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Langmuir 2009, 25, 4104-4110
From Specular Reflectivity to Time-Resolved Grazing Incidence X-ray Scattering out of the Specular Plane (GISAXS): Equilibrium and Nonequilibrium States of Organic/Inorganic Monolayers at Liquid Surfaces† Lutz Wiegart,‡ Sean M. O’Flaherty,§ and Pierre Terech*,| European Synchrotron Radiation Facility (ESRF), BP 220, 38043 Grenoble Ce´dex 09, France, Oxford Instruments Americas, 945 Busse Road, Elk GroVe Village, Illinois 60007, and CEA-Grenoble, INAC-SPrAM, UMR5819, 17 rue des Martyrs, 38054 Grenoble Ce´dex 09, France ReceiVed October 24, 2008 The grazing incidence diffuse X-ray scattering out of the specular plane technique (GISAXS) is presented, and its capabilities are compared to the more classical X-ray specular reflectivity technique (XSR). Three experimental illustrations are given to prove the efficiency of GISAXS. First, the structure of a DPPC phospholipid monolayer is analyzed. Second, the time-resolved kinetics of lipid desorption from a monolayer interacting with a mineral gel subphase is studied. Third, the structural evolution of biomembranes at extremely low temperatures is illustrated. The GISAXS technique appears to compete efficiently with neutron reflection techniques by taking advantage of remarkably short acquisition times crucial in kinetics experiments.
Introduction Layered organic systems receive a tremendous amount of attention for the fundamental questions that they raise and the potential applications that they offer. Single- or multilayered films can be formed at air-liquid (organic or aqueous) interfaces or on solid substrates.1 Lipidic membrane molecules and amphiphilic molecules that can develop 1D self-assemblies in bulk solutions2,3 and on air-liquid interfaces4 are systems where the knowledge of structure resolved at a submolecular length scale is of utmost importance. The structural characterization of a monolayer at the air-liquid interface details the 2D aggregation mechanism. This is a prerequisite piece of information required for deciphering the structures developed in the bulk solution or in multilayered films on solid substrates. Two techniques using the intense X-ray beams delivered by modern synchrotron sources are presented in this work. Their suitability for studying monomolecular layers at liquid surfaces is demonstrated using three different situations involving a low mass amphiphile (phospholipid derivative). First, a single monolayer at the air-water interface will be studied as a function of surface pressure. Second, a time-resolved kinetics investigation of the desorption of such a monolayer from a subadjacent monolayer of charged mineral disks in a gel subphase will be presented. Third, it will be shown that the aggregation mechanism can also be studied at temperatures lower than the freezing point of water. X-rays were preferential to neutrons because the flux of photons from a synchrotron source is much larger than the flux of neutrons from a neutron source, thereby allowing much shorter acquisition † Part of the Neutron Reflectivity special issue. * Author to whom correspondence should be addressed. E-mail:
[email protected]. ‡ European Synchrotron Radiation Facility (ESRF). § Oxford Instruments Americas. | CEA-Grenoble.
(1) Decher, G. Science 1997, 277, 1232–1237. (2) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1976, 72, 1525–1568. (3) Israelachvili, J. N. Intermolecular and Surface Forces, 3rd ed.; Academic Press: London, 1992; pp 341-435. (4) Palacin, S. AdV. Colloid Interface Sci. 2000, 87, 165–181.
times. Additionally, the accessible momentum transfer (q) range available with X-rays is larger. This is an important detail given the extremely small thickness of monomolecular films as the scattered radiation generates oscillations with large q periods in reciprocal space. X-ray scattering and neutron scattering are similar with respect to the contrast scattering magnitude steps in the electron density or the neutron scattering length density of their constitutive elements, respectively. In addition, neutron reflectivity can take advantage of the different scattering powers for the isotopes of a given element (in particular, H vs D). The performance of the GISAXS technique appears to enable deeper investigations of molecular aggregation mechanisms and enables studies of the kinetics and temperature dependence of the aggregation modes for specialized molecules (e.g., gelators).5 It can also be used to perform studies typically covered with standard techniques such as specular reflectivity6-9 over much shorter time frames, thus minimizing in the X-ray case possible effects due to radiation damage. GISAXS versus XSR. X-ray specular reflectivity (XSR) is established as a powerful technique for studying surfaces and probing the structure of single- and multilayered thin film materials of hard and soft condensed matter.10-14 The electron density profile along the surface normal, comprising the layer thicknesses, the average electron density of the layers, and the root-meansquare (rms) amplitude of the interface roughness (which is indistinguishable from the transitional density thickness of a (5) Langmuir 2009, 25(7). Langmuir special issue. (6) Kuzmenko, I.; Rapaport, H.; Kjaer, K.; Als-Nielsen, J.; Weissbuch, I.; Lahav, M.; Leiserowitz, L. Chem. ReV. 2001, 101, 1659–1696. (7) Gregory, B. W.; Vaknin, D.; Gray, J. D.; Ocko, B. M.; Stroeve, P.; Cotton, T. M.; Struve, W. S. J. Phys. Chem. B 1997, 101, 2006–2019. (8) Tachibana, T.; Hori, K. J. Colloid Interface Sci. 1977, 61, 398–400. (9) Vaynberg, K. A.; Wagner, N. J.; Ahrens, H.; Helm, C. A. Langmuir 1999, 15, 4685–4689. (10) Russell, T. P. Mater. Sci. Rep. 1990, 5, 171–271. (11) Synchrotron Radiat. News 1999, 12(2). (12) Tolan, M. X-ray Scattering from Soft-Matter Thin Films; Tracts in Modern Physics; Springer: Berlin, 1999; p 148. (13) Holy, V.; Pietsch, U.; Baumbach, T. High-Resolution X-ray Scattering from Thin Films and Multilayers. Springer: Berlin, 1999; Vol. 149. (14) Daillant, J., Gibaud, A., Eds.; X-ray and Neutron ReflectiVity: Principles and Applications; Springer Verlag: Berlin, 1999.
10.1021/la803547j CCC: $40.75 2009 American Chemical Society Published on Web 02/12/2009
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graded interface) can be readily extracted from experimental reflectivity data. Essentially there are two methods for obtaining X-ray specular reflectivity data: (1) at a fixed wavelength with simultaneous variation of incidence (Ri) and scattering (Rf) angles where Ri ) Rf and both angles are measured from the surface plane, commonly referred to as the θ -2θ technique, and (2) at fixed incident and scattering angles with variation of the wavelength (energy dispersive reflectivity).15-17 In general, the θ -2θ technique requires from tens of minutes to hours to scan a reflectivity curve point by point. Thus, a study of structural changes that occur over time frames of several minutes becomes impossible. In addition, the incident beams’ footprint on the sample varies as a function of the angle of incidence; therefore, each observation on the reflectivity curve is a result of averaging over different respective surface areas. This becomes important when the sample is not homogeneous across the surface or/and the footprint length becomes larger or smaller than the transverse coherence length of the incident X-ray beam. Although the energy-dispersive reflectivity technique in principle overcomes footprint effects, it nontrivially requires an incident beam with a large spectrum of wavelengths. Usually, to build a reflectivity curve in the region of interest, measurements are performed at several angles of incidence, thus limiting the capability to study structural transformations of thin films on short time scales. In addition, energy-dispersive measurements often suffer from normalization, rendering absolute measurements difficult. Mora et al.18 used grazing incidence diffuse X-ray scattering to determine the elastic properties of Langmuir films.19 On the basis of the theoretical development of diffuse X-ray scattering, the technique of grazing incidence diffuse X-ray scattering out of the specular plane (GIXOS or GISAXS as denoted here) has been developed.20,21 GISAXS allows the collection of a reflectivity-like curve for thin films on liquid surfaces for measurement times of less than 30 s with the intensity of a high-brilliance synchrotron X-ray source. The reflectivity R(qz) of a graded interface perpendicular to the z direction can be written within the kinematic (first Born) approximation as22,23
|∫ ( ) |
R(qz) ) RF(qz)
∞
0
dF(z) iqzz 2 dz e ) RF(qz) S(qz) dz
(1)
where F(z) describes the normalized electron density profile of the interface and S(qz) is the accompanying structure factor. Equation 1 is valid for qz > 3qc, with the critical wavevector transfer qc. RF(qz) denotes the Fresnel reflectivity, which is replaced by R(qz) ) RF(qz) exp(-σ2qz2) in the case of a rough surface, with σ being the rms roughness. For rough surfaces, the lateral fluctuations give rise to diffuse scattering for which the theoretical framework within the distorted wave Born ap(15) Neissendorfer, F.; Pietsch, U.; Brezesinski, G.; Mo¨hwald, H. Meas. Sci. Technol. 1999, 10, 354–361. (16) Felcher, G. P.; Hilleke, R. O.; Crawford, R. K.; Haumann, J.; Kleb, R.; Ostrowski, G. ReV. Sci. Instrum. 1987, 58, 609–619. (17) Pietsch, U.; Grenzer, J.; Geue, T.; Neissendorfer, F.; Brezsesinski, G.; Symietz, C.; Mohwald, H.; Gudat, W. Nuclear Instrum, Methods Phys. Res., Sect. A 2001, 467, 1077–1080. (18) Mora, S.; Daillant, J.; Luzet, D.; Struth, B. Europhys. Lett. 2004, 66, 694–700. (19) Gaines, G. L. Insoluble Monolayers at the Liquid-Gas Interface; WileyInterscience: New York, 1966. (20) Wiegart, L.; O’Flaherty, S. M.; Struth, B. Langmuir 2005, 21, 1695– 1698. (21) Wiegart, L.; Struth, B.; Tolan, M.; Terech, P. Langmuir 2005, 21, 7349– 7357. (22) Buff, F. P.; Lovett, R. A.; Stilling, F. H. Phys. ReV. Lett. 1965, 15, 621– 623. (23) James, R. W. The Optical Principles of the Diffraction of X-rays. Ox Bow: Woodbridge, CT, 1982.
proximation (DWBA) was developed by Sinha et al.24 Holy et al. expanded this result to deliver an expression for layered systems.25 For totally correlated interfaces, all oscillations observed in the specular reflectivity are also found in the diffuse scattering.26 The height-height correlation function for the capillary-wave-induced interfacial roughness can be written for organic films as27
g(r) )
∑ σi2 + BγE + B ln( κr2 )
(2)
i
where the sum is extended over the number of interfaces, γE is the Euler constant, and κ is the wavevector cutoff for capillary waves. B is given as kBT/πγ, where γ denotes the interfacial tension. For a single interface, the effective specular reflectivity can be calculated as28 2
2
R(qz, qxy ) 0) ) RFqzeqz σeff
)
(
2 2 1 1-η 1 - η 1 qxy ξ F , , 1 1 2 2 4π2 √π 2 (3)
(
)
where η is given by Bqz2/2 and ξ denotes the cutoff length originating from the effective coherence length along the surface.24,28 1F1 is the Kummer function of the first kind, which is equal to unity for the specular condition qxy ) 0. The factor 1/π Γ(1- η/2) is unity except for η.1. The reflectivity RP(qz) expressed by the Parratt algorithm29 can be used as an exact solution for the reflectivity of a stratified system with an arbitrary 2 2 number of interfaces and to replace the term RF(qz)eqz σeff in eq 3. For off-specular conditions (qxy * 0), the Fresnel reflectivity in eq 3 is according to the DWBA formalism24 replaced by the Fresnel transmission coefficients T(ki) and T(kf) of the incoming and outgoing beams, respectively. Consequently, the reflectivity reads in this case
R(qz, qxy) )
RP(qz) 1 1-η F × |T(ki)|2|T(kf)|2 Γ RF(qz) 2 1 1 √π
(
(
)
)
2 2 1 - η 1 qxy ξ (4) , , 2 2 4π2
where for fixed angle of incidence Ri and varying exit angle Rf the corresponding transmission coefficients are constant and given by the Vineyard function,30 respectively. It is noteworthy that beyond the rather simple model presented here other formalisms have been developed to include, for instance, the bending rigidity or the diffuse scattering due to inhomogeneities in the layer (e.g., Mora et al.).18 The major difference between XSR and GISAXS is evident from eq 4: the intensity measured by the detector is proportional to a conventional reflectivity signal normalized by the Fresnel reflectivity. Consequently, the typical decay in the scattering intensity in XSR with qz-4 does not apply to GISAXS measurements. Therefore, the long acquisition times required by XSR for reliable counting statistics do not apply to the GISAXS technique. This is demonstrated in Figure 1, which displays an XSR curve and a GISAXS spectrum for phospholipid monolayers on the surface of aqueous clay mineral dispersions (hydrosols). (24) Sinha, S. K.; Sirota, E. B.; Garoff, S.; Stanley, H. B. Phys. ReV. B 1988, 38, 2297–2311. (25) Holy, V.; Baumbach, T. Phys. ReV. B 1994, 49, 10668–10676. (26) Sinha, S. K. J. Phys. III 1994, 4, 1543–1557. (27) Basu, J. K.; Sanyal, M. K. Phys. ReV. Lett. 1997, 79, 4617–4620. (28) Sanyal, M. K.; Sinha, S. K.; Huang, K. G.; Ocko, B. M. Phys. ReV. Lett. 1991, 66, 628–631. (29) Parratt, L. G. Phys. ReV. 1954, 95, 359–369. (30) Vineyard, G. H. Phys. ReV. B 1982, 26, 4146–4159.
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Figure 1. (O) GISAXS spectrum for DPPC on a hydrosol subphase (1.8 wt % laponite) at a surface pressure of 55 mN/m. (2) Specular reflectivity for DSPC on a hydrosol subphase (0.2 wt % montmorillonite) at a surface pressure of 22 mN/m.
A detailed description of the systems can be found in refs 31 and 21. The positive charge in the zwitterionic headgroup of the phospholipids interacts with the negative surface charge of the mineral platelets, leading to the formation of a mineral adsorption layer underneath the phospholipid monolayer. The XSR curve was obtained for such an organic/inorganic hybrid layer consisting of the phospholipid DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine, CAS no. 816-94-4) on a 0.2 wt % dispersion of the natural clay mineral montmorillonite at a surface pressure of 22 mN/m. The GISAXS spectrum was recorded from a DPPC (1,2dipalmitoyl-sn-glycero-3-phosphocholine, CAS no. 63-89-8) monolayer on a 1.8 wt % dispersion of laponite (the synthetic analog of montmorillonite) at a surface pressure of 55 mN/m. The dispersed minerals have a disklike shape with a thickness of about 9.8 Å and diameters of 2500 Å (montmorillonite) and 250 Å (laponite). The most striking difference in the data shown in Figure 1 is that the intensity of the XSR curve decays by about 8 orders of magnitude over the qz range while the intensity of the GISAXS spectrum oscillates within 1 order of magnitude over the same qz range. The GISAXS technique utilizes the advantages of measuring at a fixed angle of incidence, fixed beam footprint area, fixed wavelength, and thus a controlled penetration depth of the beam into the sample. However, because of the limited penetration depth on the order of 100 Å in the grazing incidence geometry, the technique can be applied only to appropriately thin films. Combined with the requirement of conformal roughness, the technique is particularly powerful for thin films on liquid surfaces that require extremely long acquisition times using conventional XSR. GISAXS has been applied as an alternative not only to conventional XSR but also to metastable systems20 and radiationsensitive samples32,33 such as phthalocyanines, which form 1D supermolecular structures upon compression at the air-liquid interface. (31) Struth, B.; Rieutord, F.; Konovalov, O.; Brezesinski, G.; Gru¨bel, G.; Terech, P. Phys. ReV. Lett. 2002, 88, 25502. (32) O’Flaherty, S. M.; Wiegart, L.; Konovalov, O.; Struth, B. Langmuir 2005, 21, 11161–11166. (33) O’Flaherty, S. M.; Wiegart, L.; Struth, B. J. Phys. Chem. B 2006, 110, 19375–19379.
Figure 2. (a) Sketch of the scattering geometry. The incident beam hits the sample surface under the grazing angle Ri, and the diffuse scattering is recorded at an in-plane angle R simultaneously for a whole set of existing angles Rfn simultaneously by a position-sensitive linear detector (PSD). (b) Wavevector transfers for the geometry depicted in part a. For an in-plane scan in the classical reflectivity geometry (φ ) 0), qy ) 0 and qxy ) qx.
Experimental Setup GISAXS measurements presented here were performed at the ID10B beamline at the ESRF, Grenoble, France.34 A monochromatic synchrotron X-ray beam was achieved by using a diamond (111) double-crystal monochromator at symmetric Bragg-Bragg reflection conditions. Higher harmonics were filtered from the fundamental X-ray mode using a palladium-coated double mirror. The downstream mirror was used to deflect the X-ray beam down from the horizontal plane to the liquid surface. The X-ray beam was collimated before the sample with 100 µm (vertical) and 300 µm (horizontal) slits. The incident flux of the X-ray beam on the sample was on the order of 1010 photons/s. A sketch of the measurement geometry is depicted in Figure 2a. The angle of incidence at the liquid surface was Ri ) 0.1°, and the wavelength was λ ) 1.55 Å (8 keV X-ray energy). GISAXS spectra were recorded at φ ) 0.37°, where the intensity of the scattering signal was about 7 orders of magnitude smaller than that of the incident beam. The significant flux difference between the incident and scattered radiation justifies the indispensable need for synchrotron X-ray sources. The scattered beam was detected with a 150-mm-long (1024 channels) position-sensitive linear detector (PSD, Gabriel) oriented perpendicular to the liquid surface. The PSD records an intensity profile as a function of the vertical scattering angle Rf. A pair of slits 150 mm × 0.3 mm and 150 mm × 0.5 mm separated by a 620mm-long evacuated flight path and placed in front of the PSD provided a horizontal angular resolution of 0.1°. The wavevector transfer qz perpendicular to the surface is given by qz ) 2π/λ(sin Ri - sin Rf). The in-plane components are given as qx ) 2π/λ(cos Rf cos φ cos Ri) and qy)2π/λ(cos Ri sin φ) from which the total wavevector transfer qxy parallel to the surface is derived as qxy ) (qx2 + qy2)1/2. The PSD in this geometry allows simultaneous acquisition of the qz spectrum from 0 Å-1 < qz < 0.55 Å-1 in reciprocal space. The wavevector transfers as a function of the detection angle are plotted in Figure 2b for a typical experimental setup. GISAXS for the DPPC Monolayer on Pure Water. Langmuir monolayers of the phospholipid DPPC on a water surface can be (34) Smilgies, D. M.; Boudet, N.; Struth, B.; Konovalov, O. J. Synchrotron Radiat. 2005, 12, 329–339.
Organic/Inorganic Monolayers at Liquid Surfaces
Figure 3. GISAXS spectra for DPPC at surface pressures of (a) 6, (b) 9, (c) 12, (d) 15, (e) 18, (f) 21, (g) 24, (h) 27, (i) 30, (j) 33, (k) 36, (l) 39, and (m) 42 mN/m. The solid black lines represent a two-box model according to eq 4, and the parameters are quoted in Table 1. The data sets have been shifted relative to one another for clarity. The arrow is intended as a guide to the eye to look approximately along the shifting position of the minimum.
used to demonstrate the applicability of the GISAXS technique for studying the structure of ultrathin films. The structure of this system as a function of the surface pressure has been well studied and is understood,35,36 making it an ideal system for testing the technique. A custom-designed Langmuir trough equipped with a moveable single barrier was used to prepare the monolayers. The surface pressure of the monolayers was measured using a Wilhelmy balance (Riegler) with a 3-mm-wide filter paper plate and was kept constant during the measurement. The vessel containing the trough was filled with a flow of water-saturated helium to reduce both evaporation on the liquid surface and parasitic air scattering. Substitution of air with helium in the trough was monitored by the level of oxygen (oxygen generally below 0.1 vol %) measured with a oxygen meter (Dra¨ger microPac). All measurements were performed at room temperature (20 °C). The phospholipid DPPC was spread from a 0.27 mmol/L chloroform solution by means of a Hamilton microlitre syringe. The GISAXS spectra collected from the DPPC monolayer are presented in Figure 3 where the acquisition time for each spectrum was 30 s. The surface pressures for the spectra were (a) 6, (b) 9, (c) 12, (d) 15, (e) 18, (f) 21, (g) 24, (h) 27, (i) 30, (j) 33, (k) 36, (l) 39, and (m) 42 mN/m. Qualitatively, it can be noted that all curves exhibit an oscillatory nature. The period of these oscillations is defined by the layer thickness. In general, it can be observed that the positions of the minima and maxima move toward smaller qz with an increase in surface pressure, in accordance with the expected growth of the film under compression. This behavior is well known for this system from standard reflectivity experiments.35,36 The arrow in Figure 3 is intended as a guide to the eye to approximately map the shift of the minima throughout the compression. The solid lines in Figure 3 correspond to calculations using eq 4 where the reflectivity has been calculated using a two-box model (one of which represents the polar head groups of the molecules and the other for the aliphatic chains or the tails) typical of the DPPC monolayer system. Good agreement between the calculation and the experimental data was achieved in all cases. All parameters used in the calculations are quoted in Table 1. In general, it can be seen that with increasing surface pressure and decreasing area per molecule, the layer thicknesses increase and the electron densities increase or remain equivalent. The headgroup-tail (σ2) and tail-atmosphere interface roughness (σ3) are found to increase during the compression. In Figure 4, a brief literature overview of the total thickness reported for DPPC monolayers, comprising the sum of the head layer thickness (35) Daillant, J.; Bosio, L.; Harzallah, B.; Benattar, J. J. J. Phys. II 1991, 1, 149–170. (36) Thoma, M.; Schwendler, M.; Baltes, H.; Helm, C. A.; Pfohl, T.; Riegler, H.; Mo¨hwald, H. Langmuir 1996, 12, 1722–1728.
Langmuir, Vol. 25, No. 7, 2009 4107 and the tail layer thickness, dt + dh, as a function of surface pressure is presented. In this Figure, the filled circles represent the data collected in this study using the GISAXS analysis presented in Figure 3 whereas the other studies were conducted using regular θ-2θ specular reflectivity. Thoma et al.36 reported the specular reflectivity of DPPC monolayers at 20 °C on water for surface pressures of 2, 8, and 35 mN/m. At 35 mN/m, they found that the thicknesses of the head and tail layers were 7.4 and 16.8 Å, respectively. This compares well with the values of 7.1 and 16.6 Å found here, implying a difference of 0.5 Å in the combined film thickness. Daillant et al.35 have also reported a specular reflectivity study of DPPC monolayers on water through a range of surface pressures spanning from 10 to 45.8 mN/m at 23 °C. At 29 mN/m, they reported that the thicknesses of the head and tail layers were 6 and 16.8 Å, respectively, a 1.1 Å thinner head layer than that reported here at 30 mN/m. Their total monolayer thickness of 22.8 Å is slightly thinner than the 23.3 Å found in this study (30 mN/m). Similarly, at 8 mN/m Thoma et al. reported 10.8 and 14.7 Å for the head and tail layers compared to 6.7 and 14.7 Å found at 9 mN/m in our study. The thickness of the head layer that we present is noticeably smaller, and in fact, in Figure 4 it can be seen that the total thickness extracted from this observation at 8 mN/m does not follow the general trend exhibited by the other data. Daillant et al. found that the head and tail layers at 10 mN/m were 5 and 15 Å, respectively, exhibiting good agreement between the tail layers whereas their head layer is somewhat thinner than that reported here. At 42 mN/m, Daillant et al. reported 6 and 17 Å for the thicknesses of the head and tail layers, respectively, where we have found 7.1 and 16.9 Å. Thus, our total layer thickness is approximately 1 Å greater at this surface pressure. The ratio of the electron densities of the layers to the water is also comparable to that reported by Thoma et al., with the most significant differences in the tail layers where they reported δt/δw ) 0.9 for 8 and 35 mN/m and we have found 0.97 and 0.99 for 9 and 36 mN/m, respectively. Daillant et al. have reported δt/δw ) 0.96 for 10 and 37 mN/m, in good agreement with our findings. At 35 mN/m, Thoma et al. found δh/δw ) 1.3, again in good agreement with 1.28 found at 36 mN/m in our study, with both of these being somewhat lower in magnitude than δh/δw ) 1.5 at 37 mN/m reported by Daillant et al. If one considers Figure 4 and the total monolayer thickness, then it can be seen that the data extracted from the diffuse scattering spectra is in good agreement with the data previously determined using specular reflectivity, thus providing verification of the technique, with the essential difference being that our entire series of 13 spectra was collected over a total of 6.5 min of X-ray exposure. Kinetics from Time-Resolved GISAXS Spectra. The in situ observation of adsorption or desorption processes of monomolecular thin layers at the liquid/gas interface is a difficult task because of the very limited amount of scattering material. Apart from the necessary sensitivity, the technical requirement for a time-resolved study is the ability to measure the parameters of interest with a sampling time much shorter than the time scales on which the sample evolves. GISAXS with its sampling time on the order of 1 min enables the study of processes with typical time scales on the order of tenths of minutes. We have studied the decomposition process of the organic/inorganic hybrid layer system described in the Introduction. The desorption of the mineral discs from the lipid monolayer can be induced by the screening of the electrostatic interaction between the organic and inorganic entities.37 A relatively small and negatively charged molecule can diffuse into the interlayer between the adsorbed minerals and the lipid headgroups to trigger the desorption process. In accordance with the medical analog of this hybrid system,38 we have chosen citric acid, which is used as an active agent in the arthritic pattern,39 to dissolve the hybrid layer. A DPPC monolayer was prepared on an aqueous suspension of laponite (0.2 wt %) at a surface pressure of 25 mN/m. Figure 5, (37) Wiegart, L.; O’Flaherty, S. M.; Terech, P.; Struth, B. Soft Matter 2006, 2, 54–56. (38) Wierzbicki, A.; Dalal, P.; Madura, J. D.; Cheung, H. S. J. Phys. Chem. B 2003, 107, 12346–12351. (39) VanLinthoudt, D.; Salani, I.; Zender, R.; Locatelli, P.; Ott, H.; Schumacher, H. R. J. Rheumatol. 1996, 23, 502–505.
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Table 1. Parameters for the GISAXS Analysis of DPPC Monolayers on Water during Compressiona (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) (m)
Π (mN/m)
area (Å2)
δh/δw
δt/δw
σ1 (Å)
σ2 (Å)
σ3 (Å)
dh (Å)
dt (Å)
6.0 9.0 12.0 15.0 18.0 21.0 24.0 27.0 30.0 33.0 36.0 39.0 42.0
74.4 55.0 51.7 50.2 49.1 48.2 47.5 46.8 46.1 45.5 44.7 44.1 43.6
1.11 1.26 1.27 1.27 1.27 1.27 1.27 1.27 1.28 1.28 1.28 1.28 1.28
0.95 0.97 0.98 0.98 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99
3.4 3.4 3.4 3.4 3.4 3.4 3.4 3.4 3.4 3.4 3.4 3.4 3.4
4.0 4.6 4.9 4.7 4.5 4.5 4.8 4.7 4.9 5.3 5.3 5.3 5.2
2.9 2.9 3.1 3.3 3.4 3.6 3.6 3.8 3.8 4.0 4.1 4.3 4.5
6.7 6.7 6.9 6.9 7.0 7.1 7.1 7.1 7.1 7.1 7.1 7.1 7.1
12.0 14.7 15.3 15.5 15.6 15.6 16.0 16.1 16.2 16.5 16.6 16.6 16.9
a Π is the surface pressure, area implies area per molecule, δh/δw and δt/δw imply the ratio of the electron densities of the head layer and tail layer to water, respectively (the known electron density of water was kept fixed during the data refinement), σ1, σ2, and σ3 are the interfacial roughnesses for the water-head, head-tail, and tail-atmosphere interfaces, respectively, and dh and dt are the head and tail layer thicknesses, respectively.
Figure 4. Literature overview of the total thickness of DPPC monolayers comprising the sum of the head layer thickness and the tail layer thickness, dt + dh, as a function of surface pressure. The filled circles (•) represent the data collected in this study using GISAXS, and the other data have been collected using regular specular reflectivity from Daillant et al.35 (0) and Thoma et al.36 (3).
Figure 5. GISAXS spectra and fits for DPPC on a 0.2 wt % sol subphase: (a) equilibrium state before the injection of citric acid solution into the subphase, (b) 40 min after the injection, and (c) final equilibrium state after 1317 min.
curve a shows the GISAXS spectrum obtained after reaching an equilibrium state. The corresponding electron density profile is well described by a four-layer model: in addition to the headgroup and tail layer, this model consists of a water interlayer of about 3 Å thickness between the lipid headgroup and the adsorbed minerals
Figure 6. Decrease in the normalized electron density δmineral/δsubphase as a function of time. t ) 0 corresponds to the injection of citric into the subphase. The dashed line is an exponential fit to the data.
and the actual mineral adsorption layer itself. Subsequently, 10 vol % of the subphase was replaced by an aqueous buffered solution of citric acid trisodium salt (CAS no. 68-04-2, pH 7.6) without any disturbance to the surface. Figure 5, curve b displays the GISAXS spectrum 40 min after the exchange whereas curve c corresponds to the new equilibrium state reached after 1317 min. The reduced sample thickness for the final state is clearly evident from the increased period of the intensity oscillations over qz. The reconstruction of the electron density profiles reveals a reduction of the total sample thickness by about 9.5 Å whereas the thickness of the headgroup layer was found to be slightly increased. The results are consistent with the desorption of the mineral discs, which exhibit a nominal thickness of 9.8 Å, and the electrostatic adsorption of citric acid molecules around the headgroups of the lipids. In the intermediate time regime where the desorption of the minerals takes place, the system is well described by a four-layer model with an unchanged layer thickness but a decreasing electron density in the mineral layer as a function of time. Figure 6 displays the obtained normalized electron densities δmineral/δsubphase for the first 250 min. The exponential decrease in the electron density of the mineral layer (dashed line in Figure 6) is compatible with a Langmuir desorption model40 that exhibits an inverse exponential time dependence. The electron density in the mineral layer is directly proportional to the number of mineral particles in that layer. As the mineral particles desorb, the corresponding electron density decreases in magnitude, from which the kinetic constants of the desorption process can be calculated.37 Evolution of Biomembranes on Cryoprotective Subphases during Cooling. Self-assembled organic monolayers, such as biomembranes of phospholipids, have been widely explored at the (40) Langmuir, I. J. Am. Chem. Soc. 1916, 38, 2221–2295.
Organic/Inorganic Monolayers at Liquid Surfaces liquid/gas interface.6,41 Almost all studies use pure water or waterbased buffers as a subphase; consequently, the investigation of the low-temperature phase behavior and morphology of these systems is restricted to temperatures around or above the freezing point of water at 0 °C. However, questions about the stability and functionality of biomembranes at low temperatures arise, for example, in the context of life under extreme conditions (extremophiles). Although the phase diagram of fatty acids is generic in the sense that it scales toward low temperature by increasing the length of the alkane chain,42 the polymorphism of phospholipids is more complicated. Similar to fatty acids, the phospholipid alkane chain length is related to temperature scaling; however, the phase behavior is affected by other molecular modifications such as the headgroup, the number of alkane chains, and chain branching.43 To date, a unified generic phase diagram for phospholipid monolayers does not exist, necessitating case-by-case studies. We have investigated the phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) on cryoprotective subphases (glycerol- and alcohol-based) below 0 °C and have also performed some comparisons with a fatty acid (eicosanoic acid). The GISAXS technique has been used to monitor the structure of these systems during the cooling process to provide the first insight into layer stability and morphology from the reconstruction of the electron density profiles. Because of the fast data acquisition, the radiation dose is much lower than that using conventional specular reflectivity. The diffusion of surfactants at the surface and selfhealing capabilities of the monolayer depend on the subphase viscosity.44 Particularly at low temperatures, the viscosity of the glycerol-based subphases is orders of magnitude higher than that of water, enabling dynamic changes in the sample. The GISAXS technique therefore offers various advantages over conventional measurement techniques for monitoring the varying layer structure of such systems. The cryoprotective antifreeze agents used glycerol (Sigma-Aldrich, purity g99%) and ethanol (Sigma-Aldrich, HPLC grade) in a mixture with ultrapure water (Elga Purelab Classic). The binary eutectic mixture of glycerol/water is 67/33 by weight and exhibits a freezing point of Tf ) -46 °C. In combination with sodium chloride (NaCl), the freezing point of the ternary mixture NaCl/glycerol/water (5/ 73/22 wt %) is lowered to Tf ) -64 °C.45 Monolayers were also investigated on a subphase of pure glycerol (Tf ) 17 °C).46 However, because of the tendency of glycerol to supercool, much lower temperatures can be reached without crystallization. For the subphases described above, stable surfactant monolayers can be prepared by spreading from a microliter syringe at ambient temperature, benefiting from the high surface tension of both glycerol and water (63.0 and 72.7 mN/m, respectively, at 20 °C). Because of the lower surface tension of ethanol (22.4 mN/m at 20 °C), the maximum amount of ethanol in the binary mixture ethanol/water was limited to 25 vol %, resulting in a freezing point of Tf ) -12 °C. At higher ethanol concentrations, surfactant molecules sank into the subphase, and the samples were found to be unstable over time. Samples were prepared in a sample chamber specifically designed for low-temperature studies of surfactant monolayers at the liquid/gas interface. The chamber consists of a double-walled cell where the inner cell hosts a 68mm-diameter trough cooled by Peltier elements whereas the outer cell provides an insulation vacuum toward the ambient environment. The inner cell was flushed with helium gas to minimize parasitic air scattering. At low temperatures, cold He gas from a liquid-helium Dewar was used (after reheating if necessary) in order to maintain thermal equilibrium between the liquid and the atmosphere. With in situ monitoring of the surface pressure, monolayers were spread (41) Kaganer, V. M.; Mo¨hwald, H.; Dutta, P. ReV. Mod. Phys. 1999, 71, 779– 819. (42) Kaganer, V. M.; Peterson, I. R.; Kenn, R. M.; Shih, M. C.; Durbin, M.; Dutta, P. J. Chem. Phys. 1995, 102, 9412–9422. (43) Bringezu, F.; Dobner, B.; Brezesinski, G. Chem.sEur. J. 2002, 8, 3203– 3210. (44) Kang, Y. S.; Majda, M. J. Phys. Chem. B 2000, 104, 2082–2089. (45) Pegg, D. E. Cryo-Letters 1983, 4, 259–268. (46) The Merck Index, 12th ed.; Merck & Co.: Whitehouse Station, NJ, 1996.
Langmuir, Vol. 25, No. 7, 2009 4109
Figure 7. Typical GISAXS spectra obtained with an acquisition time of 180 s at a sample temperature of T ) -20 °C: (∆) eicosanoic acid on a subphase of NaCl/glycerol/water, Π ) 4.5 mN/m and (O) DPPA on a subphase of glycerol/water, Π ) 30 mN/m.
Figure 8. Normalized electron density profiles obtained from the GISAXS spectra shown in Figure 7. The upper panel shows a one-box model for eicosanoic acid, and the lower panel shows a two-box model for DPPA.
from chloroform solutions at concentrations of 0.29 mmol/L (DPPA, CAS no. 169051-60-9) and 0.60 mmol/L (eicosanoic acid, CAS no. 506-30-9), respectively. The monolayers were cooled at a cooling rate of 0.5 °C/min, and sufficient time was allowed to reach thermodynamic equilibrium prior to data acquisition. Figure 7 shows two typical GISAXS spectra for DPPA at -20 °C on glycerol/water at 30 mN/m and for eicosanoic acid on NaCl/ glycerol/water at 4.5 mN/m with an acquisition time of 180 s per spectrum. The spectra for DPPA were fitted using a two-layer model corresponding to the headgroup and tails of the lipid. By contrast, because of the much smaller carboxylic acid hydrophillic headgroup group ([-COOH]), eicosanoic acid can be well described by a onelayer model. Figure 8 shows the two electron density profiles corresponding to the GISAXS spectra shown in Figure 7. The profiles were calculated using an error-function model.12 Figure 9 displays the thickness dt of the DPPA monolayers on the different subphases as a function of temperature. The thickness variations of the headgroup layer are quite inconsequential (5.2 ( 0.2 Å), and the evolution of the system is more or less entirely reflected by changes in the tail layer. For the DPPA monolayer on pure glycerol, dt does not show a clear temperature dependence. The increase of dt at -40 °C goes along with a significant increase in the roughness at the headgroup-tails (from 2.9 to 5.9 Å) interface whereas the roughness at the tails-atmosphere interface remains almost constant (∼4.2 Å). Although the reconstructed electron density profiles, even at the lowest temperature (-38 °C), point toward an
4110 Langmuir, Vol. 25, No. 7, 2009
Wiegart et al. subtle functions of biologically relevant membranes, such as transport mechanisms, at low temperature.
Summary/Outlook
Figure 9. Thickness of the tail layer as a function of temperature for different subphases. Solid symbols represent DPPA at Π ) 30 mN/m, and open symbols represent eicosanoic acid at Π ) 4.5 mN/m. (In the latter case, dt corresponds to the total layer thickness (see the text)). (∆), (2) NaCl/glycerol/water, (() glycerol, (0) ethanol/water, and (1) glycerol/water. Dashed lines are linear fits to the data and are intended to be a guide to the eye.
intact layer structure, the findings of increased interfacial roughnesses may indicate a commencement of crystallization of the supercooled glycerol subphase. Such behavior has been reported for supercooled water, where surfactant-induced freezing has been observed for alcohol monolayers.47 For the ethanol/water and glycerol/water surfaces, there is a clear trend toward increased layer thickness at lower temperatures. This would imply conformational changes in the alkane chain layer as a result of a temperature reduction and the accompanying increase in the subphase viscosities. It is noteworthy in this context that on pure water DPPA adopts a phase with hexagonal in-plane ordering of the chains and an upright chain conformation for surface pressures >20 mN/m.48 The expected layer thickness of 20.4 Å corresponding to a methyl-group-terminated C16 alkane chain in an all-trans configuration is exclusively reached on the glycerol/ water subphase at -20 °C. However, the possible origin of a reduced layer thickness such as a tilt of the alkane chain relative to the surface normal or a kink within the chain itself cannot be revealed from the electron density profiles. The interface roughnesses are almost identical for both subphases ethanol/water and glycerol/water (σ2 ≈ 2.5 Å, σ3 ≈ 3.9 Å) and temperature-independent. For the ethanol/water subphase, there is no evidence of crystallization down to the nominal freezing temperature of -12 °C, whereas at -20 °C the surface was too rough to allow for any meaningful GISAXS observations. The DPPA monolayer on the NaCl/glycerol/water subphase exhibits on average the smallest tail layer thickness, which appears to be somewhat temperature-independent over 22 to -40 °C. By contrast, the eicosanoic acid monolayer prepared on the same subphase exhibits a clear trend toward a larger layer thickness at lower temperatures. Assuming that the alkane chain of the molecule is tilted at the rather low surface pressure of 4.5 mN/m, the increase in layer thickness can possibly be caused by a reduction in the tilt angle upon cooling. The subphase-dependent evolution of the electron density profile of the monolayers upon cooling suggests the importance of specific interactions between the subphase and the surfactant molecules. This requires further investigations by surface X-ray diffraction methods, for example. However, the results obtained in this study allow us to confirm the existence of intact monolayers on cryoprotective subphases far below the freezing threshold of water. As far as the functionalities of the biomembranes are concerned, the ability to separate liquid volumes is most likely maintained with the intact layer structure. Further investigations are required to draw conclusions concerning the more (47) Popovitzbiro, R.; Wang, J. L.; Majewski, J.; Shavit, E.; Leiserowitz, L.; Lahav, M. J. Am. Chem. Soc. 1994, 116, 1179–1191. (48) Wiegart, L.; Struth, B. Physica B 2005, 357, 126–129.
We have presented the recently developed technique of grazing incidence X-ray scattering out of the specular plane (GISAXS) that provides complementary results to standard neutron and X-ray reflectivity. The capabilities and limitations of the GISAXS technique have been discussed, with GISAXS being very well suited to the study of thin films at air-liquid interfaces, where it enables reflectivity-like measurements with time resolution on the order of tens of seconds. By extrapolation, a relaxation of the monochromaticity of the X-ray beam (the pink-beam mode at a synchrotron facility) and the accompanying gain in incident flux could allow for the investigation of changes in the electron density profile of thin films on subsecond time scales. Although the studies presented here are focused on the membrane aspect of amphiphilic molecules at air-liquid interfaces, the motivation for the study of molecular thin films goes far beyond this facet, including bola amphiphiles and their self-assembly into supramolecular 1D structures. Above a critical overlap concentration in bulk solutions, the overlap of these self-assembled 1D aggregates generates a giant interconnected solidlike network with associated viscoelastic features. The resulting materials can be molecular hydrogels49 or molecular organogels50 The most important challenge in the field consists of designing a priori, from molecular engineering approaches, a low-mass molecule that would 1D self-associate and form rods or fibers in sol or gel phases. To achieve this goal, accurate knowledge of the molecular aggregation mechanism is crucial. GISAXS could serve as an adequate high-throughput screening technique for identifying potential molecules and their 2D organization. For instance, it is known that ordered arrays of metallosupramolecular 1D species can be transferred as Langmuir-Blodgett films.51 The resulting functional materials may open applied avenues in optoelectronics, electrochromophorism, and so forth.52 Pressure-induced transitions from isotropic to 2D-ordered phases could also gain fundamental input from studies at the liquid-air interface.53 The exploitation of self-assembly in soft matter in bulk solution to produce nanotechnological devices frequently involves transfers of the structures to solid surfaces.54 In such a context, the investigation of the structural evolution from structures developed at liquid interfaces where the thermal energy is a dominant parameter for their ordering to solid substrates or Langmuir-Blodgett films is a key piece of information that can be gained from GISAXS. GISAXS can thus nicely complete time-of-flight nonspecular neutron reflectivity approaches55 (or more classical ones)56 or can proceed further into shorter time scales of kinetic processes that are today not accessible by neutron reflectivity. Acknowledgment. We thank the ESRF for providing access to the X-ray beam. L.W. acknowledges grants for a Ph.D. thesis from CEA Grenoble and the ESRF. We are deeply indepted to B. Struth for his contribution to the presented work, helpful discussions, and the XR data shown in Figure 1. LA803547J (49) Estroff, L. A.; Hamilton, A. D. Chem. ReV. 2004, 104, 1201–1217. (50) Terech, P.; Weiss, R. G. Chem. ReV. 1997, 97, 3133–3159. (51) Schutte, M.; Kurth, D. G.; Linford, M. R.; Colfen, H.; Mo¨hwald, H. Angew. Chem., Int. Ed. 1998, 37, 2891–2893. (52) Kurth, D. G. Sci. Technol. AdV. Mater. 2008, 9, 014103–014128. (53) Kim, F.; Kwan, S.; Akana, J.; Yang, P. D. J. Am. Chem. Soc. 2001, 123, 4360–4361. (54) Hamley, I. W. Angew. Chem., Int. Ed. 2003, 42, 1692–1712. (55) Salditt, T.; Munster, C.; Mennicke, U.; Ollinger, C.; Fragneto, G. Langmuir 2003, 19, 7703–7711. (56) Cubitt, R.; Fragneto, G.; Ghosh, R. E.; Rennie, A. R. Langmuir 2003, 19, 7685–7828.
