Evidence for a Reverse U-Shaped Conformation of Single-Chain

Institute of Chemistry, Martin-Luther-UniVersity Halle-Wittenberg, Mühlpforte 1, 06108 Halle, Germany,. Max-Planck Institute of Colloids and Interfac...
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Langmuir 2007, 23, 6063-6069

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Evidence for a Reverse U-Shaped Conformation of Single-Chain Bolaamphiphiles at the Air-Water Interface Annette Meister,† Marcus J. Weygand,‡ Gerald Brezesinski,‡ Andreas Kerth,† Simon Drescher,§ Bodo Dobner,§ and Alfred Blume*,† Institute of Chemistry, Martin-Luther-UniVersity Halle-Wittenberg, Mu¨hlpforte 1, 06108 Halle, Germany, Max-Planck Institute of Colloids and Interfaces, Research Campus Golm, 14476 Potsdam, Germany, and Institute of Pharmacy, Martin-Luther-UniVersity Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, 06120 Halle, Germany ReceiVed January 5, 2007. In Final Form: February 26, 2007 Infrared reflection absorption spectroscopy and X-ray reflectivity have been used to elucidate the molecular orientation and hydrocarbon chain conformation and packing of the symmetric long-chain bolaamphiphiles dotriacontane-1,1′diyl-bis-[2-(trimethylammonio)ethylphosphate] (PC-C32-PC) and dotriacontane-1,1′-diyl-bis-[2-(dimethylammonio)ethylphosphate] (Me2PE-C32-Me2PE) at the air-water interface. At low surface pressures, these bipolar amphiphiles are found to lie flat on the water surface with a disordered chain. With increasing surface pressure, the alkyl chain becomes more ordered. Concomitantly, the chain is bent pointing into the air, whereas both polar headgroups keep contact with the water subphase. At an area of 0.9-1.1 nm2 per molecule, a surface pressure plateau is reached for both bolaamphiphiles, where the molecules adopt a reverse U-shaped conformation with a strongly tilted alkyl chain. Further compression leads to the formation of 3-D aggregates.

Introduction In recent years, various symmetrical and unsymmetrical bipolar amphiphiles (bolaamphiphiles) have been synthesized to study their quite unusual supramolecular self-assembling properties in aqueous media and at the air-water and liquid-solid interface.1-3 Bolaamphiphiles are composed of one or two long hydrophobic spacers, in many cases alkyl chains, and two polar headgroups attached to their ends. The lyotropic properties of these compounds were investigated in great detail to understand the role of bipolar tetraether lipids in membranes of archaebacteria,4 the formation of lipid nanotubes (LNTs) as novel host structures,5 or the design of new, effective small molecule hydrogelators.6 The investigation of the interfacial properties of bolaamphiphiles has attracted considerable interest mainly due to the possibility of providing a fundamental model for the molecular orientation and packing mode of those molecules at the air-water interface and to study interactions with various substrates from the water subphase.7-9 The interfacial aggregation and the surface morphology of the structures of bolaamphiphiles at the air-water interface were elucidated using a variety of surface sensitive methods such as surface pressure-area (π-A) isotherms, Brewster angle microscopy (BAM), fluorescence microscopy, epi-fluorescence spectroscopy, and X-ray diffraction (XRD) measurements. The * Corresponding author. E-mail: [email protected]. † Institute of Chemistry, MLU Halle-Wittenberg. ‡ Max-Planck Institute of Colloids and Interfaces. § Institute of Pharmacy, MLU Halle-Wittenberg. (1) Benvegnu, T.; Brard, M.; Plusquellec, D. Curr. Opin. Colloid Interface Sci. 2004, 8, 469-479. (2) Fuhrhop, J.-H.; Wang, T. Chem. ReV. 2004, 104, 2901-2937. (3) Meister, A.; Blume, A. Curr. Opin. Colloid Interface Sci., accepted. (4) Benvegnu, T.; Re´thore´, G.; Brard, M.; Richter, W.; Plusquellec, D. Chem. Commun. 2005, 5536-5538. (5) Shimizu, T.; Masuda, M.; Minamikawa, H. Chem. ReV. 2005, 105, 14011443. (6) Estroff, L. A.; Hamilton, A. D. Chem. ReV. 2004, 104, 1201-1217. (7) Sun, X.-L.; Biswas, N.; Kai, T.; Dai, Z.; Dluhy, R. A.; Chaikof, E. L. Langmuir 2006, 22, 1201-1208. (8) Guo, P.; Liu, M.; Nakahara, H.; Ushida, K. Chem. Phys. Chem. 2006, 7, 385-393. (9) Jiao, T.; Cheng, C.; Xi, F.; Liu, M. Thin Solid Films 2006, 503, 230-235.

investigations show a strong dependence of the observed surface structures on the size and type of the headgroups and the length, chemical structure, and stiffness of the hydrophobic linker.7,10-12 Basically, two possible orientations are described for bolaamphiphiles in monolayers at the air-water surface, where either one or both polar headgroups are immersed in the water. The case where only one headgroup was in contact with water was reported for bolaamphiphiles with a very short or an extremely rigid hydrophobic region, which is oriented perpendicular to the water surface.13-15 Many bolaamphiphiles adopt a reverse U-shaped or horseshoe-like conformation,14-18 where both hydrophilic chain ends are immersed in the water. Whereas some information about the possible conformation and packing of the molecules can be deduced from the molecular area at the film collapse, evidence about the molecular orientation can only be obtained using X-ray, neutron reflectivity, or Infrared reflection absorption spectroscopy (IRRAS) measurements. Recently, we reported the self-assembly behavior of the symmetric long-chain bolaamphiphiles dotriacontane-1,1′-diylbis-[2-(trimethylammonio)ethylphosphate] (PC-C32-PC)19,20 and dotriacontane-1,1′-diyl-bis-[2-(dimethylammonio)ethylphos(10) Mizoshita, N.; Seki, T. Langmuir 2005, 21, 10324-10327. (11) Mizoshita, N.; Seki, T. Soft Matter 2006, 2, 157-165. (12) Matsuzawa, Y.; Kogiso, M.; Matsumoto, M.; Shimizu, T.; Shimada, K.; Itakura, M.; Kinugasa, S. J. Mater. Chem. 2004, 14, 3532-3539. (13) Bo¨hme, P.; Hicke, H.-G.; Boettcher, C.; Fuhrhop, J.-H. J. Am. Chem. Soc. 1995, 117, 5824-5828. (14) Liu, M.; Cai, J. Langmuir 2000, 16, 2899-2901. (15) Patwardhan, A. P.; Thompson, D. H. Langmuir 2000, 16, 10340-10350. (16) Di Meglio, C.; Rananavare, S. B.; Svenson, S.; Thompson, D. H. Langmuir 2000, 16, 128-133. (17) Jonkheijm, P.; Fransen, M.; Schenning, A. P. H. J.; Meijer, E. W. J. Chem. Soc., Perkin Trans. 2 2001, 1280-1286. (18) Lee, J.; Joo, H.; Youm, S. G.; Song, S.-H.; Jung, S.; Sohn, D. Langmuir 2003, 19, 4652-4657. (19) Ko¨hler, K.; Fo¨rster, G.; Hauser, A.; Dobner, B.; Heiser, U. F.; Ziethe, F.; Richter, W.; Steiniger, F.; Drechsler, M.; Stettin, H.; Blume, A. Angew. Chem., Int. Ed. 2004, 43, 245-247. (20) Ko¨hler, K.; Fo¨rster, G.; Hauser, A.; Dobner, B.; Heiser, U. F.; Ziethe, F.; Richter, W.; Steininger, F.; Drechsler, M.; Stettin, H.; Blume, A. J. Am. Chem. Soc. 2004, 126, 16804-16813.

10.1021/la070029h CCC: $37.00 © 2007 American Chemical Society Published on Web 04/21/2007

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Figure 1. Chemical structure of the bolaamphiphiles PC-C32-PC with R ) CH3 and Me2PE-C32-Me2PE with R ) H (at pH ) 5) consisting of two bulky headgroups connected by a C32 alkyl chain.

phate] (Me2PE-C32-Me2PE)21 (see Figure 1) in bulk water and at the air-water interface.22 The different space requirements between the large headgroups and the long hydrocarbon chain lead to the formation of a dense network of helical structured fibers in bulk water but also to interesting temperature-dependent aggregation properties at the air-water interface. The shapes of the π-A isotherms indicate for both bolaamphiphiles a reverse U-shaped conformation when the surface pressure reaches upon compression a plateau region. Further compression to lower areas in the plateau region leads to fibrous domains for PC-C32-PC observable by BAM. In contrast, Me2PE-C32-Me2PE shows a higher tendency to form more extended 3-D aggregates due to the additional attractive interaction via hydrogen bonds between the headgroups. A more or less complete film of microcrystals is observed on the water surface. The aim of this study was to provide an unambiguous analysis of the surface pressure (π)-molecular area (A) isotherms by combining X-ray reflectivity (XR) and IRRAS measurements to obtain information about the molecular orientation and the hydrocarbon chain packing of these bolaamphiphiles at the airwater interface. To our knowledge, this is the first time that a combination of these methods was used for the characterization of bolaamphiphiles at the air-water interface. We will show that these two methods have their separate strengths and in combination give a unifying picture on the conformation, orientation, and packing of amphiphilic molecules at the air-water interface. Materials and Methods Materials. PC-C32-PC and Me2PE-C32-Me2PE were synthesized as described previously.19,23 Chloroform and methanol were purchased from Roth (Karlsruhe, Germany). Sodium acetate, acetic acid (100%), sodium carbonate, and sodium hydrogencarbonate (p.a.) were purchased from Merck (Darmstadt, Germany), Riedel-de Hae¨n (Seelze, Germany), Acros (Geel, Belgium), and Solvay Alkali GmbH (Rheinsberg, Germany), respectively. For all film balance measurements, deionized water with a resistivity of 18.2 MΩ cm (SG Wasseraufbereitung and Regenerierstation GmbH, Barsbu¨ttel, Germany) was used. BAM. The morphological changes of the monolayer film were observed using a MiniBAM instrument (Nanofilm Technology GmbH, Go¨ttingen, Germany). To avoid scattering of light from the trough bottom, a black glass plate was put into the trough below the microscope. A high-powered diode laser (30 mW, 688 nm) was used as a source of light, and the images were taken using the integrated CCD camera. The lateral resolution of the microscope is approximately 20 µm. IRRAS Measurements. Monolayer Preparation. All experiments were performed with a Wilhelmy film balance (Riegler and Kirstein, Berlin, Germany) using a filter paper as a Wilhelmy plate. (21) Ko¨hler, K.; Meister, A.; Fo¨rster, G.; Dobner, B.; Drescher, S.; Ziethe, F.; Richter, W.; Steiniger, F.; Drechsler, M.; Hause, G.; Blume, A. Soft Matter 2006, 2, 77-86. (22) Ko¨hler, K.; Meister, A.; Dobner, B.; Drescher, S.; Ziethe, F.; Blume, A. Langmuir 2006, 22, 2668-2675.

Two Teflon troughs of different sizes (300 mm × 60 mm × 3 mm and 60 mm × 60 mm × 3 mm for the reference trough) were linked by three small water-filled bores to ensure equal height of the airwater interface in both troughs. The temperature of the subphase was maintained at 20 ( 0.5 °C. The movement of the Teflon barriers of the larger sample trough was computer controlled. Measurements were performed in the large trough, the smaller one usually serving as a reference trough. A Plexiglas hood covered both troughs to minimize evaporation of water. Both troughs were filled with pure water for PC-C32-PC or a buffer solution for Me2PE-C32-Me2PE (pH 5: 10 mM acetate buffer). The monolayer films of the bolaamphiphiles were formed by directly spreading the respective solution (∼1 mM) in a mixture of chloroform and methanol (3:1) onto the subphase. After a waiting period of at least 15 min for the evaporation of the solvent, the π-A isotherms were recorded at a constant compression speed of 2 Å2 molecule-1 min-1. IRRAS. Infrared spectra were recorded with an Equinox 55 FTIR spectrometer (Bruker, Karlsruhe, Germany) connected to an XA 511 reflection attachment (Bruker) with an external narrow band MCT detector using the trough system described previously. The IR beam was focused by several mirrors onto the water surface, and different angles of incidence can be adjusted. A computer controlled rotating KRS-5 polarizer (>98% degree of polarization) was used to generate parallel and perpendicularly polarized light. The trough system was positioned on a moveable platform to be able to shuttle between the sample and the reference trough. This shuttle technique diminishes the spectral interferences due to the water vapor absorption in the light beam.24 The monolayer films were compressed to a desired area per molecule, the barriers were then stopped, and the IRRA spectra were recorded. In all experiments, the angle of the incident infrared beam with respect to the normal of the water surface was 40°. Perpendicular or parallel polarized radiation was used. Spectra were recorded at a spectral resolution of 8 cm-1 using Blackman-Harris-4-Term apodization and a zero-filling factor of 2. For each spectrum, 2000 or 4000 scans were co-added over a total acquisition time of about 6-9 min. The single-beam reflectance spectrum of the reference trough surface was ratioed as a background against the single-beam reflectance spectrum of the monolayer on the sample trough to calculate the reflection absorption spectrum as -log(R/R0). Spectral calculations were performed using a Visual Basic program with an implementation of the formalism published by Mendelsohn et al. and Flach et al.25,26 Scattering Experiments. Monolayer Preparation. Monolayers of bolalipids were spread from a mixture of chloroform and methanol (3:1) onto the subphase in a LB trough. For the measurements of PC-C32-PC, the subphase was water, whereas for the pH sensitive Me2PE-C32-Me2PE, it consisted of a 10 mM buffer solution of pH (23) Ziethe, F. Synthese und Physikochemische Charakterisierung von Modellsubstanzen der Archaebakterienlipide. Ph.D. Dissertation, University of Halle-Wittenberg, Halle, Germany, 2003. (24) Flach, C. R.; Brauner, J. W.; Taylor, J. W.; Baldwin, R. C.; Mendelsohn, R. Biophys. J. 1994, 67, 402-410. (25) Mendelsohn, R.; Brauner, J. W.; Gericke, A. Ann. ReV. Phys. Chem. 1995, 46, 305-334. (26) Flach, C. R.; Gericke, A.; Mendelsohn, R. J. Phys. Chem. B 1997, 101, 58-65.

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Figure 2. (A) Surface pressure vs area isotherm of PC-C32-PC on water. Horizontal arrows (I-III) point to positions where the surface pressure was held constant for XR measurements. The inset shows a BAM image at A ) 52 Å2. (B-D) IRRA spectra (a-e) of the PC-C32-PC film at the respective areas of the π-A isotherm in panel A. All spectra have been recorded at an angle of incidence of 40° and with p-polarized (B and C) or s-polarized (D) light. (B) OH and CH2 stretching region of spectra taken at the beginning (c) and the end (e) of the surface pressure plateau. The dashed line represents a simulated IRRA spectrum of the experimental spectrum c (see text for details). (C and D) CH2 stretching region of spectra a-e. The wavenumber shift of the antisymmetric (open squares) and symmetric (filled squares) methylene stretching vibration (ν(CH2)) during film compression is shown in the insets. 5. The subphase was thermostated to 20 °C, and the monolayers were compressed at a constant rate of 5 Å2 molecule-1 min-1 while continuously monitoring the surface pressure. The XR experiments were performed on the liquid surface diffractometer installed at the synchrotron undulator beamline BW1 in HASYLAB, DESY (Hamburg, Germany). The experimental setup and evaluation procedures are described in detail elsewhere.27,28 The setup consisted of a sealed, thermostated box that was purged with flowing helium. The box contained a Langmuir trough that was equipped with a single movable barrier and a Wilhelmy plate for monitoring the surface pressure. In all the experiments, the wavelength of the X-ray radiation was 1.3038 Å. The reflectivity measurements probed the vertical electron density profile across the interface by varying the incident angle (Ri) and exit angle (Rf ) Ri) simultaneously. The specular reflectivity was measured by a NaI scintillation detector. The measured reflectivity data at different surface pressures are inverted by applying a model-independent approach29 to yield the electron density F(z).30 (27) Als-Nielsen, J.; Jacquemain, D.; Kjaer, K.; Leveiller, F.; Lahav, M.; Leiserowitz, L. Phys. Rep. 1994, 246, 252-313. (28) Kjaer, K. Phys. B 1994, 198, 100-109. (29) Petersen, J. S. J. Appl. Crystallogr. 1994, 27, 36-49. (30) Jensen, T. R.; Kjaer, K. In NoVel Methods to Study Interfacial Layers; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 2001; p 205.

Results and Discussion π-A Isotherms and IRRAS. PC-C32-PC. The π-A isotherm of PC-C32-PC on pure water at 20 °C is plotted in Figure 2A. Upon compression starting at a large molecular area of about 250 Å2, a surface pressure increase is observed up to a value of 20 mN/m, where a surface pressure plateau is reached at a molecular area of 95 Å2 per molecule.22 By extrapolating the linear part of the surface pressure increase to zero, a limiting area of approximately 145 Å2 is obtained. This value corresponds to more than twice the space requirements for a phosphocholine headgroup, and it indicates a possible reverse U-shaped conformation of the bolaamphiphile. The limiting molecular area of the lipid l,2-dipalmitoyl-glycero-3-phosphocholine (DPPC), for instance, which bears additionally the glycerol backbone, amounts to 57 Å2 per molecule at 25 °C. The following preliminary interpretation of the isotherm was suggested in our previous publication:22 at a surface pressure of almost zero and large molecular areas, the molecules lie isolated on the water surface with the alkyl chain almost parallel to the water surface. Upon compression, the molecules get in contact, and the flexible C32 chains begin to bend upward. At a certain molecular area, the further approach of the polar headgroups becomes energetically

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Figure 3. (A) Surface pressure vs area isotherm of Me2PE-C32-Me2PE on pH 5 buffer solution. Horizontal arrows (I-III) point to positions where the surface pressure was held constant for XR measurements. The inset shows a BAM image at A ) 40 Å2. (B-D) IRRA spectra (a-e) of the Me2PE-C32-Me2PE film at the respective areas of the π-A isotherm in panel A. All spectra have been recorded at an angle of incidence of 40° and with p-polarized (B and C) or s-polarized (D) light. (B) OH and CH2 stretching region of spectra taken at the beginning (c) and end (e) of the surface pressure plateau. The dashed line represents a simulated IRRA spectrum of the experimental spectrum c (see text for details). (C and D) CH2 stretching region of spectra a-e. The wavenumber shift of the antisymmetric (open squares) and symmetric (filled squares) methylene stretching vibration (ν(CH2)) during film compression is shown in the insets.

unfavorable due to headgroup repulsion. Further compression induces the formation of multilayers or aggregates, or dissolution of PC-C32-PC into the subphase can take place. The formation of different aggregate structures, depending on the temperature of the subphase, was verified by BAM. The inset of Figure 2A shows a BAM image of the PC-C32-PC film at 20 °C, recorded at 52 Å2, which proves the formation of blurred stripes at the air-water interface, but no domains could be found at the beginning of the surface pressure plateau at 95 Å2. To extend our knowledge about the molecular orientation and hydrocarbon chain packing of PC-C32-PC at the air-water interface, IRRAS measurements were performed. The compression of the film was stopped at selected points of the isotherm, and IRRA spectra were recorded. In the surface pressure plateau region, the surface pressure decreased slightly during data accumulation due to the relaxation of the film and the formation of 3-D aggregates. The IRRA spectra in Figure 2B-D monitor the OH stretching band at about 3560 cm-1, and the methylene stretching modes at about 2923 and 2850 cm-1 in the region of the pressure increase and the plateau, thus correlating the surface pressure with structural and conformational information. From the intensity of the OH stretching vibrational band, the absolute film thickness can be calculated in certain cases (A.

Kerth, A. Gericke, A. Blume, unpublished results). For a quantitative interpretation, the optical constants of the film have to be known.31,32 In Figure 2B, the OH stretching region and the methylene stretching modes are presented at the beginning (trace c) and the end (trace e) of the surface pressure plateau. Supposing a monomolecular coverage of the water surface at 95 Å2 with bolaamphiphiles in the reverse U-shaped conformation and densely packed headgroups, a band shape simulation was performed using the approach described by Flach et al.26 A value for the refractive index of 1.4 for the symmetric CH2 stretching vibrational band was used for the estimation of the layer thickness. With an effective length of approximately 20 Å for the alkyl chain in the U-shaped bolaamphiphile conformation, the best agreement between experimental and simulated spectra is obtained for a tilt angle of 60° with respect to the surface normal, assuming a uniaxial distribution of the molecules at the air-water interface. This leads to a thickness of approximately 10 Å for the hydrophobic part of the monolayer of the U-shaped alkyl chains (31) Kerth, A. Infrarot-Reflexions-Absorptions-Spektroskopie an Lipid-, Peptidund Flu¨ssigkristall-Filmen an der Luft/Wasser-Grenzfla¨che. Ph.D. Dissertation, MLU Halle-Wittenberg, Halle, Germany, 2003. (32) Hussain, H.; Kerth, A.; Blume, A.; Kressler, J. J. Phys. Chem. B 2004, 108, 9962-9969.

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Figure 4. IRRA spectra (c-e) of the Me2PE-C32-Me2PE film in the CH2 stretching and bending region and phosphate PO2- stretching region at the respective areas of the π-A isotherm in Figure 3A. All spectra have been recorded at an angle of incidence of 40° and with p-polarized light.

(effective length cos θ ) layer thickness). The simulated IRRA spectrum (dashed trace) is given in Figure 2B for the OH stretching band as well as in the inset for the symmetric CH2 stretching band. It will be shown next that the remarkably high tilt angle determined from the analysis of the OH stretching band could be confirmed by XR measurements. The underlying reason for the high tilt angle of the chains is the following: the headgroups of PC-C32-PC with their larger cross-sectional area prevent close contacts of the alkyl chains when they are oriented perpendicular to the air-water interface. To maximize the van der Waals contacts, the chains consequently have to tilt. This phenomenon is well-known in bulk systems. For instance, for DPPC, a tilt angle of ca. 30° is observed in the LR phase. In DPPC, there is a value of 2 for the ratio of alkyl chains to headgroup. For the bolaamphiphile, this ratio is 1; therefore, a larger tilt angle is to be expected. This is confirmed by other experiments with different lipids. Yaseen et al. calculated a tilt angle of 56° for a Gibbs monolayer of the single alkyl chain surfactant hexadecylphosphocholine (C16PC) at 20 mN/m and 53 Å2 per molecule from neutron reflectivity measurements.33 The authors also determined a layer thickness of 13.5 Å for the hydrophobic part and a number of water molecules of 15 per headgroup. Representing half of a PC-C32-PC molecule, it is not surprising that under the condition of the same surface pressure of 20 mN/m, C16PC orients in a similar way at the air-water interface, namely, with strongly tilted alkyl chains but without an additional chain loop. The slightly higher tilt angle for PC-C32-PC may be due to the (t)ggtgg(t) conformation of the loop, which was described for cyclic (CH2)36 by X-ray and IR spectroscopic studies.34,35 The formation of a loop with four gauche bonds will cost an energy of approximately 4.2 kJ/mol per bond (i.e., ca. 16-17 kJ/mol in total). However, the chains at low surface pressures are already thermally disordered. The work of compression as judged from the experimental isotherm in Figure 2 is ca. 24 kJ/mol up to a (33) Yaseen, M.; Lu, J. R.; Webster, J. R. P.; Penfold, J. Langmuir 2006, 22, 5825-5832. (34) Lee, K.-S.; Wegner, G.; Hsu, S. L. Polymer 1987, 28, 889-896. (35) Trzebiatowski, T.; Dra¨ger, M.; Strobl, G. R. Makromol. Chem. 1982, 183, 731-744.

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surface pressure of 20 mN/m. During the compression, the number of gauche conformers is even reduced. Therefore, it can be understood that the formation of the loop does not cost any additional energy and that this conformation is the one with the lowest energy before the formation of the 3-D aggregates. As was already mentioned, the compression of the PC-C32PC monolayer at the air-water interface also involves conformational changes of the alkyl chains, which are reflected in the frequencies of the antisymmetric (νas) and symmetric (νs) methylene stretching modes at about 2923 and 2850 cm-1, respectively. Because of their conformational sensitivity, these modes can be empirically correlated with the trans/gauche ratio of the alkyl chains. The wavenumber and bandwidth increase with an increasing number of gauche conformers, and lower wavenumbers are characteristic for an ordered all-trans conformation of the chains.36-38 Figure 2C,D shows the IRRA spectra (a-e) of the PC-C32-PC film at the respective areas of the π-A isotherm in Figure 2A. The intensity of both CH2 stretching modes increases with a decreasing molecular area, which is expected when the surface concentration of the molecules increases upon compression of the film. However, this intensity increase may be additionally indicative of orientational changes of the alkyl chains relative to the water surface. Simulations of the CH2 bands as a function of tilt angle show clearly that the intensity of the CH2 stretching bands decreases with an increasing tilt of the alkyl chains with respect to the normal of the water surface. This is due to the fact that with increasing the tilt angle, the transition dipole moment for the CH2 stretching vibrations, which is oriented perpendicular to the chain axis, changes its orientation with respect to the angle of the incident light.39 As is evident from the insets of Figure 2C,D, the frequencies of the band maxima are shifted to lower wavenumbers upon compression of the film. At the beginning of the surface pressure plateau, where a monomolecular film of U-shaped molecules is assumed, the frequencies still indicate a high amount of gauche conformers. The lowest frequencies (νas(CH2) ) 2920 cm-1 and νs(CH2) ) 2849 cm-1 for p-polarized light and νas(CH2) ) 2918 cm-1 and νs(CH2) ) 2850 cm-1 for s-polarized light) are typical for an all-trans conformation of the alkyl chains and were observed at the end of the surface pressure plateau at a low molecular area, where 3-D aggregates are present. Unfortunately, it is not possible to elucidate the molecular arrangement within the aggregates due to their large dimensions as compared to the size of the individual molecules and the size of the 6-7 nm thick fibers observed in bulk water.19 But similar to the aggregate formation in the bulk phase, we expect strong hydrophobic interactions also for the domain formation at the air-water interface since intermolecular H-bonds between the bolaamphiphile molecules are not possible. Several arrangements are conceivable: under the imposed pressure of the barriers of the Langmuir trough, molecules are squeezed out from the monomolecular film into the third dimension (see increasing intensity of the OH stretching band in Figure 2B). During this process, the U-shaped molecules could either maintain their bent conformation or they could relax to an extended conformation. The frequencies of the CH2 stretching bands indicate at 35 Å2 an almost all-trans conformation (36) Snyder, R. G.; Aljibury, A. L.; Strauss, H. L.; Casal, H. L.; Gough, K. M.; Murphy, W. F. J. Chem. Phys. 1984, 81, 5352-6361. (37) Snyder, R. G.; Strauss, H. L.; Elliger, C. A. J. Phys. Chem. 1982, 86, 5145-5150. (38) Snyder, R. G.; Hsu, S. L.; Krimm, S. Spectrochim. Acta, Part A 1978, 34, 395-406. (39) Tung, Y.-S.; Gao, T.; Rosen, M. J.; Valentini, J. E.; Fina, L. J. Appl. Spectrosc. 1993, 10, 1643-1650.

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Figure 5. (A and C) Specular XR normalized by Fresnel reflectivity, R(qz)/Rf(qz), of a PC-C32-PC film (A) at π ) 4 mN/m (I), 12 mN/m (II, shifted for clarity with a factor of 0.005), and 20 mN/m (III, shifted for clarity with a factor of 0.001); of a Me2PE-C32-Me2PE film (C) at π ) 0.5 mN/m (I), 9 mN/m (II, shifted), and 14 mN/m (III, shifted); and best fits to the data (solid lines). (B and D) Electron density profiles along the surface normal z. In panel B, the alkyl tail region (dashed line) at π ) 20 mN/m has been calculated with the assumption of a symmetrical electron density distribution and accounting for the number of electrons in the chain and the area per chain. Table 1. Summary of Data for Molecular Area and Film Thickness of Bolaamphiphiles Deduced from Molecular Structure, Langmuir Isotherms, and XR Measurements M (g mol-1)

#eleca

π(mN m-1)

A(Å2 molecule-1)

hchainb (Å)

PC-C32-PC

812

450

Me2PE-C32-Me2PE

784

434

4 12 20 0.5 9 14

150 120 95 175 120 95

6.6 8 9.6 5.8 8.5 10.2

bolaamphiphile

a

Number of electrons in one molecule. Number of electrons for the alkyl chain is 256. b Fwhm of the chain electron density distribution.

of the alkyl chains (see insets in Figure 2C,D) so that the latter scenario could be envisaged. Me2PE-C32-Me2PE. The characterization of the second investigated bolaamphiphile Me2PE-C32-Me2PE, a dimethylammonio analogue of PC-C32-PC, was performed in a similar way using a pH 5 buffer solution as a subphase. At this pH, both functional groups are charged, and Me2PE-C32-Me2PE is in a zwitterionic state. Figure 3A shows the π-A isotherm of Me2PE-C32-Me2PE at 20 °C. As in the case of PC-C32-PC, the long-range surface pressure increase at large molecular area is followed by a region where the pressure is essentially constant. However, the onset of the surface pressure increase of Me2PEC32-Me2PE is shifted to lower molecular areas by approximately

20 Å2 per molecule,22 and the plateau pressure is lower for Me2PE-C32-Me2PE in comparison to its trimethylammonio analogue PC-C32-PC (14 mN/m for Me2PE-C32-Me2PE as compared to 20 mN/m for PC-C32-PC under the same conditions at the beginning of the plateau). It was assumed that the orientation of Me2PE-C32-Me2PE is comparable to PC-C32-PC with a stretched or slightly bent conformation of the molecules parallel to the water surface at large molecular areas (>200 Å2) and a reverse U-shaped conformation upon further compression. The beginning of the plateau is found at 105 Å2 per molecule, and the limiting molecular area of approximately 135 Å2 is slightly lower than for PC-C32-PC. However, completely different domain structures are observed by BAM at low molecular areas (42 Å2). Here, a

ReVerse U-Shaped Conformation of Bolaamphiphiles

homogeneous granular film consisting of small microcrystals is formed (see inset of Figure 3A). The formation of the different aggregate structures can be explained by the possibility of Me2PE-C32-Me2PE forming intermolecular hydrogen bonds between the headgroups, whereas for PC-C32-PC, this is not possible due to a missing hydrogen bond donor. In Figure 3B, the analysis of the IRRA spectra in the OH stretching region shows a continuous increase of the film thickness, which is consistent with the idea that the chains obtain a more upright orientation with a concomitant increased bending of the alkyl chains upon film compression. A spectrum calculated using the same assumptions as for PC-C32-PC is given in Figure 3B for the OH vibrational band and in the inset for the symmetric CH2 band. The spectrum fits to the experimental spectrum c which was recorded at the beginning of the plateau at 95 Å2. The calculated tilt angle of the alkyl chains amounts to 60°, the same value as was determined for PC-C32-PC. For the CH2 stretching bands, similar spectroscopic features were observed as before. However, the inversion of the intensities found for the antisymmetric and symmetric stretching bands is significantly enhanced at low molecular areas because the antisymmetric CH2 band becomes very broad with low frequency shoulders. Obviously, there is a correlation of the intensity inversion with the formation of a homogeneous granular film of small microcrystals. In the insets of Figure 3C,D, the frequencies of the CH2 stretching vibrational region indicate for s-polarized light the presence of gauche conformers even at low molecular areas where 3-D aggregates are present. The bands recorded with p-polarized light are difficult to analyze due to the extreme broadening of the antisymmetric band. This broadening, however, may indicate that the characteristic bend in the chains is still present.40 In contrast to PC-C32-PC, we could obtain because of better signalto-noise ratios in these measurements additional information from the analysis of the CH2 scissoring band δ(CH2) (see Figure 4). Starting from 54 Å2, a sharp band at 1471 cm-1 appears, which indicates a triclinic packing of the hydrocarbon chains.41 This single sharp band is indicative of ordered solid-like phases with strong interchain interactions, as in triclinically packed polymethylene chains of crystalline hydrocarbons.42,43 Whereas the CH2 bands probe the hydrophobic alkyl chain, the phosphate PO2- stretching modes are useful markers for the degree of hydration of the headgroups. The position of the antisymmetric and symmetric PO2- stretching vibrations is very sensitive to hydrogen bonding. The PO2- stretching bands of Me2PE-C32-Me2PE at the air-water interface are also shown in Figure 4. For aqueous suspensions of Me2PE-C32-Me2PE, both bands are positioned at very low wavenumbers, namely, at 1216 and 1077 cm-1, indicating a strong hydration of the phosphate groups and/or intermolecular hydrogen bonds between the phosphate oxygens and the proton from the HN(CH3)+ group. The frequencies for the antisymmetric and symmetric PO2- stretching vibrational bands are with 1213 and 1070 cm-1 even lower than for the bulk system, indicating stronger hydrogen bonds to the phosphate groups in the 3-D aggregates at the air-water surface. XR. Figure 5A,C shows XR data of PC-C32-PC and Me2PE-C32-Me2PE at selected points (I-III) of the π-A isotherms in Figures 2A and 3A. They clearly demonstrate the formation (40) Tuchtenhagen, J. Kalorimetrische und FT-IR-spektroskopische Untersuchungen an Phospholipidmodellmembranen. Ph.D. Dissertation, Universita¨t Kaiserslautern, Kaiserslautern, Germany, 1994. (41) Mantsch, H. H.; Madec, C.; Lewis, R. N. A. H.; McElhaney, R. N. Biochim. Biophys. Acta 1989, 980, 42-49. (42) Snyder, R. G. J. Mol. Spectrosc. 1961, 7, 116-144. (43) Casal, H. L.; Mantsch, H. H.; Cameron, D. G.; Snyder, R. G. J. Chem. Phys. 1982, 77, 2825-2830.

Langmuir, Vol. 23, No. 11, 2007 6069

Figure 6. Schematic illustration of the proposed monolayer structures of PC-C32-PC and Me2PE-C32-Me2PE at the air-water interface at the respective areas in points I-III of the π-A isotherms in Figures 2A and 3A.

of homogeneous films at the beginning of the surface pressure plateau for both bolaamphiphiles. The electron density profiles along the surface normal of the films at the air-water interface, as given in Figure 5B,D, were obtained by model-free inversion of the XR data. The profiles give unambiguous evidence for a U-shaped conformation of the bolaamphiphiles PC-C32-PC and Me2PE-C32Me2PE at the air-water interface, where both headgroups point into the subphase. Assuming a symmetrical chain region and constraining the number of electrons in the chains (#elec) and the molecular area of a bolaamphiphile molecule obtained from the π-A isotherm results in a unique electron density distribution. The value for the full width at half-maximum (fwhm) of the chain electron density hchain can be determined, which translates directly into the thickness of the alkyl chain part of the corresponding film (see Table 1). This thickness of the hydrophobic part of the film increases for both bolaamphiphiles on compression, which can be easily understood if we imagine an increased bending of the alkyl chain with increasing surface pressure and a more upright orientation. Under the condition of densely packed alkyl chains at point III of the isotherm, the effective length of the alkyl chains in the U-shaped conformation was estimated to be 20 Å from which the tilt angle θ ) 60° for the alkyl chains could be calculated for both bolaamphiphiles (θ ) arccos(layer thickness/effective length) of the U-shaped alkyl chains). This calculated tilt angle is in excellent agreement with the value obtained from the analysis of the OH stretching bands of the IRRA spectra. On the basis of these data, the arrangement of the bolaamphiphiles at the air-water interface can be summarized in the scheme shown in Figure 6. It shows the change of the bolaamphiphile conformation and orientation with increasing surface pressure from an extended to a strongly bent conformation with a final tilt angle of 60° of the molecule relative to the surface normal. The three states correspond to the points in the π-A isotherm where reflectivity measurements were performed.

Conclusion The main findings from this work are outlined schematically in Figure 6. The combination of IRRAS and XR measurements provides a reliable experimental approach to determine the conformation, orientation, and packing of bipolar amphiphilic molecules at the air-water interface. An unambiguous analysis of the surface pressure-molecular area isotherms was performed for the symmetrical long-chain bolaamphiphiles PC-C32-PC and Me2PE-C32-Me2PE. At low surface pressures, the molecules were found to lie flat on the water. With increasing surface pressures, the alkyl chain began to bend pointing into the air, whereas both polar headgroups kept contact with the water subphase. At the surface pressure plateau, both bolaamphiphiles adopted a reverse U-shaped conformation with a strongly tilted alkyl chain. Acknowledgment. This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. LA070029H