ARTICLE pubs.acs.org/Langmuir
Polymorphism of Natural Fatty Acid Liquid Crystalline Phases Helene Fay,†,§ Steven Meeker,‡ Juliette Cayer-Barrioz,§ Denis Mazuyer,§ Isabelle Ly,† Frederic Nallet,† Bernard Desbat,|| Jean-Paul Douliez,^,# Virginie Ponsinet,*,† and Olivier Mondain-Monval† †
Universite de Bordeaux, Centre de Recherche Paul Pascal, UPR CNRS 8641, 115 avenue Schweitzer, 33600 Pessac, France Universite de Bordeaux, LOF, CNRS, LOF, and Rhodia, UMR 5258, F-33600 Pessac, France § Laboratoire de Tribologie et Dynamique des Systemes, Ecole Centrale de Lyon, UMR CNRS 5513, 36 avenue Guy de Collongue, 69134 Ecully, France University of Bordeaux, CBMN, UMR CNRS 5248, IPB, allee Geoffroy Saint Hilaire, 33600 Pessac, France ^ quipe Interfaces et Systemes Disperses, rue de la Geraudiere, Biopolymeres Interactions Assemblages, UR INRA 1268, E 44316 Nantes, France
)
‡
bS Supporting Information ABSTRACT: We study the phase behavior in water of a mixture of natural long chain fatty acids (FAM) in association with ethylenediamine (EDA) and report a rich polymorphism depending on the composition. At a fixed EDA/FAM molar ratio, we observe upon dilution a succession of organized phases going from a lamellar phase to a hexagonal phase and, finally, to cylindrical micelles. The phase structure is established using polarizing microscopy, SAXS, and SANS. Interestingly, in the lamellar phase domain, we observe the presence of defects upon dilution, which SAXS shows to correspond to intrabilayer correlations. NMR and FF-TEM techniques suggest that these defects are related to an increase in the spontaneous curvature of the molecule monolayers in the lamellae. ATR-FTIR spectroscopy was also used to investigate the degree of ionization within these assemblies. The successive morphological transitions are discussed with regards to possible molecular mechanisms, in which the interaction between the acid surfactant and the amine counterion plays the leading role.
1. INTRODUCTION Surfactants which are environmentally friendly and may be extracted from renewable sources (animal, marine, and vegetable oils) have attracted increasing attention in the last decades.1 They are produced to replace petrochemical products and are generally derivatives of unsaturated fatty acids. Most often obtained by hydrolysis of oils from the aforementioned oleochemical sources, they have the advantage of being biocompatible. However, their solubility in water is low, especially for fatty acids with chains longer than 16 carbons. This has motivated the chemical industry to produce derivatives in order to increase their aqueous solubility.2,3 However, such derivatives require chemical modifications and further purifications (and in some cases, the use of solvents), which represents an additional cost. Fatty acids can also be used with inorganic48 (with simple sodium or potassium counterions or in hydroxide or hydrochloric acid solutions) or organic (as tetramethylammonium9,10 or various amines11,12) bases to ensure their solubility via ionpairing. Depending on the nature of the base that, once protonated, plays the role of counterion, such mixtures yield a broad polymorphism including micelles, bilayers, tubes,11 and vesicles13 at low concentrations and crystals or liquid crystal phases at higher concentrations.58,14,15 The molar ratio fatty r 2011 American Chemical Society
acid/counterion and the nature of the counterion are of particular importance in such systems since they regulate the mean degree of charge and consequently the spontaneous curvature of the fatty acid layers, on the one hand, and the type of interactions between them on the other hand. Indeed, the fatty acid is a weak acid, and there may be a coexistence of fatty acids under their carboxylic COOH and carboxylate COO forms. The ratio of these two forms may change the curvature at the polar interface12 and hence the polymorphism. Recently, in diluted systems, diamines have been used as counterions, leading to pseudogemini structures when coupling occurs between one diamine molecule and two fatty acid chains.16 By contrast with the gemini case, however, coupling between the amine and the carboxylic group is reversible and may be tuned by the diamine/fatty acids ratio. Still, little has been established, so far, on the phase diagrams of such systems in concentrated regimes, although the relevance for applications is high. In this work, we study the morphologies of the self-assembled structures of a mixture of fatty acids (further referred to as FAM), Received: September 30, 2011 Revised: November 26, 2011 Published: November 28, 2011 272
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Table 1. Composition of the Fatty Acids Mixture (FAM) components
and circular apertures, with a beam diameter at the sample of 7.6 mm. The neutron wavelength was set to 12 or 4 Å with a mechanical velocity selector (Δλ/λ ≈ 0.1), the 2D detector (128 128 pixels, pixel size 5 5 mm2) being positioned at 4.4 or 1.4 m, respectively. The scattering wave vector q then ranges typically from 0.1 to 4.4 nm1, with a significant overlap between the two configurations. The samples, prepared with deuterated water, were held in flat quartz cells with a 1 or 2 mm optical path length. The azimuthally averaged data were corrected for the solvent cell, as well as for background noise, and were normalized to the incoherent H2O signal. 2.5. Freeze Fracture (FF). FF replica preparation was performed by putting a drop of the sample onto a gold planchette, then freezing the sample by quickly plunging the holder into liquid propane that was held at the temperature of liquid nitrogen. The freezing step must be fast in order to vitrify the sample and avoid structure disruption due to crystallization. In order to check for freezing artifacts, samples containing 30 wt.% glycerol in the water phase were prepared to compare with our neat samples (this point is discussed later). Frozen samples were then introduced in the freeze-fracture enclosure of a BAF 060 Balzers apparatus and held at 125 C and a pressure of 108 mBar. The samples were fractured using a knife held at a temperature of 200 C. The fractured surface was immediately shadowed by the successive deposition of platinum at an angle of 45 and carbon at 90. The gold planchettes were immersed in a 50/50 water/ethanol solution for 3 h to detach the replicas from the sample, then in three consecutive 1 M KOH solutions for 30 min each, and finally in pure water for a few more hours. This long process was necessary to completely remove the fatty acids from the replicas. The replicas were finally collected on 200 μm-mesh copper grids and dried before TEM imaging. 2.6. Transmission Electron Microscopy (TEM). TEM was performed with a HITACHI H 600 TEM. 2.7. Solid-State NMR. Deuterium solid-state NMR experiments were performed at room temperature on a 400 MHz Bruker spectrometer operating at 61 MHz for deuterium using a static double-channel probe. The sample coil of the probe was adapted to load a 7 mm rotor such as those used for magic angle spinning probes equipped with a stretched stator. Typically, a volume of ca. 700 μL was transferred into the rotor, which was sealed and then end-capped. A Hahn quadrupolar echo sequence was used with an interpulse delay of 40 μs.18 A total of 8 K points in 1 K accumulations (every 2 s) were done with a 90 pulse and a spectral width of 8 μs and 250 kHz. Free induction decay signals were zero-filled to 16 K points prior to Fourier transform after a broad line exponential multiplication of 200 Hz. For deuterium spectroscopy, the general theory for lipid systems can be found in the literature.19,20 Briefly, the deuterium NMR signal is composed of doublets with a splitting, Δν, which depends on the orientation between the C-D bond and the magnetic field. In an anisotropic but disoriented medium, all orientations are allowed, and these doublets are superimposed to form a powder spectrum having two main peaks with an increased intensity corresponding to the 90 orientation, separated by Δν90. The edge of the spectrum corresponds to the 0 orientation, with a splitting, Δν0, equal to 2Δν90. In the case of perdeuterated systems, the spectrum is composed of the superimposition of signals from each labeled position. When working with oriented samples, however, the spectrum obviously no longer exhibits a powder shape: it is composed of doublets corresponding to the given orientation, one for each labeled position.
weight percent
oleic acid (1 unsaturation)
32%
linoleic acid (2 unsaturations) and other C18 fatty acids
55%
fatty acids of shorter chain length (typically C16)
10%
free rosin acids
max 3%
which is of industrial relevance, and ethylenediamine (EDA). We vary the global weight fraction in water and the molar ratio of diamine/fatty acids. The structures of the different phases are studied using several experimental techniques: polarizing optical microscopy, NMR, small-angle X-ray and neutron scattering, and freeze-fracture transmission electron microscopy. When the concentration of the solution at fixed diamine/FAM ratio is increased, we observe the formation of a phase of wormlike micelles, a direct hexagonal phase, a lamellar phase with defects and, finally, a defect-free lamellar phase. The detailed analysis of the different phases leads us to propose that their successive positions in the phase diagram are related to a variation of the spontaneous curvature of the FAM monolayers, driven by the interaction between the acid head and the amine counterion. After a presentation of the experimental details, we describe and discuss the detailed structure of the successive phases.
2. MATERIALS AND METHODS 2.1. Sample Preparation. Ethylenediamine (EDA) was purchased from Aldrich and used directly as received. The biobased fatty acids come as a mixture (FAM), kindly provided by Rhodia, with the composition described in Table 1. The main components are oleic and linoleic acids, which are unsaturated fatty acids with a chain of 18 carbon atoms and a carboxylic COOH group as a polar head. The samples were prepared using an “equivalent” molar mass Meq = 280.8 g/mol. The composition of each sample is described by two parameters: the total weight fraction in FAM, XFAM (%), and the ethylenediamine to FAM molar ratio, x = nEDA/nFAM. When necessary the volume fraction of fatty chains ϕFAM is also used, which is slightly different from the weight fraction XFAM.17 The samples are prepared by vigorously stirring the components at a temperature of 80 C. Once homogeneous, the samples are cooled slowly to room temperature before study. 2.2. Polarizing Optical Microscopy (POM). POM was used to observe the samples birefringent textures and to establish the phase diagram. 2.3. Small-Angle X-ray Scattering (SAXS). SAXS profiles were acquired in transmission on a Nanostar-U instrument (Bruker AXS) with a copper anode source, Goebel mirrors and a HiSTAR 2-D detector permitting scattering vector q values from 0.4 to 8.0 nm1 to be reached. The resulting 2-D images are found to be isotropic, and the data are azimuthally averaged to yield curves of the scattering intensity corrected from experimental background versus q = (4π/λ) sin θ, where λ = 1.54 Å is the wavelength of the Cu Kα radiation and θ is half the scattering angle. The SAXS intensities I(q) present peaks corresponding to the lattice structure factor S(q), from which one may obtain the interdomain spacing d. The nonoriented (powder) samples were studied in cylindrical Lindemann-type capillaries, while oriented samples were studied in Vitrodynamics flat capillaries of 200-μm optical path. In both cases, the signal of an empty capillary was subtracted from the data. 2.4. Small-Angle Neutron Scattering (SANS). SANS experiments were performed at the Laboratoire Leon-Brillouin (laboratoire mixte CEA/CNRS, Saclay, France) on the PAXY spectrometer. The neutron beam was collimated by appropriately chosen neutron guides
2.8. Polarized Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR). Spectroscopic experiments were performed on a Nexus 6700 spectrometer equipped with a single germanium reflection ATR cell MIRacle (Pike Technology). Samples were spread on the ATR germanium crystal. For each sample the two polarizations p and s were recorded after coaddition of 400 scans at 8 cm1 of spectral resolution. A linear baseline correction was performed on each spectrum. The p and s polarizated spectra are sensitive to the 273
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Figure 2. Phase diagram of the FAM/EDA/water system. The different domains correspond to given values of the weight fraction FAM (XFAM) and of the molar ratio (x = nEDA/nFAM) where nEDA and nFAM are the amounts of EDA and FAM in moles respectively.
Figure 1. ATR-FTIR spectrum of a concentrated sample (XFAM = 40.6%) performed on a germanium crystal. The band corresponding to the COOH vibrations at 1710 cm1 is not visible, which means that all fatty acids are deprotonated. This is also observed for the less concentrated samples (down to XFAM = 5%).
values are stable with time. The IR data, on the other hand, cannot provide conclusive information on the form of the amine functions because the characteristic bands of NH2 and NH3+, although visible (close to 3350 and 1600 cm1 respectively), are not isolated from other signals and cannot be quantitatively analyzed. However, the concentrations in RNH2 and RNH3+ functions can be deduced using the electroneutrality requirement and mass conservation. In all samples, we find that RNH3+ and COO are the dominant ionized functions in solution, with [RNH3+] = [COO] + [OH]. In Figure 2, we present the general phase diagram, displaying the morphology of the system as a function of x and XFAM. This was obtained using polarized optical microscopy (POM), which can provide23 a clear indication of the morphology of liquid crystal phases thanks to specific textures. In the studied range (1.5 < x < 3), the value of x appears to have a limited effect on the phase behavior, which is mostly determined by the total FAM weight fraction. As XFAM is increased at fixed x > 1.5 (horizontal lines in the diagram of Figure 2), we successively observe an isotropic phase (I domain), an isotropic phase becoming birefringent under flow (BUF domain), a hexagonal phase (H domain), a lamellar phase (L domain), and a mixture of lamellar phase and crystallites. In the following, we first describe our observations concerning the lamellar phase (high concentration) and then discuss the hexagonal domain and finally the lower concentration range. We also discuss the main features of each phase transition. 3.2. Lamellar Domain. At 38% < XFAM < 65% wt and x = 2.6, the textures observed with POM (not shown here) are characteristics of a lamellar phase. SAXS measurements performed in this domain are presented in Figure 3. Two successive peaks, located at q0 and 2q0, confirm the presence of a lamellar order. The SAXS measurements were performed at various values of FAM concentration throughout the lamellar domain. The extracted lamellar period d, varying between 2.5 and 5.5 nm, is plotted as a function of the inverse of the FAM volume fraction (see Figure 4a). Oddly enough, the observed dilution law does not follow the geometrical law often encountered in lamellar phases, which is drawn as a full line in Figure 4a
orientation and the transition moment value of each vibrations and the spectrum sum, p + s, is only characteristic of the transition moment value of each vibration. More details on the experiments can be found in previous publications.21
3. RESULTS AND DISCUSSION 3.1. Phase Diagram. We have prepared samples varying the total weight fraction in FAM, XFAM (%), and the ethylenediamine to FAM molar ratio, x = nEDA/nFAM. First, we observed that samples are not homogeneous for ratios x < 1, i.e. with less than one diamine per fatty acid chain. In such cases, we note the presence of crystallites in equilibrium with an isotropic liquid. As a consequence, we have restricted our study to the domain where x g 1 and more precisely to the domain 1.5 e x e 3. Even though this system comprises only two components in water (fatty acids and the counterion), each of them may be present under the basic or acid form, depending on the pH. Fatty acids can be protonated (carboxylic form) or under their carboxylate form and their apparent pKa may reach a value higher than 8 depending on the assembly in which they are embedded.4,7 Ethylenediamine has two pKa with values 10.7 and 7.6. The different forms (protonated or not) of the protagonists imply different types of interactions between them. Indeed, the protonated amine and the deprotonated acid would strongly interact via Coulombic attraction, whereas the uncharged amine and the protonated acid may associate through hydrogen bonding.22 Since the EDA molecule bears two amine functions, different stoichiometries of interactions may also occur: diamineacid coupling may be 1:1, leaving one amine function free on the EDA molecule, or 1:2, with both amine functions involved in the interaction. Infrared spectroscopy was useful in assessing the predominant forms for each of the molecular species. In all studied samples, from XFAM = 5 to 60% (and at constant x = 2.6), the signals display no clear vibrational band at 1710 cm1, showing that all fatty acid molecules are under the deprotonated form COO. This is illustrated in Figure 1 at XFAM = 40.6%. It is consistent with the fact that, for the samples in the domain 1.5 e x e 3, the measured pH of the mixture is between 10.5 (at low XFAM and x) and 13.5 (at high XFAM and x), always significantly higher than the pKa of the carboxylic groups. Note that these pH
dlam ¼
2π 2v ¼ q0 ϕFAM Σ
ð1Þ
where dlam is the lamellar repeating distance, v the molecular volume of the amphiphiles, ϕFAM the FAM volume fraction and 274
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in which Σ, the area per amphiphile polar head, does not change upon dilution. The discrepancy shown in Figure 4a could be due to variations of Σ upon dilution, which could be related to an actual change of the area per polar head of the amphiphile, to undulations of the bilayers,24 or to the proliferation of defects in the bilayers.25 All three effects can be represented by a varying “apparent” Σ. Using eq 1 we can extract the apparent value of Σ from the SAXS data and plot it (Figure 4b) as a function of XFAM in the lamellar domain (a similar calculation will also be performed in the hexagonal domain). The apparent area per fatty acid head presents significant and nonmonotonic variations along the lamellar dilution. Such large variations were described for fatty acid heads in other systems. In particular, nonmonotonic variations of the area per polar head were related to variations of the ratio COO/COOH in fatty acid lamellar phases.4 This explanation does not hold in our system, however, since the fatty acid heads are mostly deprotonated, as shown by the FTIR results, all along the dilution line. We suggest that the observed behavior can be understood as two different regimes. In the most concentrated regime of the studied concentration range, when XFAM is increased
from 55 to 65%, Σ very steeply increases from 40 to 50 Å2. These values of XFAM corresponds to very high molar fraction of EDA that varies from ∼40% (XFAM = 55%) to 100% (XFAM = 65%) in the water phase. At such high concentrations (when the EDA molecules are strongly confined between the layers) the increase in area per polar head might be due to the steric hindrance of the non coupled EDA molecules, which are much bigger than water molecules and jam the volume between the adsorbed EDA molecules. This leads to a lateral stretching of the bilayers (through an increase of Σ), which may minimize the potential energy associated with the repulsive interactions between neighboring bilayers. However, this possible explanation is only speculative and would require further investigations. In the less concentrated part of the lamellar domain (XFAM between 38 and 48%), we suggest that the variations of the area per polar head could be related to the evolution of the complexation between the negatively charged acid heads and the cationic amine functions from the EDA molecules. This will be further commented in the article, after the description of more experimental results. In the lamellar domain and below the concentration X/FAM ≈ 48%, the first order peak of the SAXS signal becomes significantly wider (see Figure 3). Inspecting the successive peaks of Figure 3, one clearly notes the appearance, for XFAM < X/FAM, of a shoulder, at a q value lower than the main peak position, which becomes more and more pronounced as the sample is diluted. For the lowest ϕFAM values, the peak shoulder seems to become a wide separate peak (see the arrow pointing at the shoulder of the peak in the spectrum of the most dilute lamellar system at XFAM = 38.2%). This observation is reminiscent of previous work performed on other lamellar systems, in which such a peak was attributed to the presence of defects in the lamellar phase.26,27 We used SAXS on oriented samples to study this wide peak more precisely. The samples were inserted in 200 μm-thick flat glass capillaries, which were sealed and left to equilibrate at room temperature for several hours. With time, some parts of the sample spontaneously align homeotropically with respect to the glass surfaces (with the layers oriented parallel to the glass surface, corresponding to the dark-colored domain in Figure 5a). In other parts of the capillary, planar domains are observed where the layers spontaneously orient perpendicular to the glass surface (the lightcolored and strongly textured domains in Figure 5a). Though the size of the different domains cannot be experimentally controlled, they are large enough to allow for illumination with the X-ray beam. The results obtained with the oriented sample at
Figure 3. X-ray spectra performed in the lamellar domain. The thin peak observed at q0 in the concentrated regime (XFAM g X/FAM ≈ 48%, see text) becomes broader as XFAM decreases and a shoulder appears at low q (see the black arrow pointing at the peak shoulder for XFAM = 38.2%). The curves are vertically shifted for the sake of visibility.
Figure 4. (a) Lamellar phase period d as a function of the inverse of FAM volume fraction 1/ϕFAM. The dashed line is the best possible straight line following eq 1 with constant Σ. (b) Evolution of the apparent area per acid head Σ, as extracted from the SAXS data and eq 1 and 4, in the hexagonal (H) and lamellar (L) domains. The molecular volume v is taken as 465 Å3. The hatched zone corresponds to the “defected” lamellar domain. 275
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Figure 5. (a) POM picture of the capillary tube containing the mixture at XFAM = 40.6%. 2D X-ray scattering patterns obtained (c) in the planar region (light-colored and textured zone) and (d) in the homeotropic region (dark colored domain). (b) SAXS spectra obtained for the non oriented lamellar mixture (continuous light gray line) and for the oriented mixture in the two regions (XFAM = 40.6%): homeotropic domain (continuous black line) and planar domain (dashed dark gray line).
XFAM = 40.6% are presented in Figure 5b, together with the signal obtained with the same composition when no particular orientation occurs. The scattering signal from the nonoriented sample (light gray full line in Figure 5b) presents an intense peak at q0 and a shoulder at a slightly smaller wavevector value qd. When the X-ray beam is scattered from a planar zone, the signal is strongly anisotropic (Figure 5c), showing the good alignment of the lamellar domains in the probed zone: two main peaks (dark gray dashed line in Figure 5b) correspond to the interlamellae correlations at qz,0, along a wavevector normal to the layers. These peaks are thin, with no observable shoulder. By contrast, when the X-ray beam probes a homeotropic zone (Figure 5d), the signal is isotropic, consistent with the homeotropic orientation of the lamellar structure. Once azimuthally averaged (black full line in Figure 5b), the signal I(q) presents a wide bump, which we interpret as the superposition of the “defect” bump at qd and a very small interlamellae correlation peak at q0, probably due to small remaining planar subdomains. Because the “defect” bump is absent from the (planar) data along qz, we conclude that it is related to correlations within the lamellar plane, i.e., along q . Moreover, we can deduce the characteristic length of this intralamellar correlation 2π/q ,d = 6.3 nm. When the FAM concentration is increased from 38.2 to 42.2%, we obtain an evolution of this intralamellar correlation length from 6.2 to 6.4 nm. This correlation length is hardly larger than the lamellar period (here d = 5.2 nm for XFAM = 40.6%), which raises questions about the geometry of the defects. Previous authors suggested several possibilities for lamellar defect topology: holes in the membrane, disks, or ribbon-like defects.27,28 Techniques complementary to SAXS are needed to extract more local geometrical information. We studied these possible defects further using FF-TEM and NMR. In Figure 6, we present some of the FF-TEM pictures obtained for the lamellar phase at three concentrations. For XFAM = 57.6% (Figure 6a), we observe a typical lamellar phase with very regularly spaced steps. The texture of the surface of the lamellae is quite smooth (see the white arrow that points to the surface of the layers) with a roughness that reflects the average size (ca. 2 nm) of the PtC grains used to form the metal freeze-fracture replica.29 As the concentration is decreased, first to 44.6% (Figure 6b) then )
)
to 40.6% (Figure 6c), the roughness on the lamellae surface becomes more and more visible. At XFAM = 40.6%, we observe a corrugated texture on the lamellae surface (white arrows in Figure 6c). However, the characteristic size for these corrugations (difficult to evaluate precisely but around 1020 nm in Figure 6c) is significantly larger than the intralamellar correlation length, as deduced from the SAXS study. Nevertheless, the difference in texture between the concentrated lamellar domain and the more dilute one is striking. As the dilute lamellar phase contains a larger amount of water than the concentrated one, artifacts due to water crystallization could be suspected a priori but were ruled out since identical textures were obtained when the water was replaced by a 70/30 water/glycerol solution, which does not crystallize under the used FF-TEM preparation conditions. Finally, the FF-TEM study confirms the presence of intralamella structural features but does not allow a more detailed understanding of the geometry of these features. It is interesting to compare the lamellar phases FF-TEM images with those obtained for the hexagonal phase (see Figure 6d). The lamella edges, which are straight at large concentration, become more and more irregular as the concentration is reduced. A certain similarity can be observed between the defected lamellar phase and the hexagonal one. It is then tempting to suggest that the dilute, supposedly defected, lamellar phase shows an “intermediate” texture between the regular lamellar phase and the hexagonal phase, and that there exists some continuity in the transition between the two phases. We performed deuterium solid state NMR experiments in order to get additional information. Two different types of experiments were conducted. In the first experiment, we doped the system with a deuterated fatty acid and probed the dynamics of the alkyl chains. More precisely, a small amount of selectively labeled (deuterated on position 9 and 10) oleic acid was added to the FAM system: oleic acid being already present in the mixture, the modification of the system is very modest. Indeed, using POM and SAXS, we checked that doping the system with 30 mg/mL of oleic acid does not significantly modify the succession of phases upon dilution. We also observed that the variation of the lamellar repeating distance upon dilution is unaffected by the doping (results not shown). 276
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Figure 6. FF-TEM pictures of the lamellar and the hexagonal phases. (a) Lamellar phase at XFAM = 57.6%, the surface of the layer plane, indicated by the white arrow, is smooth and the layer stacking is very regular. (b) Lamellar phase at XFAM = 44.6%, the surface of the layers (see white arrows) becomes a little more corrugated. (c) “Defected” lamellar phase at XFAM = 40.6%, the surface of the layers (see white arrows) is no longer smooth but presents a rather wavy texture. The layer stacking appears much more perturbed than in panel a. (d) Hexagonal phase at XFAM = 25.6%. Scale bar: 100 nm.
Several regions of the phase diagram were investigated: the concentrated lamellar domain, the “defected” lamellar domain close to the lamellar-hexagonal phase transition, and the hexagonal domain. Selected spectra are plotted in Figure 7. The doped systems present one broad isotropic line due to the signal of water, since we used nondepleted deuterated water. In addition, we obtained two broad spectra composed of superimposed powder patterns, which correspond to the signal of the two deuterons along the alkyl chain of the selectively labeled oleic acid. On each powder pattern, one clearly sees the doublet which stands for the Pake peaks, between which we can measure the quadrupolar splittings, Δν1 and Δν2 (see Figure 7).19 The signal for each deuteron (position 9 or 10) cannot be assigned to a given powder pattern. However, it is generally assumed that the lower the quadrupolar splitting, the faster the molecular dynamics. Moreover, the dynamics of the alkyl chain increases when going toward the methyl position. The value of Δν2 was
plotted as a function of FAM concentration and is shown in Figure 8. The border between the previously determined hexagonal and lamellar domains is also shown. Clearly, for the most concentrated samples, between 53 and 46%, Δν2 does not vary significantly upon dilution. This shows that in this regime, the dynamics of the fatty chains embedded in the lamellar bilayer is not affected by dilution. However, when entering the “defected” lamellar domain, a break in the curve occurs and Δν2 significantly decreases upon further dilution. The crossover concentration (∼45%) below which Δν2 starts decreasing is not far from that corresponding to the appearance of the peak shoulder on the small-angle X-ray spectra (∼4749%). The subsequent decrease of Δν2 reflects the increase in the dynamics and a loss of anisotropic ordering of the fatty chains, which may occur from an increase in the bilayer undulations or from the formation of regions of strong local curvature in the lamellar phase, when approaching the hexagonal phase. This increase also rules out the possibility of a “ripple phase”, 277
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Figure 7. NMR spectra of a hexagonal (bottom) and two lamellar mixtures doped with 30 mg/mL of deuterated oleic acid. Figure 8. Evolution of the doublet splitting (a) for the mixture with deuterated oleic acid (Δν2) and (b) in D2O (Δν2D2O) as a function of the FAM weight fraction XFAM. The vertical black line separates the hexagonal and lamellar domains and the hatched zone corresponds to the “defected” lamellar zone.
which can be found in some phospholipids lamellar phases, since these phases with intralamella corrugations have mostly frozen chains.30 On the other hand, this increase in the system curvature is remarkably progressive and never leads to a hexagonal-lamellar phase separation. The NMR spectra of the most diluted lamellar phases (close to the hexagonal domain) are indeed characteristic of single phase spectra, whereas a phase separation would have led to the superposition of a lamellar spectrum and a hexagonal one. This result suggests a rather continuous lamellar-hexagonal transition. It is surprising that the transition seems continuous even though the change of symmetry implies a first-order transition. Note that lamellar-hexagonal transitions in surfactant or copolymer systems are usually described32 with either a two-phase region, i.e., by a first-order transition, or with the presence of intermediate mesophases (rectangular, monoclinic, cubic, etc.). None of these clearly applies here. It is of course possible that it is a “slightly firstorder” transition with a narrow coexistence domain, therefore undetected experimentally. In a second set of experiments, D2O was used as the deuterium source to probe the dynamics of water within the ordered phases. In this case, we observe a single powder pattern (not shown), the quadrupolar splitting of which is much smaller than for the selectively labeled oleic acid. This is mainly due to the higher dynamics of water compared to the alkyl chains. The signal results from the fast exchange between the water molecules more or less bound to the fatty acid polar heads and those between the bilayers,31 further away from the membranes. The first contribution yields a powder pattern because of the anisotropy of the system and the second one an isotropic contribution. The resulting signal is a powder pattern, with a quadrupolar splitting modulated by the ratio between the two types of water molecules (bound and unbound). We observe that, upon dilution, the D2O doublet splitting Δν2D2O decreases, which is simply related to the fact that the ratio of water between the bilayers (isotropic contribution) increases (see Figure 8b). By contrast
with the results shown in Figure 8a, this decrease can be considered regular and no abrupt change is seen in the curve. This absence of abrupt change tends to rule out the possibility that the “defects” connect adjacent water layers with holes. We can thus deduce that, in our case, regions of strong curvature appear in the systems, most probably as bumps on the surface of the lamellae as suggested by the FF-TEM pictures. However, these regions of strong curvature do not lead to the formation of holes connecting the water layers through the fatty acids membranes as was observed in the decanol/sodium dodecyl sulfate/water system.27,28 In previously investigated defected lamellar phases, it is usually considered that the appearance of all types of bilayer defects is due to an increase in the spontaneous curvature of the surfactant monolayers (either through a variation of cosurfactant or ionic species concentration).27,28 In our case, the experimental results suggest that such an increase in monolayer spontaneous curvature is also involved in appearance of the defected phase, and could be related to an increase in the average charge density of the bilayer surfaces, via variations of the amount of protonated EDA molecules condensed on the COO monolayers. Indeed, the coupling of charged EDA molecules with COO heads can be described as a complexation equilibrium of the type ðRCOO EDA þ Þ a RCOO þ EDA free þ As dilution occurs (at constant x), the concentration of “free” EDA+ decreases in the aqueous phase domain that is comprised between the surfactant layers. As a consequence the equilibrium is displaced toward the decomplexation of EDA+ from the bilayer, 278
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~, Figure 9. Schematic representation of the evolution of the assemblies from the lamellar to the hexagonal phases: (a) via a centered undulated phase L (b) via uncorrelated bilayer modulations: Note that there is no geometrical relation between the modulations in adjacent bilayers.
)
which leads to an increase in the density of charged polar carboxylic heads and to an increased repulsion between these heads. This induces an increase in the area per polar head Σ as well as a curvature frustration, which, below a certain threshold concentration XFAM ≈ 48%, could be relaxed by a static modulation of the bilayers. The existence of an undulated ~) phase, with a peristaltic modulation of the bilayer lamellar (L thickness (as shown in Figure 9a), was proposed as an intermediate phase in the lamellar-hexagonal phase transition for other systems.32 It could be tempting to correlate the “defect” peak, found at wavevector q ,d in the SAXS data, to the wavelength of the thickness modulations in such a structure. ~ phase would not However, due to symmetry reasons, the L provide a correlation peak at a smaller wavevector qd than the main structure peak q0, as obtained in the SAXS patterns shown in Figure √ 3, but rather a higher order peak, typically between q0 and 3q0. On the other hand, the experimental SAXS patterns could be interpreted as the signal of a lamellar phase with peristaltic modulations of the bilayer thickness uncorrelated between adjacent layers, as shown in Figure 9b. In terms of symmetry, such a phase is equivalent to the so-called “sliding columnar phase” described in hybrid systems of colloidal rods (DNA fragments) confined in between the bilayers of a phospholipid lamellar phase.33 Upon dilution, the lamella modulations increase and induce a smooth transition toward the hexagonal structure when the area per polar head attains a maximal value of ∼47 Å2 in the hexagonal domain (where the curvature is maximal). In this defected lamellar phase, which we can tentatively visualize as a lamellar phase with a peristaltic modulation of the bilayer thickness, the apparent area per polar head, extracted from eq 1, would depend on the amplitude of the modulation of the bilayers due to a projection effect, even if the actual area per polar head would not change. In our system, the measured increase of the apparent area per polar head Σ when dilution occurs in the defected lamellar domain (hatched in Figure 4b), could then be the combination of an actual increase of the polar head area due to an evolution of the acid-amine interaction and an effect of the increase of the thickness modulation amplitude. 3.3. Intermediate Concentration Domain (5% < XFAM < 38%, x = 2.6). When the concentration is decreased below 38%,
Figure 10. SAXS spectra of the system at various FAM weight fractions in the hexagonal domain. The curves are vertically shifted for the sake of visibility.
the system reaches the hexagonal phase: the mixture suddenly becomes highly viscous and shows a typical birefringent texture of the hexagonal phase under POM (not shown here). This is confirmed by SAXS measurements which are presented in Figure 10. The observed Bragg peaks can be easily attributed to the presence of a hexagonal order since they appear at √ successive positions q0, 3q0, and 2q0. The hexagonal domain extends over a concentration range from 13% to 38% (for x = 2.6), above which the system turns into the previously discussed lamellar phase. From the SAXS spectra performed at various FAM concentrations in the hexagonal phase, one can deduce the values of the triangular array length a through the relation 4π 1 a ¼ pffiffiffi 3 q0
ð3Þ
a is plotted as function of the volume fraction of water and EDA ϕ = ϕwater + ϕEDA = (1 ϕFAM) in Figure 11a. In Figure 11b, we present the different possible hexagonal organization for these systems. 279
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Figure 11. (a) Evolution of the lattice parameter a (black filled circles), deduced from the SAXS measurements shown in Figure 10, through eq 3 as a function of the volume fraction of water and EDA. The three lines correspond to the three possible hexagonal packings discussed in the text (eqs 4α, 4β and 4γ) and schematized in panel b. (b) Different possible hexagonal packings. Direct hexagonal phase: (α). The fatty acids chains form the cylinders in the continuous water phase. Inverse hexagonal phases: the fatty acids chains form the continuous phase, and the water and EDA domains are either cylindrical (β) or hexagonal (γ).
For these three different hypotheses, geometrical arguments provide the respective expressions of the dilution laws, which are ðαÞ : a ¼ lC =½31=2 =ð2πÞ:ð1 ϕÞ1=2 ðβÞ : a ¼ 2lC =½1 ð2:31=2 =π:ϕÞ1=2 ðγÞ : a ¼ 2lC =ð1 ϕ1=2 Þ
ð4Þ
The only unknown parameter is the carbon chain length lC. Choosing a reasonable value for lC (1.9 nm), we obtain the three lines in Figure 11a, which we compare to the measured values (points). From this comparison, it is clear that the structure of the hexagonal phase is of the direct type, as also observed by Cistola et al. in 1:1 fatty acid/fatty acid soap mixtures.8 The data can then be analyzed more precisely in terms of area per polar head Σ by modifying equation (4α) into !1=2 2v 2π pffiffiffi ð5Þ a¼ Σ 3ð1 ϕÞ
Figure 12. SANS spectra of the different mixtures for 5% < XFAM < 26%. Evolution of q4I(q)/XFAM as a function of q. The solid black line is the form factor of monodisperse cylinders of radius R = 1.8 nm (eq 6 in the text).
where v is the molecular volume of the fatty acid. The values of Σ extracted from the experimental data using eq 5 are displayed on the hexagonal domain of the Figure 4. The obtained values of Σ vary continuously from the hexagonal domain to the lamellar domain. The area per polar head is high in the hexagonal phase, which is consistent with a high curvature and a relatively low amount of condensed EDA molecules on the FAM cylinders. It changes only slightly at the H f L transition because the lamellae are strongly undulating, but decreases significantly during the further concentration increase as the bilayers become progressively smoother. Upon further dilution, POM first shows the appearance of an isotropic phase becoming birefringent under flow for concentrations 12% < XFAM < 15% (here referred to as the BUF phase) and then an isotropic phase for XFAM < 12%, in which no birefringence could be detected by POM. SAXS measurements of the mixtures have also been performed as the FAM concentration is decreased toward the BUF phase and into the isotropic domain. The SAXS spectra (not shown here, see the Supporting Information) show no significant changes when passing through the boundary
between the BUF and the isotropic domains. The structure of the isotropic and BUF phases were elucidated using SANS and cryo-TEM. For the SANS experiments, samples were prepared with D2O replacing water. The benefit of SANS here is to simplify the form factor of the FAM domains, as it can be reduced to a single contrast value between the D-rich aqueous domains and the H-rich fatty domains. In Figure 12, we plot the product I(q)q4 (Porod representation) obtained using SANS, as a function of q for various FAM concentrations ranging from 5% (isotropic phase) to 26% (hexagonal phase). In this Figure, the form factor P(q) for infinite and monodisperse cylinders of radius R is also plotted as a continuous line for comparison34 " PðqÞ ¼ cste q 280
1
J1 ðqRÞ qR
#2 ð6Þ
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Langmuir where J1(qR) is the first order Bessel function and taking R = 1.8 nm. From this representation, it clearly appears that the experimental data related to the BUF, the isotropic and the HI domains present very similar form factors, compatible with cylindrical objects of radius R ≈ 1.8 nm. This is also in good agreement with the carbon chain length value of 1.9 nm, which was deduced from the dilution law in the hexagonal domain. All three domains seem to contain the same types of self-assembled objects, differing only by the degree of order. The BUF phase can be understood as consisting of elongated micelles of radius R, which are organized in an isotropic manner at rest and become aligned when submitted to a shear flow. In the isotropic domain, the average distance between the cylinders increases and the system no longer organizes under flow. Cryo-TEM observation of the isotropic phase supports our hypothesis, (see Supporting Information). Thus, we believe that the birefringence under flow that is observed between 12 and 15% of FAM is due to the cooperative alignment of wormlike micelles that occurs when such objects become close enough to each other in the solution. As XFAM increases above 15%, the elongated objects correlate over larger distances on a triangular packing, which leads to the appearance of the hexagonal phase. It is important to note that, as opposed to other studied systems,35 the transition to the H phase appears at relatively low concentration, which was also observed by Antunes et al. in the simple sodium oleate/water mixture,5 and probably means that the interaction between the cylinders is rather long-range. The transition from the BUF domain to the hexagonal domain occurs when the periodic array length is between 9 and 8 nm. Subtracting 3.6 nm for the diameter of one cylinder, we get a value of the distance between cylinders of 4.4 nm < dcyl < 5.4 nm at the boundary between the BUF and the H domains. Taking such a range as due to the screening of electrostatic interactions in a solution of monovalent ions, one would deduce a ions concentration of the order of 0.02 mol/L, which is about six times smaller than the value calculated from the infrared spectroscopy results shown before. Thus, the interaction between cylinders probably combines steric and electrostatic effects, but is difficult to evaluate precisely. In fact, because the carboxylic acid functions are all deprotonated (as was shown with IR spectroscopy, see section 3.1), the outer surface of the cylinders is highly charged. This high charge density may provide stiffness and the large persistence length may generate long-range steric repulsions.35 Moreover, it is known that some (typically one-third) of the counterions (mostly monovalent H2NCH2 CH2NH3+ salt of ethylenediamine) are condensed on the charged surface, in order to decrease the Coulombic energy associated with a high charge density36,37 and may contribute with steric effects.
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bilayers induce a steep increase in the area per fatty acid head. In the third and least concentrated subdomain, the existence of corrugations on the bilayer surfaces, as shown by FFTEM, of intralamellar structural correlations, as evidenced by SAXS, and of an increase in the fatty chains disorder, as shown by NMR, all are consistent with the hypothesis of a lamellar phase with peristaltic modulations of the bilayer thickness uncorrelated between adjacent layers (sliding phase) as an intermediate structure in the lamellar-to-hexagonal transition. The transition from one phase to the other thus appears to be very progressive with an increase in the spontaneous curvature of the surfactant layers. This increase in spontaneous curvature may be due to a decrease in the amount of EDA molecules condensed on the highly charged COO monolayers. (ii) At lower concentration, a hexagonal domain appears and we demonstrate, using SAXS, that the hexagonal phase is direct. The transition toward a birefringent-under-flow phase occurs at surprisingly low concentrations, which we attribute to a possible electrosteric effect induced by both the important charge density of the objects and the presence of EDA molecules that are condensed at the micelles surface or long-range repulsions between highly charged cylinders. (iii) In the lowest concentration regime, we identified an isotropic phase and SANS measurements show that it is constituted of long cylinders of fatty acid molecules, with roughly the same diameter as the cylinders encountered in the hexagonal phase. Close to the transition toward the hexagonal phase, the cylinders start to orient when submitted to shearing, which leads to the appearance of birefringence. Our study shows the richness of the phase diagram encountered in this fatty acid system, in which the interaction between the acid surfactant and the amine counterion tunes the spontaneous curvature and therefore controls the morphologies.
’ ASSOCIATED CONTENT
bS
Supporting Information. Polarized optical micrographs of the hexagonal and lamellar phases and SAXS and cryo-TEM results on the diluted phases. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Present Addresses
4. CONCLUSION We have studied the phase behavior of water/fatty acid/diamine mixtures. First, we showed that an excess of diamine is required to solubilize the fatty acids in the aqueous phase. Then, at fixed and sufficiently large diamine/fatty acid molar ratio, we have studied the succession of liquid crystalline phases. (i) At large fatty acid concentration, we observe the presence of a lamellar phase. The lamellar domain can be separated in three subdomains. In the two first subdomains, at high concentration (more than 45% of fatty acids), the lamellae are flat. In the very concentrated subdomain, (more than 52% of fatty acids), increased steric repulsions between
#
Universite de Bordeaux, UMR 1332 Biologie du fruit et Pathologie, F-33140 Villenave d’Ornon, France. INRA, UMR 1332 Biologie du fruit et Pathologie, F-33140 Villenave d’Ornon, France.
’ ACKNOWLEDGMENT We thank the Rhodia Company and the Conseil Regional d’Aquitaine for financial support. Fruitful conversations with O. Diat, G. Sigaud, P. Le Cornec, S. Deroo, A. Bourdette, and M. In are gratefully acknowledged. We also thank A. Vacher for the Cryo-TEM experiments and L. Noirez and the Laboratoire Leon 281
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Brillouin, CNRS-CEA, Saclay, France, for access to the smallangle neutron scattering facility PAXY.
(35) Berret, J. F. Rheophysics of Wormlike Micelles. In Molecular Gels; Weiss, R. G., Terech, P., Eds.; Springer: Dordrecht, The Netherlands, 2005; pp 235275. (36) Manning, G. S. J. Phys. Chem. B 2007, 111, 8554–8559. (37) Hunter, R. J. Zeta Potential in colloid Science; Academic Press: New York, 1981.
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