Monolayers of mono- and bipolar palmitic acid derivatives

Colloids and Surfaces A: Physicochem. Eng. Aspects 250 (2004) 57–65

Monolayers of mono- and bipolar palmitic acid derivatives Xiuli Yuea , Paul Steffena , Bodo Dobnerb , Gerald Brezesinskia , Helmuth M¨ohwalda,∗ a

Max Planck Institute of Colloids and Interfaces, Am M¨uhlenberg 1, D-14476, Golm/Potsdam, Germany b Institute of Pharmaceutical Chemistry, Martin-Luther University, D-06120, Halle/S., Germany Received 6 February 2004; accepted 8 March 2004 Available online 22 September 2004

Abstract Monopolar and bipolar derivatives of hexadecanoic acid (HA), 16-hydroxyhexadecanoic acid (HHA), methyl hexadecanoate (MH) and methyl 16-hydroxyhexadecanoate (MHH) have been investigated on pure water and NaCl solutions with different ion concentrations (1, 2 and 3 mol l−1 ). Surface pressure area isotherms show that HA forms a fully condensed monolayer on pure water at 20 ◦ C [E. Teer, C.M. Knobler, S. Siegel, D. Vollhardt, G. Brezesinski, J. Phys. Chem., B104, 43, 2000, pp. 10053–10058] whereas in the case of the corresponding bipolar HHA the hydroxy group as a second polar moiety leads to a destabilization of the monolayer. The presence of two relatively strong hydrophilic polar groups at opposite ends of the chain prevents the formation of condensed films. The esterification of the carboxyl group (MH) changes the phase sequence from L2 –Ov–LS for HA to L2 –LS. Inserting a hydroxy group at the end of the chain (MHH) shifts the liquid expanded/liquid condensed (LE/LC) phase transition to higher surface pressures but does not change the phase sequence, however it increases the chain tilt. The pressure of the first-order phase transition LE/LC is strongly temperature dependent for MH, while the transition pressure of MHH is almost temperature independent. The phase behavior of MHH and MH on pure water was further studied by surface potential, Brewster angle microscopy (BAM), fluorescence microscopy and grazing incidence X-ray diffraction (GIXD) measurements. The LC domains of MHH on pure water are so small that no inner texture can be observed by BAM in contrast to the LC domains of MH. 3M NaCl in the subphase does not change the MH textures, while it increases the size of the LC domains of MHH. The influence of the hydroxy group on the monolayer behavior is discussed in terms of the formation of hydrogen bonds. The presence of NaCl in the subphase expands the monolayers. The results obtained are explained by changes in monolayer–monolayer and monolayer–subphase interactions. © 2004 Elsevier B.V. All rights reserved. Keywords: Mono- and bipolar amphiphiles; Monolayers; Isotherms; BAM; GIXD

1. Introduction Over the past few years, bolaamphiphiles, which consist of two polar headgroups connected to each other by one or more hydrophobic spacer groups, [2] have progressively gained importance because of their ability to offer several advantages for the formation of monolayer lipid membranes and for the construction of advanced liposomes that are characterized by high mechanical and thermal stability due to the organization of the membrane [3–7]. Monolayers of chemically well defined bipolar amphiphiles are important for understanding of structure and function of biological and reconstituted mem∗

Corresponding author. Tel.: +49 331 567 9200; fax: +49 331 567 9202. E-mail address: [email protected] (H. M¨ohwald).

0927-7757/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2004.03.035

branes. Different bipolar amphiphiles were investigated in order to obtain information about molecules with different headgroups and polar substituents in the hydrocarbon chain and their intermolecular interaction in monolayers [8–17]. For the tailor-made synthesis of bipolar amphiphiles for special requirements, it is interesting to know the effect of specific polar groups on the monolayer properties. Studies of simpler bipolar molecules are necessary in order to obtain a clearer view on the effect of the hydrophilicity of the attached second polar group. As typical substances, fatty acid derivatives with a hydroxy substituent would seem to be ideal for this purpose, since the hydroxy group is small. While monolayers of monopolar molecules on the basis of palmitic acid and its esters are well investigated with regard to formation, stability and properties, much less is known about

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the structures of amphiphiles with two polar groups [18–20]. Furthermore, the presence of sugar hydroxyl groups should contribute to the stabilization of supramolecular aggregates through cooperative hydrogen bonding, as observed in natural archaeal glycolipids [21]. The interest in amphiphiles with hydroxy groups as a second polar moiety within an amphiphilic structure is not only because they are versatile model compounds, [22–24] but they could also be industrially important materials [25] and of extensive interest in technology including drug encapsulation [26–28] and gene delivery [29,30]. It would also be very interesting to know how electrolytes modify the structure and stability of such monolayers because electrostatic forces cause a change of the interactions within the monolayer as well as its interactions with the subphase. Addition of electrolytes has important biological consequences, such as influence on the transport of ions through a membrane or on oriented nucleation of minerals on organic surfaces [31]. Also, theoretical efforts have been made to explain the effect of ions on the structure and morphology of charged monolayers [32–34]. Ionized monolayers are of interest for the deposition on solid substrates which plays also an important role in many industrial applications [35,36]. In order to learn more about the stabilization or destabilization of the alkyl chain packing induced by the insertion of a second polar group that can form intra- and inter-molecular hydrogen bonds, monolayers of hexadecanoic acid (HA) and methyl hexadecanoate (MH) are compared with monolayers of their corresponding bolaamphiphiles 16-hydroxyhexadecanoic acid (HHA) and methyl 16-hydroxyhexadecanoate (MHH) on different subphases and at different temperatures. The direct observation of the formation of condensed phases was performed by fluorescence microscopy and Brewster angle microscopy (BAM). Additionally, investigation of monolayers at the air–water interfaces with grazing incidence X-ray diffraction (GIXD) enables a direct insight into the monolayer structure at molecular level. Variations in the phase behavior between MH and MHH are expected to correlate directly with distinct interactions between the polar and non-polar molecular moieties.

2. Experimental section 2.1. Materials NaCl, hexadecanoic acid (HA), 16-hydroxyhexadecanoic acid (HHA), methyl hexadecanoate (MH) were purchased from Sigma (approximately 99%) and used without further purification. The spreading solvent was chloroform obtained from Merck. Methyl 16-hydroxyhexadecanoate (MHH) was synthesized according to the following procedure: 16-Hydroxyhexadecanoic acid (0.01 mol, 2.72 g) was dissolved in methanol/ether (10/1). Then an ether solution of diazomethane was added with stirring until the nitrogen evolution has finished and the reaction mixture becomes pale

yellow. Acetic acid was dropped into the solution to destroy the excess diazomethane. The mixture was then evaporated and the residue was dissolved in ether. The ether phase was two times extracted with 20 ml 5% cold potassium hydroxide and washed with water and ammonia chloride solution until the water phase became neutral. After drying (sodium sulfate) and evaporation the crude ester was purified by chromatography using hexane/chloroform (8:2). Yield: 1.92 g (67.2%), mp: 39–41◦ C, ESI-MS: 309.4 [M+Na], 1 H-NMR (CDCl3 , 400 MHz): ␦ ppm): 1.11–1.24 (m 22H, CH2 ), 1.43–1.54 (m,4H, CH2 ) 2.18–2.22 (t, 2H, CH2 COOCH3 ), 3.52–3.57 (m, 6H, HO CH2 COOCH3 ) Found: C: 71.28; H: 11.62 (calc: C: 71.04, H: 11.96) 2.2. Film balance and surface potential measurements, Brewster angle and fluorescence microscopy Surface pressure-area isotherms (π–A isotherms) were measured on a Teflon trough (Riegler&Kirstein, total surface area of 144 cm2 ). The surface pressure was recorded with a continuous Wilhelmy-type pressure measuring system using a filter paper as plate with accuracy better than 0.1 mN/m. The substances were spread from 1 mM chloroform solutions onto the water surface using a 100 ␮L sy˚ 2 per ringe. The monolayers were compressed at a rate of 5 A −1 molecule min after allowing about 10 min for evaporation of the solvent. All experiments were performed using ultrapure water obtained from a Millipore system with a specific resistance above 18 Mcm as a subphase. The pH value of the subphase was slightly below six. Surface potential–area (Ψ –A) isotherms were recorded using the above described trough. During monolayer compression, π and Ψ were recorded simultaneously. The surface potential sensor consists of a Kelvin probe vibrating electrode model KP2 (NIMA Technology, England), which is suspended above the monolayer. The morphology of the monolayers was visualized using the Brewster angle microscope BAM-2 (NFT; G¨ottingen). The optical system and the Langmuir film balance are mounted on an antivibration table. A p-polarized beam of a diode laser is reflected at a mirror and strikes the water surface under the Brewster angle to give zero reflection. The monolayer changes the optical properties of the interface so that it can be visualized by the reflected light. An analyzer in the reflected beam enables optical anisotropy of the film to be distinguished. Monolayer compression or expansion is recorded on videotape, and frames are grabbed and digitized by a computer. Fluorescence microscopy images were generated from the light emitted by the 2-[12-(7-nitrobenz-2-oxa-1,3-diazol4-yl)dodecanoyl]-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-C12-HPC) fluorescent probe (Molecular Probes Inc. Eugene, USA), which was added to the spreading solution in small quantities (1 mole.%). The NBD molecules preferentially partition into the fluid phase, causing a bright image in the microscope. As the phase separation occurs and

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round domains are formed, NBD molecules are squeezed out of the solid phase, and the domains appear as dark regions in the image. Monolayers were viewed from above by a microscope (Zeiss Axiotron). The light source was a mercury lamp (HBO 50 W), where the excitation beam was obtained through a filter transmitting in the range of 450–490 nm. The image was collected by a SIT camera (Hamamatsu C2400) and viewed on the display screen. The measurements were performed on the film balance described above. The images were stored by means of a video recorder during compression. 2.3. Grazing incidence X-ray diffraction (GIXD) Grazing-incidence X-ray diffraction was performed using the liquid-surface diffractometer on the undulator beamline BW1 at HASYLAB, DESY, Hamburg, Germany [37,38]. ˚ A monochromatic beam with a wavelength of 1.481 A was adjusted to strike the surface with an angle of incidence 0.85␣c (␣c = 0.13◦ is the critical angle for total reflection). The intensity of the diffracted beam is detected by a linear position-sensitive detector (PSD) (OED-100-M, Braun, Garching, Germany) as a function of the vertical scattering angle ␣f . The resolution of 0.009◦ of the horizontal scattering angle 2 is given by a Soller collimator located in front of the PSD. According to the geometry of diffraction, the scattering vector Q has an in-plane component Qxy = (2π/␭) (cos2 ␣i + cos2 ␣f − 2cos ␣i cos ␣f cos2)1/2 and an out-of plane component Qz = (2π/␭) (sin ␣i +sin ␣f ). The positional correlation length can be estimated from the full width at half-maximum (fwhm). For an exponential decay of positional correlation as observed in liquid crystals (corresponding to a Lorentzian as a Bragg peak profile), it is ␰ = 2/fwhm(Qxy ). The accumulated position-resolved scans were corrected for polarization, footprint area, and powder averaging (Lorentz factor). Model peaks taken to be Lorentzian in the in-plane direction and Gaussian in the out-of-plane direction were fitted to the corrected intensities. The lattice spacings are obtained from the in-plane diffraction data. The lattice parameters can be calculated from the lattice spacings.

3. Results and discussion 3.1. Isotherms on pure water Fig. 1 presents the π–A isotherms of HA, HHA, MH and MHH on pure water at 22 ◦ C. All π–A isotherms show a pressure increase on reduction of the molecular area. HA and MH form fully condensed monolayers. In contrast, the bipolar amphiphile 16-hydroxyhexadecanoic acid (HHA) is unable to form a stable monolayer at the air–water interface whereas the corresponding bipolar ester exhibits a clear first order transition from a liquid-expanded (LE) to a stable condensed (LC) state. Obviously, adding a hydroxy group to HA increases the

Fig. 1. Surface pressure-area isotherms at 22 ◦ C for HHA, MHH, HA and MH on pure water.

˚ 2 per solubility, therefore the very small area (less than 20 A molecule) obtained for HHA can be explained either by dissolution of HHA molecules into the subphase or the formation of disordered 3-D aggregates. On the other hand, the presence of two relatively strong hydrophilic groups (hydroxy group and acid group) at opposite ends of the hydrophobic chain of the HHA molecule prevents the formation of an upright conformation. However, esterification of the carboxyl group (MHH) decreases the hydrophilicity of this group sufficiently so that the hydroxy group can now be regarded as the primary hydrophilic group. A second point is that the stability of the bulk phase is significantly smaller in the case of the ester compared with the acid. Therefore, the combination of these two factors favors a condensed monolayer phase for the ester MHH whereas this is impossible for the acid HHA, which was proved by the observation that ␣,␻-dicarboxylic acids do not form a stable condensed phase, but their diethyl esters do. MHH appears to be more expanded at lower pressure compared to other single-chain amphiphiles. The transition into the LC phase starts around 5.5 mN/m at a very large molecular ˚ 2 . The plateau represents the LE–LC coexistence area of 98 A region. A further sharp increase of π corresponds to the formation of a compact LC phase. Obviously, the presence of two hydrophilic head groups has a clear expanding effect. A possible explanation for the conformational changes occurring during the film compression is that bolaamphiphiles are bent with both hydrophilic groups attached to the water surface in the liquid-expand region at large molecular areas. According to the bipolar structure the formation of a condensed monolayer phase requires the removal of one polar group from the water surface in the two-phase coexistence region [14,15,24]. The relationship between interfacial behavior and organization at the surface is best indicated by the dipole moments; i.e., any change in the orientation should be clearly seen in the surface potential values. The vertical component of the dipole moment ␮, can be estimated from measured surface potential using the Helmholtz equation V = ␮/␧ ␧0 A, where ␧0 and ␧ are the relative permittivities in vacuum and

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Fig. 3. ␲–A isotherms of MH (top) and MHH (bottom) on pure water at different temperatures (indicated). Fig. 2. Surface pressure (solid line) and surface potential (dotted line)–area isotherms of MH (top) and MHH (bottom) on pure water at 25 ◦ C.

air and A is the average area/residue. Fig. 2 shows the surface potential–area isotherms measured simultaneously with the π–A isotherms for MH and MHH at 25 ◦ C on pure water. At ˚ 2 per molecule), the onset-pressure of the LE phase (∼73 A the surface potential of MH increases suddenly to ∼243 mV and rises slowly with film compression. At the onset of the ˚ 2 per molecule), V is about LE–LC phase transition (∼56 A 280 mV. The increase in V is much more pronounced in the condensed phase indicating drastic changes in the orientation of the molecule. There are no clear differences between the surface potential measurements of MH and MHH. Additional information about the effect of the hydroxy group is provided by the temperature dependence of the phase transition pressure. Isotherms have been measured in the temperature range between 16 and 36 ◦ C (Fig. 3). The pressure of the LE/LC phase transition shifts to higher values with increasing temperature. The molecular area at the onset of ˚ 2 for the LE/LC phase transition increases from 45 to 55 A 2 ˚ for MHH. Fig. 4 represents the MH and from 85 to 102 A comparison of the phase transition pressure πt as function of temperature. Both MH and MHH monolayers exhibit an approximately linear dependence of the surface pressure on temperature. Based on these results, the temperature coefficient dπt /dT is determined and presented in Table 1. The temperature coefficients are quite different: 0.47 mN/km for MH and 0.102 mN/Km for MHH. The transition enthalpy was

Fig. 4. Temperature dependence of the main phase transition pressure ␲t for MH (dashed line) and MHH (solid line).

Table 1 Thermodynamic data derived from isotherm measurements. The surface pressure π is given in mN/m, the characteristic temperatures T0 and Tc in ◦ C, the transition enthalpy H in kJ/mol, the transition entropy S in J/mol K and dπt /dT in mN/km compound

dπt /dT

T0

dH/dT

dS/dT

Tc

MH MHH

0.470 0.102

19.9 −31.8

−1.222 −0.065

−4.359 −0.286

43.7 203.1

X. Yue et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 250 (2004) 57–65

Fig. 5. Temperature dependence of the transition enthalpy H of the LE/LC phase transition of MH (
) and MHH (䊉).

calculated by the area change during the LE/LC transition applying a modified Clausius–Clapeyron equation dπt /dT = H/T(AC − AE ), where πt is the plateau pressure at temperature T, AE is the molecular area in the liquid-expanded state at πt and Ac is the molecular area in the condensed phase at πt . Fig. 5 shows the transition enthalpy versus temperature. Additionally, the entropy change S calculated by S = H/T is presented in Fig. 6 as function of subphase temperature. The values of H and S for MHH are very weakly temperature dependent and relatively small because of the weak temperature dependence of πt . This could mean that either the liquidexpanded phase of MHH is more ordered or the condensed phase is more disordered. Thus, MH shows a considerable change in conformation during film compression as deduced from the high transition enthalpy and entropy values, whereas in the case of MHH the enthalpy and entropy values of LE and LC phases must be more similar. The extrapolation to zero transition pressure yields the temperature T0 , above which a plateau in the isotherm can be observed and below which no LE phase exists. The critical temperature Tc , above which the monolayer cannot be compressed into a condensed state, can be observed by extrapolating H to zero. In the case of MH, the monolayer is fully condensed below 20 ◦ C and no

Fig. 6. Temperature dependence of the entropy change of the LE/LC phase transition of MH (solid line) and MHH (dashed line).

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condensed phase can be obtained above 44 ◦ C. The extrapolated values of MHH (T0 = −32 ◦ C and Tc = 203 ◦ C) can never be reached experimentally. Consequently, from these calculations we conclude that the hydroxy groups change the interactions between the molecules in the condensed film drastically, most probably by forming a hydrogen bonding network. To visualize possible differences in the shape of condensed domains in MH and MHH monolayers, Fig. 7 shows Brewster angle micrographs of the corresponding monolayers taken in the plateau region of the π–A isotherms. The bright domains in the images obtained from BAM measurements represent a condensed phase that appeared during film compression at the onset of the plateau region. These condensed phase domains are surrounded by the expanded phase within the twophase coexistence region [39,40]. The area fraction of the condensed phase increases continuously during compression according to the lever rule for a first order phase transition. MH forms very large round domains with 6 segments first observed by Knobler and coworkers [41]. Regions of different reflectivity indicate that the molecules are tilted and areas with different tilt directions exist. The bipolar MHH forms many much smaller condensed phase domains compared to MH. The small domains are circular and do not show good contrast so that it is impossible to observe possible inner textures. This is due to a high nucleation rate and a much smaller growth rate of MHH in comparison to MH. In the case of HHA, we don’t see any contrast in the BAM micrographs during compression of the monolayer. Therefore, it is most likely that HHA dissolves in the subphase, which explains the small molecular areas observed in the isotherm. Because of higher resolution fluorescence microscopy could give additional insight into the growth of condensed phase domains. The fluorescence probe (NBD-C12 -HPC) is soluble in the fluid phase and squeezed-out from the condensed phase. Therefore, the bright areas in the fluorescence micrograph represent the expanded phase. At the concentration used (1 mol.%) there was no observable effect on the size or shape of the condensed phase domains. A typical fluorescence micrograph is given in Fig. 8. At the onset of the transition, the first solid nuclei are observed in a homogeneous fluid phase media. The initial non-uniform domain size

´˚ 2 per Fig. 7. Brewster angle micrographs of monolayers of a) MH (30 A ´ 2 ◦ ˚ per molecule) on water at 25 C. molecule) and b) MHH (30 A

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´˚ 2 per molecule) Fig. 8. Fluorescence micrograph of MHH monolayer (30 A at 22 ◦ C using NBD-C12 -HPC as fluorescence label.

distribution is a consequence of simultaneous nucleation and growth. The faster the compression rate the greater the nucleation rate. Whereas at larger areas per molecule the domains gave no indication of long-range interactions, at smaller areas per molecule they show a much more uniform superstructural packing, indicating long-range electrostatic repulsion. The monolayer behavior affected by ions dissolved in the subphase at concentrations from 1 to 3M of NaCl was also studied for these amphiphiles (Fig. 9). Compared with pure water (∼pH 6), a significant expansion is observed in all cases and increases with increasing sodium chloride concentration. The LE phase is stabilized even for HHA. However, the observed plateau region does not correspond to the phase transition into a condensed phase but to a collapse of the monolayer. This is supported by BAM observations (Fig. 10). The high intensity of the reflected light suggests the presence of more than one molecular layer. The presence of ions decreases the solubility of HHA molecules and leads to the formation of multilayers in the plateau region. The phase behavior of the monolayers depends on the overall interactions, such as intermolecular forces between adjacent monolayer molecules and between these molecules and the subphase molecules. The presence of ions changes the dissociation degree of the acid head group. This effect can be understood in terms of the Gouy–Chapman theory relating the ionization degree with the bulk pH [42]. Using PM-IRRAS experiments, it was observed that the pH gap between the interface and the subphase depends on the type of counterion and amounts to pH = −3.4 for arachidic acid on a subphase containing Na+ [43]. Increasing counterion concentration at a fixed bulk pH increases the ionization degree. The electrostatic repulsion between charged head groups leads to the observed fluidization of the HHA monolayer. In the case of MH and MHH, no effect is expected due to ionization of head groups because the pH of the subphase is too low to observe any measurable hydrolysis of the ester. However, there is a small increase of the phase transition pressure between LE and LC due to increasing salt concentration. The reason for the small influence of NaCl on the behavior of the hexadecanoic acid methyl ester monolay-

Fig. 9. Surface pressure-area isotherms at 22 ◦ C for HHA (top), MH (middle) and MHH (bottom) at different NaCl concentrations (indicated).

ers should be the increased ionic strength, which influences polarity and interactions of the water molecules with the carboxyl group as well as between water–water molecules. The domains of MH on 3 M NaCl solution are similar to the ones on pure water while the domains of MHH on 3 M NaCl are much bigger than the domains on pure water. Additionally, these domains on NaCl subphases are subdivided into several segments of different reflectivity. However, the size of the domains is still small and it is very difficult to define the exact number of segments (which might be 5 or 6). No variation between different domains can be observed. To obtain further information about the structure of the condensed phases at a molecular level, the monolayers have been studied by GIXD. The X-ray measurements of HA and MH at 20 ◦ C indicate that HA exhibits a phase

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´˚ 2 per molecule, 25 ◦ C), (b) MHH (33 A ´˚ 2 per molecule, 25 ◦ C) and c) HHA (60 A ´˚ 2 per Fig. 10. Brewster angle micrographs of monolayers of (a) MH (30 A molecule, 22 ◦ C) on 3M NaCl.

Fig. 11. Contour plots of the corrected intensities as function of the in-plane scattering vector component Qxy and the out-of-plane scattering vector component Qz for MHH on pure water at 20 ◦ C and different surface pressures (indicated).

sequence L2 (NN (nearest neighbor) tilted orthorhombic phase)—Ov (NNN (next-nearest neighbor) tilted orthorhombic phase)—LS (untilted hexagonal) [1] whereas the methyl ester MH exhibits only a phase sequence L2 –LS. At 20 ◦ C, the transitions occur at 16.5 and 22.4 mN/m for HA and at around 10 mN/m for MH. Fig. 11 shows selected contour plots of the diffracted intensity as a function of the in-plane scattering vector component Qxy and the out-of-plane scattering vector component Qz for MHH at different surface pressures. The positions of the Qxy and Qz maxima are given in Table 2. At low pressures, two diffraction peaks were observed. One peak is located at zero Qz and the other Bragg peak at Qz > 0. Such an intensity distribution corresponds to a centered rectangular unit cell and indicates a tilt direction of the alkyl chains toward nearest neighbors (L2 ). Increasing surface pressure shifts the diffraction peaks to lower Qz and larger Qxy values, indicating a decrease of the tilt angle t and a closer packing in the water plane (decreasing molecular area Axy ). Further compression leads to a diffraction pattern typ-

ical for a hexagonal packing of untilted chains (LS). Since the condensed isotherms exhibit a linear relation between pressure and molecular area: Axy = K1 −K2 π, where Axy = A0 /cos(t) with A0 as cross-sectional area of the chains and t as chain tilt angle, plotting of 1/cos(t) as a function of π allows the determination of the tilting phase transition pressure πt by extrapolation towards 1/cos(t) = 1. For the transition from L2 to LS a pressure of 18.9 mN/m has been determined. Comparison of the monopolar and the bipolar esters MH and MHH, respectively, shows that the bipolar MHH is more tilted than MH at low lateral pressure. The transition pressure L2 –LS is also shifted to higher values. This indicates that the tilted L2 phase is stabilized at the expense of the untilted LS phase. A comparison of the substances with respect to the ˚ for MH at 22 ◦ C positional correlation length ␨ gives 180 A ˚ at 20 ◦ C and and 20 mN/m and a much smaller value of 87 A 20 mN/m for MHH. The positional correlation length ␨ is a measure of the lattice order. It is also interesting that the ˚2 cross-sectional area per molecule is reduced from 19.9 A

Table 2 GIXD data with fwhm in brackets, lattice parameters a and b, tilt angle t, cross-sectional area A0 and tilt direction of MHH on water at 20◦ C ␲ (mN/m) 8 11.5 14.5 20

˚ −1 ) Qxy (A

˚ −1 ) Qz (A

˚ a (A)

˚ b (A)

˚ 2) A0 (A

t (◦ )

Tilt direction

1.545 (0.020) 1.458 (0.033) 1.538 (0.018) 1.490 (0.031) 1.537 (0.019) 1.491 (0.030) 1.529 (0.023)

0 0.423 0 0.309 0 0.290 0

5.081

8.129

19.5

18.9

NN

4.923

8.169

19.5

13.6

NN

4.918

8.177

19.6

12.8

NN

4.745

8.218

19.5

0

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˚ 2 for MHH. From this observation one can for MH to 19.5 A conclude that the additional hydroxy group is not disturbing the chain packing but leads to an even tighter packing of the chains most likely due to the formation of hydrogen bonds between the molecules. This could also explain the smaller correlation length of the lattice because hydrogen bonds are directed bonds with a relatively fixed distance and therefore not so flexible to react on compression of the layer. If we assume that the generic phase diagram of the bipolar hydroxyfatty acid methyl esters is similar to the one of monopolar fatty acid methyl esters one can additionally conclude that the transition into the untilted orthorhombic S phase should occur at higher temperatures for MHH compared with MH. In conclusion, the discrimination for MH and MHH at a molecular level as deduced from grazing incidence X-ray diffraction measurements correlates well with large differences in surface pressure-area isotherms.

4. Conclusions We have endeavored to explore systematically the surface behavior of hexadecanoic acid derivatives. The difference in polar group hydrophilicity plays an important role for the monolayer properties of the bolaamphiphiles. The additional hydroxyl group influences the phase behavior and the film stability in a dramatic way. HHA failed to achieve a condensed monolayer state because of equally strong hydrophilic head groups at opposite ends of the chain. But the stability of the monolayers is improved by addition of salt (NaCl) to the subphase. The monolayer structure and stability is strongly related to the formation of intra- and inter-molecular hydrogen bonds. GIXD data reveal that inserting the hydroxyl group into MH does not lead to a qualitative change of the lattice structure and the phase sequence, but leads to a stronger tilt of the molecule and shifts the phase transition into an untilted state to higher values. It also leads to a remarkable decrease of the cross-sectional area and at the same time to a reduction of the positional correlations in the LS phase compared with the monopolar MH. BAM and fluorescence microscopy investigations reveal that the hydroxy group has a disturbing effect on the growth of condensed phase domains. MHH shows a much larger expanded/condensed phase coexistence region in the π–A isotherms and forms smaller domains, while MH shows a shorter plateau and forms larger domains. Examination of the temperature dependent phase behavior of monolayers offers the possibility of determining phase transition enthalpy and entropy and thus energetic differences between the ordered (LC) and disordered (LE) phases. MHH is less sensitive to the temperature than MH, indicating that the hydroxy group has a clear influence on the properties of the monolayer through hydrogen bonds. The study of monolayers spread on salt solutions at different concentrations shows that increasing counterion concentration at a fixed bulk pH increases the ionization degree. The electrostatic repulsion

between charged head groups leads to the observed fluidization of the HHA monolayer. Only small effects are observed for MH and MHH. The reason for an influence of NaCl on the behavior of the hexadecanoic acid methyl ester monolayers should be the increased ionic strength, which influences polarity and interactions of the water molecules with the carboxyl group as well as between water–water molecules. More bolaamphiphiles chemically changed in a defined way are under examination to throw additional light on the effect of various structural order parameters.

Acknowledgement This work was supported by the Max Planck Society. We gratefully acknowledge beamtime at HASYLAB, DESY (Hamburg, Germany) and Dr. K. Kjaer for help with the beamline setup.

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