saturated part. Mycobacteria that are virulent to humans, such as M. tuberculosis and Mycobacterium bovis BCG, tend to have C26 carboxylic acid, while avirulent mycobacteria, represented by atypical acid-fast bacteria, have C24 as the shorter saturated part.1 Therefore, this minor difference in chain length may play a key role for the virulence. The characteristics of mycolic acids appear when the molecules aggregate to form monolayers. In the cell envelope of mycobacteria, some portions of mycolic acids are found to be bound to a trehalose ring via a covalent bond (trehalose dimycolate, TDM),6,7 which is further bound to the arabinogalactan layer outside the plasma (6) Lane´elle, G.; Daffe´, M. Res. Microbiol. 1991, 142, 433. (7) Almog, R.; Mannella, C. A. Biophys. J. 1996, 71, 3311.

10.1021/la011756u CCC: $22.00 © 2002 American Chemical Society Published on Web 05/15/2002

Long-Chain Fatty Acid Monolayers

membrane.5 This layer is covered by the most outer monolayer, in which an important component is free (nonbonded) TDM. In any case, mycolic acids exist in monolayers, and their packing properties are considered to be related to the regulation of drug permeability of the bacteria cells. The monolayer properties of TDM have been extensively studied, since the monolayer formation of TDM is highly stable. On the other hand, we have recently reported that mycolic acid without the trehalose ring also formed stable monolayers. In the previous study,8 the conformational changes of the most hydrophobic mycolic acid (R-mycolic acid) derived from Mycobacterium avium intracellulare complex were discussed by employing the atomic force microscopic (AFM) technique.9 The study revealed that the mero and the shorter chain parts both largely changed their conformation during the compression of the monolayer. A particular fact in this study was that the molecular folding and extending properties of the R-mycolic acid were monitored for the first time. Regardless, the molecular mechanism of the conformational changes has still been unclear, since both parts were simultaneously monitored in the study to give unresolved results. The present study, therefore, focuses only on the shorter part by a comparative study of long-chain fatty acids (C23-C26). To investigate the physical properties of the saturated long-chain fatty acid monolayers, conventional surfacechemical analyses, that is, surface pressure-area (π-A) isotherm measurements and Brewster angle microscopy (BAM), were employed.10 These surface analytical techniques suggested that the C26 monolayer had a unique molecular aggregation property, but any adequate interrelation was not suggested, since some of the data seemed inconsistent with each other. Then the IR technique was applied. Infrared external-reflection (IR-ER) spectroscopy10,11 is a technique useful for films deposited on a nonmetallic substrate. In particular, a brand-new technique recently developed is powerful to evaluate molecular orientation and optical parameters in the film, even when the film surface is not smooth.12 As a result, the C26 molecule has been found to have a uniquely strong molecular aggregation property in the monolayer in comparison to other shorter long-chain fatty acids. This uniqueness would be important to discuss the monolayer characteristics of mycolic acid, which are correlated to the virulence of mycobacteria. Materials and Methods Tricosanoic acid (C23, C22H45COOH), lignoceric acid (C24, C23H47COOH), pentacosanoic acid (C25, C24H49COOH), and hexacosanoic acid (C26, C25H51COOH) were all analytical reagents (>99% by capillary gas chromatography) purchased from Sigma Chemical Co. (St. Louis, MO), and they were used without purification. For the preparation of monomer solutions, chloroform was used for C23-C25, and a mixture solvent of methanol and chloroform (1:5, v/v) was used for C26, since C26 could not be dissolved in pure chloroform. The concentrations of C23-C25 were all 1.0 mg mL-1, while that of C26 was 0.5 mg mL-1. The solvents were spectra-grade reagents from Dojindo (Kumamoto, Japan). Surface pressure-area (π-A) isotherms were measured by a Kyowa Interface Science (Saitama, Japan) HBM LB (Langmuir(8) Hasegawa, T.; Watanabe, M.; Nishijo, J.; Funayama, K.; Imae, T. Langmuir 2000, 16, 7325. (9) Henon, S.; Meunier, J. Rev. Sci. Instrum. 1991, 62, 936. (10) Hasegawa, T.; Takeda, S.; Kawaguchi, A.; Umemura, J. Langmuir 1995, 11, 1236. (11) Brunner, H.; Meyer, U.; Hoffmann, H. Appl. Spectrosc. 1997, 51, 209. (12) Hasegawa, T.; Nishijo, J.; Umemura, J.; Theiss, W. J. Phys. Chem. B 2001, 105, 11178.

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Figure 1. π-A isotherms of four saturated long-chain fatty acid monolayers measured at 25 °C on pure water. The results of the C26 monolayer are plotted by the thick line. Blodgett) film apparatus. The electrical signals were output as voltage-proportional changes, which were collected by a handmade data acquisition apparatus comprising Dataforth (Tucson, AZ) 5B30-03 voltage-amplifier modules, a Nippon Filcon (Tokyo, Japan) JJ-JOKER (type J) A/D converter with 12 bit resolution, and an IBM Think Pad 220 personal computer with MS-DOS 5.0 and Quick Basic. The initial area of the Langmuir monolayers was 900 cm2 (14 cm width), and they were compressed by a Tefloncoated aluminum barrier at a constant rate of 14 cm2 min-1. A solution volume of 60 µL was used for the preparation of the monolayer films. The compression rate depends on the molecular weight of the sample, but it roughly corresponds to 1.4 Å2 molecule-1 min-1. The pure water used for the subphase in the LB trough was obtained by a Millipore (Molsheim, France) Milli-Q Labo water purifier equipped with a microfilter (Millipak-40) that has micropores of 0.22 µm to remove unexpected organic contaminants. The resistivity of the pure water was 18.3 MΩ cm, and the surface tension was 72.8 mN m-1 at 25 °C. For the IR-ER analysis, the C26 monolayer was transferred onto a GaAs wafer by the vertical dipping (or so-called LB) method at a surface pressure of 25.0 ( 0.3 mN m-1. The deposition ratio of the monolayer was approximately unity. The GaAs wafer was provided by courtesy of Mr. Makoto Hattori, Mitsubishi Electric Co., and it had a (100) atomically smooth surface and a coarse backside, which eliminates the multiple reflection of infrared rays in the wafer. The LB films were subjected to the IR-ER measurements on a Nicolet (Madison, WI) Magna 850 FT-IR spectrometer with a Harrick (Ossining, NY) VRA reflection attachment and a Hitachi (Ibaraki, Japan) wire-grid polarizer. The polarizer was placed perpendicularly to the reflected infrared ray after the sample, and only the p-polarization spectra were collected at the angles of incidence of 25° and 45° from the surface-normal direction. The reflected infrared ray was measured by a deuterated triglycine sulfate (DTGS) detector with a modulation frequency of 5.0 kHz. The spectral resolution was 4 cm-1, and accumulation of 300 scans was performed. For spectral analysis by use of Kim’s oscillators, simulation was carried out using a simulation software coded by Dr. Wolfgang Theiss, which was purchased from M. Theiss Hard- and Software (Aachen, Germany). The detail of the analytical method has been mentioned elsewhere.12 In the present paper, however, the analytical method will be summarized in the next section.

Results and Discussion The π-A isotherms of C23-C26 monolayers were measured as shown in Figure 1. The most striking result is that the limiting molecular areas of C23, C24, and C25 are identical to each other (0.183 nm2 molecule-1), while only the C26 monolayer has a significantly larger area (0.197 nm2 molecule-1). These results are repeatedly reproduced. These limiting molecular areas suggest that only the C26 monolayer has a different monolayer character from other monolayers, although there are clear differences in shape among the isotherms.

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The C23 monolayer exhibits a typical isotherm of a fatty acid, which is similar to that of normal chain length fatty acid, such as stearic acid.13 The two linear parts that correspond to the solid and the liquid expansion regions are connected at a kink point, 34.7 mN m-1. The solid region linearly increases up to more than 60 mN m-1, which suggests that the monolayer is highly stable. When the C24 monolayer was compressed, on the other hand, an isotherm with a significantly different shape was obtained. A clear plateau region appears approximately at 24 mN m-1, and the shape reminds us of the results of double-chain lipid monolayers, such as dipalmitoylphosphatidylcholine (DPPC).13,14 The collapse surface pressure is less than 50 mN m-1. When the linear part below the plateau is extrapolated, a larger limiting molecular area of 0.212 nm2 molecule-1 is obtained. This suggests that some portions of the molecules are lying on the water surface or have a folded conformation in the monolayer when the surface pressure begins to increase. The C25 monolayer also exhibits an isotherm with a plateau, which is roughly similar to that of C24. Nonetheless, the plateau becomes clearer and longer than that of C24, and its surface pressure appears at less than half of that of C24. The isotherm of C24 seems to be in an intermediate state between those of C23 and C25. It is interesting that they all provide the same limiting molecular area, which means similar packing, although they show clearly different surface characters in the isotherms. As a summary, the molecules of C23-C25 are readily compressed to form fully extended chains, but the molecular ordering processes are different from each other, depending on the chain length. These isotherms made us expect that the C26 monolayer should have a clearer plateau with a chain longer than C25, and the plateau surface pressure would become still lower. The result of the C26 monolayer was, however, largely different from what we expected. It has no plateau, and only a linear part appears, which seems to correspond to the solid state of a monolayer. The limiting molecular area of the C26 monolayer was obtained to be 0.197 nm2 molecule-1, which is apparently larger than the values for the rest of the long-chain compounds. A closer look shows that the slope of the isotherm of the C26 monolayer is not as sharp as that observed from the solid-state region of monolayers of C25 or shorter ones. In summary, these results strongly suggest that only C26 gives a unique monolayer property at the air-water interface. Then, the molecular structure and physical properties of the C26 monolayer were investigated by use of a novel analytical technique based on IR-ER spectroscopy. IRER spectroscopy is a powerful method to evaluate the molecular orientation in thin films when they are deposited on nonmetallic substrates. The advantage of the brandnew method is that the evaluation can be performed even when optical parameters of the film are unavailable.12 Further, thin films even with roughness and defects could be analyzed, and such an incompleteness would be evaluated through the real part of the complex refractiveindex dispersion yielded by this method. In the present study, one monolayer of C26 was transferred on a GaAs wafer by the LB method at 20 mN m-1. Since the anisotropic analysis of infrared spectra requires two independent data,12 the LB film was subjected to the IRER measurements at two angles of incidence, that is, 25° (13) MacRitchie, F. Chemistry at Interfaces; Academic Press: London, 1990. (14) Hasegawa, T.; Kawato, H.; Toudou, M.; Nishijo, J. J. Phys. Chem. B 1997, 101, 6701.

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Figure 2. IR-ER spectra (solid lines) of a C26 monolayer deposited on a GaAs wafer measured at 25° and 45° from the normal to the surface. The theoretically fitted lines (dashed lines) are overlaid on the observed spectra.

and 45°. The spectra are presented in Figure 2. The difference between the two spectra seems minor, but it has an intrinsically important difference particularly in the symmetric CH3 stretching vibration band region (near 2875 cm-1). In the spectrum of 25°, the spectral line has a round shape at the wavenumber, while the spectrum of 45° has a clear positive peak. This difference was confirmed by repeated experiments, and it is often found in a similar analysis.12 The difference in spectral shape indicates that the spectra are independent of each other, although they are arising from an identical sample. The analysis is performed by using this independency of the spectra. The analytical procedure is summarized below. Recently, a novel analytical method that enables us to evaluate both dielectric dispersion and the molecular orientation in the film has been proposed for the IR-ER spectroscopic technique.12 The new method employs Kim et al.’s anisotropic oscillators model15 as the dielectric dispersions of the film, with which reflection-absorbance is calculated by the anisotropic optical calculation under the assumption of uniaxial molecular distribution.

˜ (ν) ) ∞ +

χj )

∑j χj ) ∞ + ∑j

4πFjν0j2

(1)

ν0j2 - ν2 - iγjν

4πFjν0j2 ν0j2 - ν2 - iτjν

[ ( )]

where τj ) γj exp -Rj

ν - ν0j γj

2

(2)

Equation 1 presents a dispersion of dielectric function, ˜ (ν), against wavenumber, ν, by use of ∞, ν0j, and γj, which are the dielectric constant in the electromagnetic wave, the resonance position of the jth band, and the damping factor, respectively. The numerator of the fraction in the last term in the equation corresponds to the oscillator strength. The characteristic of Kim et al.’s oscillators is represented by the factor τ in eq 2. This is introduced to make the dispersion function more flexible, which can express an intermediate band-shape between Lorentz and Gaussian curves. The mixing rate of the curves is adjusted by R in τ. With this model, the fitting calculation needs a shorter time than the conventional Lorentz’s oscillators model, and fitting precision is largely improved. By converging the parameters in the oscillators to the optimum values with the use of the downhill-SIMPLEX (15) Kim, C. C.; Garland, J. W.; Abad, H.; Raccah, P. M. Phys. Rev. B 1992, 45, 11749.

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Langmuir, Vol. 18, No. 12, 2002 4761 Table 1. Bands Required for Convergence and Calculated Orientation Angles for the C26 Acid Monolayer on GaAs band νa(CH3)-ip νa(CH3)-op νa(CH2)

Figure 3. Dispersion curves of real (solid line) and imaginary (dashed line) parts of the refractive index against wavenumber. The left and right panels present the curves in the in-plane (x,y) and out-of-plane (z) directions, respectively.

algorithm,16 the optical anisotropic parameters are obtained as a function of wavenumber. As an initial value, ∞ was set to 2.25 ()1.52) for the calculation, but the initial value did not influence the final converged value, which indicated that the fitting calculation was stable and useful. After that, the orientation angle of each band is estimated by the next equation.12

φ ) tan-1

(

)

x2 Ωx-y Ωz

(3)

This equation uses in-plane and out-of-plane oscillator strengths (Ωx-y and Ωz) instead of using direct optical parameters such as extinction coefficient. By the use of the ratio of the oscillator strengths, their absolute values are not required. Nonetheless, the anisotropic dielectric dispersions themselves are calculated by the fitting calculation. If the dispersion of the refractive index is necessary, it is easily deduced from the dielectric dispersion, since the magnetic susceptibility can be approximated to unity in the infrared region.17

˜ (ν) ) xn˜ (ν)

(4)

In this manner, both the molecular orientation and the dispersions of optical parameters are simultaneously yielded. The fitting calculation was converged to yield the fitted curves as presented by dashed lines in Figure 2. It is found that the convergence is readily done within an acceptable error. Through eq 4, the refractive-index dispersions in the in-plane and out-of-plane directions against wavenumber were calculated, which are presented in the left and right panels in Figure 3, respectively. Two largely different dispersion curves of the extinction coefficient (k) are obtained as presented by dashed curves in the figures. The curves in Figure 3 are similar to the transmission and the reflection-absorption spectra of the LB film, respectively (data not shown). This means that the fitting calculation was readily done, and reliable dispersion curves were obtained. Since the dispersion curves are functions of oscillator strength, these curves directly provide the orientation angle for each band by use of eq 3. The calculated angles are listed in Table 1. As presented in the table, five bands were required for the adequate convergence within an acceptable fitting (16) Press, W. H.; Teukolsky, S. A.; Vettering, W. T.; Flannery, B. P. Numerical Recipes in C, 2nd ed.; Cambridge University Press: New York, 1992; pp 408-412. (17) Ibach, H.; Luth, H. Solid-State Physics: An Introduction to Principles of Materials Science; Springer: New York, 1995.

location/ orientation angle/deg cm-1 2965.0 2954.7 2917.1

10 77 83

band νs(R-CH2) νs(CH2)

location/ orientation cm-1 angle/deg 2862.3 2849.3

86 83

error. All of the band locations are assignable as in Parikh and Allara’s paper.18 With the oscillator modeling method, very minute bands such as the symmetric CH2 stretching vibration (νs(RCH2)) band arising from the R-carbon that is adjacent to the carboxylic group can be analyzed. Since the signal from this minor part in the whole molecule is very weak, the band is generally overlaid by neighbor bands. In this manner, the oscillator modeling method enables us to analyze such a minute band, which is a great advantage. In the analysis, however, the analytical precision for minute bands tends to be low due to the unclearness of the dichroic ratio and the poorness of the signal-to-noise ratio. Therefore, the orientation angles calculated only for vibrational modes with strong intensity will be discussed, while all the modes were analyzed as presented in Table 1. The antisymmetric and symmetric CH2 stretching vibration modes (νa(CH2) and νs(CH2)) are found to have orientation angles of 83° (R) and 83° (β), respectively, from the normal to the surface. The average tilt angle (γ) of the hydrocarbon chain is calculated with these orientation angles by use of the following relationship.10

cos2 R + cos2 β + cos2 γ ) 1

(5)

This equation holds since the three directions are mutually orthogonal to one another. The calculated tilt angle, γ, is 10°. In a previous study, the tilt angle of a hydrocarbon chain in compressed monolayers of cadmium stearate was reported to be approximately 14°, when a monolayer was deposited on a GaAs wafer.10 Our present result, therefore, suggests that the monolayer has a significantly highly ordered molecular conformation, which yields the unique small average tilt angle. The νa(CH2) and νs(CH2) bands are, in fact, found at 2916.7 and 2849.0 cm-1, which are very low values, and they are generally assigned to hydrocarbon chains with a highly ordered conformation.10 Full width at half-maximum (fwhm)19 values of the bands also strongly suggest that the molecules are highly ordered.20,21 The fwhm values of the νa(CH2) and νs(CH2) bands are 14.3 and 9.6 cm-1, respectively. In our former study,12 a five-monolayer LB film of cadmium stearate was measured by the same technique, and fwhm values of the bands were 16.5 and 9.8 cm-1, respectively. Since the cadmium stearate LB film is known to have highly ordered molecular architecture, the smaller fwhm of the C26 monolayer suggests that the C26 monolayer has an extraordinary ordered molecular conformation, although the monolayer is salt free. In this manner, the spectroscopic data confirm that the C26 monolayer has an apparent property that molecules are highly condensed to form a highly ordered architecture. To consider the unique (18) Parikh, A. N.; Allara, D. L. J. Chem. Phys. 1992, 96, 927. (19) Bertie, J. E. Handbook of Vibrational Spectroscopy; Griffiths, P. R., Chalmers, J. M., Eds.; John Wiley & Sons: Chichester, 2002; Vol. 5, p 3762. (20) Dluhy, R. A.; Mendelsohn, R.; Casal, H. L.; Mantsch, H. H. Biochemistry 1983, 22, 1170. (21) Casal, H. L.; Mantsch, H. H. Biochim. Biophys. Acta 1984, 779, 381.

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Hasegawa et al. Table 2. Bands Required for Convergence and Calculated Orientation Angles for the Cd(C26)2 Monolayer on GaAsa band νa(CH3)-ip νa(CH3)-op νa(R-CH2) νa(CH2) νs(CH2)FR a

Figure 4. π-A isotherms of the Cd(C26)2 monolayer on pure water measured at 25 °C and for different spread volumes.

packing property in more detail, the refractive-index dispersions were noticed. From the curves of the refractive-index dispersions presented in Figure 3, the base values of the real part of the refractive indices in the in-plane and the out-of-plane directions were obtained to be 1.37 and 1.47, respectively. The clear difference between the two values indicates that the anisotropy in the film is large, which suggests that the molecules in the film are highly oriented. The absolute values are apparently smaller than the values reported previously for cadmium stearate monolayers, which are obtained approximately at 1.5.21 Since the refractive index is calculated from the dielectric dispersion, it is influenced by the dielectric property. When organic materials are combined with metallic ions, the dielectric constant increases. It is known that, for example, the X-ray diffraction measurements are easy to perform for metallic stearate monolayers, since the electron-scattering factor increases,10 while the measurements are very difficult for stearic acid monolayers (free acid). It is understandable, therefore, that the C26 monolayer (free acid) has a low electron density around the headgroup, which causes the low refractive indices. This means that the present analytical results are reliable. As a conclusion, the C26 monolayer consists of extraordinarily organized molecules with a highly ordered conformation. If this conclusion is true, however, the limiting area of the monolayer is expected to be smaller because of the high-density packing. In other words, the large limiting area (0.197 nm2 molecule-1) by the isotherm analysis is inconsistent with the spectroscopic analysis. To investigate the inconsistency, a molecular-domain formation model was considered. The uniquely high order of the conformation suggested by the spectroscopic analysis makes us consider that the molecules are strongly aggregated by the lateral interactive forces probably due to the long chain length. To confirm this speculation, the cadmium salt of the monolayer was also investigated. The π-A isotherms of the monolayer are presented in Figure 4. It is surprising to observe that the limiting molecular areas are much larger than those for the long hydrocarbon chain acids (C23-C26). It is of further interest to find that the isotherm depends on the spread volume of the monomer solution on the water surface. Several π-A isotherms were measured with spreading volumes of 20-100 µL by 10 µL steps. The isotherms and the limiting areas for the spreading volumes of 20-40 µL were almost identical to each other, but the isotherms largely changed when the spreading volume was above 50 µL. In Figure 4, three representative isotherms are presented. When 30 µL of the solution was spread, the limiting molecular area was found at ca. 0.28 nm2 molecule-1, while it was obtained at ca. 0.25 nm2 molecule-1 for a larger volume. This phenomenon is explained by the following glacier model.

location/ orientation angle/deg cm-1 2965.0 2957.0 2927.1 2917.2 2898.0

9 85 86 75 86

band νs(CH2)FR νs(CH3) νs(R-CH2) νs(CH2)

location/ orientation cm-1 angle/deg 2888.9 2873.8 2861.1 2852.2

86 9 84 67

FR is an abbreviation of Fermi resonance band.

If the spread monolayer forms stiff domains like glaciers in the ocean surface near the arctic area, the compression of the domains would be difficult, and water surfaces uncovered with the monolayer would appear. When a small amount of volume is spread on water, large molecular domains of Cd(C26)2 would be formed, although the number of domains is estimated to be small. Once a stiff and large domain is formed, the compression of the domain is difficult, so that the shape of the domain is changed to fully cover the water surface. When a large amount of volume is spread, on the other hand, many domains with smaller size would be produced, since spreading is performed by many injections. The compression of the many domains would also be difficult, but it may be easier to decrease the uncovered area than that covered by a large domain, since the decrease can be achieved simply by the rotation of the small domains on water. This speculation strongly suggests that C26 molecules quickly form stiff molecular domains on water, in comparison to the C26 free-acid monolayer. Nonetheless, such an extraordinary stiff domain, which cannot be changed in shape by lateral compression, is new to our knowledge. Then, the monolayer formation and the pressure response of Cd(C26)2 were investigated by use of IR-ER and Brewster angle microscopy (BAM) techniques. The BAM investigation was also applied for C26 acid monolayers for comparison. The IR-ER analysis was performed in the same manner as the C26 monolayer, and the analytical results are summarized in Table 2. In this analysis, nine bands were required to have the calculation converged adequately. The minor bands at 2898 and 2889 cm-1 appeared when molecules are lying on the surface.22 Therefore, the result of the band resolution suggested that C26 molecules have a disordered conformation. In fact, the νs(CH2) band is found at a higher position (2852.2 cm-1) than that of the C26 acid monolayer. The disordered nature is reflected in the lower orientation angles obtained for νa(CH2) and νs(CH2) bands. Another important characteristic is found in the anisotropic real parts of the refractive index. The real-part values were obtained to be 1.531 and 1.529 for the in-plane and the out-of-plane directions, respectively, which meant that the monolayer was isotropic. Since the values are above 1.5, the analytical results are considered to properly reflect the metal-salt monolayer. The isotropic property strongly suggests that the Cd(C26)2 monolayer is largely disordered. Nevertheless, it is of note that the fwhm of the bands for Cd(C26)2 is less than that of the C26 monolayer. The values of the νa(CH2) and νs(CH2) bands are 12.2 and 8.8 cm-1, respectively. This result suggests that the microscopic molecular conformation of the Cd(C26)2 monolayer is further ordered in comparison to the C26 monolayer, but “film architecture” is considered to be a little disordered (22) Paudler, M.; Ruth, J.; Riegler, H. Langmuir 1992, 8, 184. (23) Yamamoto, M.; Sakurai, Y.; Hosoi, Y.; Ishii, H.; Kajiwara, K.; Ouchi, Y.; Seki, K. J. Phys. Chem. B 2000, 104, 7370.

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Figure 5. p-Polarized BAM images of C26 and Cd(C26)2 monolayers at different surface pressures.

due to the collapse of the film caused by the strong molecular aggregation property, which is reflected via band location and isotropy of the refractive index as a macroscopic property. To check the macroscopic property, the p-polarization photographs of the BAM images are presented in Figure 5. The left and right panels present images of C26 and Cd(C26)2 monolayers, respectively, on water at different surface pressures. The C26 monolayer exhibits ordered topography even at a low surface pressure, 5 mN m-1, although many lower density parts are found. When the monolayer is compressed up to 20 mN m-1, all of the surface is found to be covered with the high-density monolayer. Furthermore, when the monolayer is compressed to a collapse surface pressure, ordered topography is still observed in the photograph. This observation

suggests that the C26 molecules form a highly stable and ordered monolayer on water, which is consistent with the results by IR analysis. On the other hand, the Cd(C26)2 monolayer exhibits largely different results from those of the C26 free-acid monolayer. At low surface pressure, 5 mN m-1, highly condensed parts (bright) and bare water surface (dark) parts are simultaneously observed. This suggests that Cd(C26)2 aggregates strongly to form molecular domains, which are very stiff. The stiffness is confirmed by the BAM image of the compressed monolayer at 20 mN m-1. Even after the high compression, the domain-water topography still remains. This proves that the domains are very stiff, and the height variation cannot be leveled by the lateral compression. This stiffness was observed again when the surface pressure was increased up to the collapse pressure.

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In other words, it is suggested that the Cd(C26)2 molecules spontaneously aggregate on water immediately after the spreading, and the surface topography is not largely changed by the compression. This means that Cd(C26)2 has a uniquely strong molecular aggregation property, and the strong aggregation is considered to be caused by the long chain of the molecule. Conclusion The new IR analytical technique has been found to be powerful for the analysis of thin films with roughness. In the conventional IR analysis, the film should have perfect flatness with known thickness, since the band intensity is analyzed by the comparison to theoretical values. In the present study, however, the change in anisotropy of the film has readily been analyzed by the technique, which was supported by BAM measurements. The entire results suggest that C26 has an extraordinarily strong molecular aggregation property in comparison to other shorter longchain acids. In other words, the length of C26 has been found to be critical for the aggregation property of saturated carbon hydrates. The long-chain acid is therefore

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considered to play an important role in mycolic acid that is a major component in the cell envelope of mycobacteria. The unique characteristic of C26 in comparison to C24 would be useful to understand the difference in drug permeability of mycobacteria. Acknowledgment. The authors greatly thank Professor Masaru Nakahara, Institute for Chemical Research, Kyoto University, for allowing us to perform most of the experiments in his laboratory, and for his kind encouragements. This work was financially supported by (1) the U.S.-Japan Cooperative Science Program (99-81375/MPCR-416) promoted by the National Science Foundation and the Japan Society of the Promotion of Science, (2) a Grant-in-Aid for Scientific Research on Priority Areas (A), “Dynamic Control of Strongly Correlated Soft Materials” (No. 413/13031074), from the Ministry of Education, Science, Sports, Culture, and Technology, and (3) the Hyogo Science and Technology Association, to whom the author’s thanks are due. LA011756U