Self-assembly and emulsions of oleic acid–oleate mixtures in glycerol

Abstract

It is known that mixtures of oleic acid and sodium oleate spontaneously form vesicles in water. In this paper, we study this system in glycerol, and show that it also forms vesicles in this solvent, but with a considerably lower diameter. The critical vesicle concentration (cvc) was higher in glycerol (cvc = 27.1 mM) than in water (cvc = 0.10 mM). This finding was confirmed using an analogous system made of palmitoleic acid and palmitoleate. The vesicles in glycerol were characterized using small-angle neutron scattering (SANS) which showed that the fatty acids are embedded in a fluid bilayer phase. These fatty acid dispersions were used to produce emulsions in glycerol, using hexadecane as the oil component. We show that a higher energy is required to produce emulsions in glycerol than in water, probably because of the higher viscosity of glycerol. However, emulsions were shown to be stable in glycerol. Altogether, this shows that supramolecular self-assembly occurs in glycerol, and that emulsions can be successfully produced in this solvent.

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Introduction

When surfactants are present in aqueous solution in sufficient quantities, they are able to form supramolecular structures such as micelles, vesicles and liquid-crystalline phases.^1–5 Amongst the vast family of surfactants are fatty acids. These natural products are found especially in animal products (lard and butter) and vegetable fats (coconut oil, palm oil, olive oil, etc.).

The use of agricultural resources for industrial purposes will undoubtedly be one of the major challenges of the 21st century, both from the energy point of view (by contributing to the replacement of fossil fuels) and with respect to non-energy uses, resulting from the availability of organic biosynthons to the chemical industry. Our work on dispersions of fatty acids and hydroxylated derivatives^6,7 forms part of these efforts, in that it seeks to demonstrate the potential contribution these plant-derived compounds could make as a new class of green surfactants.

However, saturated long-chain fatty acids and their metal salts crystallize in solution below their Krafft^8,9 temperature, which hampers their use for biological and physicochemical studies, and thus for industrial applications.¹⁰ To avoid this, unsaturated fatty acids or short-chain saturated fatty acids (<C₁₂) have been used and shown to self-assemble in water, mostly as vesicles.^11–15 For instance, oleic acid (an unsaturated fatty acid) has been shown to form vesicles in water.^16,17 In these systems, fatty acids self-assemble into bilayers, depending on the pH. It has been shown that the formation of a hydrogen-bond network between the COOH and COO⁻ is essential to prevent the crystallization of the mixture.¹³ In the same way, using long-chain saturated fatty acids (>C₁₂) ion-paired with organic counter-ions allows one to produce various supramolecular assemblies, from cones to twisted ribbons and even tubes.^6,7,18–21

Supramolecular assemblies have also been shown to occur in other polar solvents such as ethylene glycol, formamide and glycerol.^22–27 Using such solvents rather than water is attractive in materials chemistry. For instance, ordered macroporous materials have been obtained using formamide to prevent the fast hydrolysis of the silica precursor in water.²⁸ Amongst these solvents, here we focus on glycerol, because it is a green solvent which presents some close similarities to water. Indeed, glycerol is highly hydrophilic, non-toxic (LD₅₀(rat) = 12600 mg kg⁻¹), very cheap (0.5 € kg⁻¹), biodegradable and abundant (1.5 Mt year⁻¹). Moreover, glycerol has a high dielectric constant (42.5), a high dipolar moment (2.60 D), a high cohesive energy (1780 nJ cm⁻²) and considerable hydrogen-bonding abilities, which are important parameters for the self-assembly of surfactants.

The additional interest of using glycerol rather than water is in the field of organic chemistry. By using glycerol as a solvent, one can reach higher reaction temperatures, since it has a boiling point of 290 °C. Recently, we have developed a procedure using emulsions in glycerol for the ring-opening of epoxides (and Heck coupling reactions) in the temperature range 90–120 °C.^29–31

Here, we study the self-assembly of fatty acids in pure glycerol and use these dispersions to produce emulsions. We focus on the oleic acid–sodium oleate system because it has been widely studied in water. As in water, this system forms vesicles in glycerol, as confirmed by both phase contrast microscopy and light scattering. Fluorescence was successfully used to determine the critical vesicle concentration (cvc). The fatty acid bilayers were characterized by using SANS.

Materials and methods

Sample preparation

Oleic acid (Sigma-Aldrich) was weighed in a tube. Sodium oleate (Sigma-Aldrich) was added to reach a 1 : 1 molar ratio, and solvent (water or glycerol, 99%, Sigma-Aldrich) was added so that the concentration was 10 mg mL⁻¹. Samples were heated at 70 °C for at least 10 min and frozen at −20 °C. This procedure was repeated 3 times, and the samples were stored at −20 °C. Prior to being used, each sample was heated at 60 °C for 10 min. The same procedure was used for the palmitoleic acid (Sigma-Aldrich) system. However, sodium palmitoleate is not commercial, and was first prepared in water by addition of a 1 M stock solution of NaOH (molar ratio palmitoleic acid–NaOH = 1 : 1). The solution was frozen at −80 °C and then lyophilized.

Phase contrast microscopy

Observations were made at room temperature at 20× magnification using an optical microscope in the phase contrast mode (Nikon Eclipse E-400, Tokyo, Japan) equipped with a 3-CCD JVC camera allowing digital images (768 × 512 pixels) to be collected. A drop of the lipid dispersion (about 20 μL) was deposited on the glass slide surface (76 × 26 × 1.1 mm, RS France) and covered with a cover slide (22 × 22 mm, Menzel-Glaser, Germany). The glass slides were cleaned prior to use with ethanol and acetone.

Determination of the cvc by fluorescence

All fluorescence spectra were measured using a Hitachi fluorometer (F-4500) at 25 °C. A stock solution of the fluorescent probe (pyrene) at a concentration of 1.0 × 10⁻⁴ M in THF was prepared. To determine the cmc, a series of surfactant solutions were prepared and a small amount of the fluorescent probe was added. The concentration of the fluorescent probe was fixed at 1.0 × 10⁻⁶ M. The samples were incubated for 12 h in the dark at room temperature before measuring the fluorescence. The excitation wavelength was 340 nm.

Small-angle neutron scattering (SANS)

Small-angle neutron scattering (SANS) experiments were performed at Laboratoire Léon-Brillouin (laboratoire mixte CEA/CNRS, Saclay, France) on spectrometer PAXY. The neutron beam was collimated by appropriately chosen neutron guides and circular apertures, with a beam diameter at the sample position of 7.6 mm. The neutron wavelength was set to 4 or 8 Å with a mechanical velocity selector (Δλ/λ ≈ 0.1), the 2D detector (128 × 128 pixels, pixel size 5 × 5 mm²) being positioned at 1.4 or 6.7 m, respectively. The scattering wave vector, Q, typically ranges from 0.005 to 0.4 Å⁻¹, with a significant overlap between the two configurations. The samples, prepared with deuterated water or glycerol, were held in flat quartz cells with a 2 mm optical path, and temperature-controlled by a circulating fluid to within ±0.2 °C. The azimuthally-averaged spectra were corrected for solvent, cell and incoherent scattering, as well as for background noise. The general theory for fitting the SANS data can be found in the literature,³² and our data were fitted with models previously described for other fatty acid systems.^19,33

Dynamic light scattering (DLS)

The size and the polydispersity of oleic acid–sodium oleate vesicles were obtained by dynamic light scattering (DLS). The measurements were carried out using a Zetasizer Nano ZS (Malvern Instruments, UK) consisting of a 4 mW He–Ne laser (λ = 633 nm). The scattering cell was thermostatted at 25.0 ± 0.1 °C. All the experiments were performed at a scattering angle of 173°, with the solvent viscosity being 934 mPa s, and the solvent refractive index being 1.47. Hydrodynamic average radius and intensity-weighted size distribution were calculated by the cumulant and CONTIN method, respectively, using programs provided by the manufacturer. The cumulant analysis was performed by gathering the intensity autocorrelation function over a period of 30 s (repeated 6 times). The mean hydrodynamic radius was calculated according to the Stokes–Einstein relationship. Great care was taken to avoid the presence of dust in every step of the preparation of the vesicles, which were analysed without any pre-treatment.

Emulsions

Emulsions were prepared with 30 vol% n-hexadecane (Sigma Aldrich) as the oil phase and 75 vol% of the aqueous or glycerol phase (oleic acid–oleate dispersion). In all cases, a fixed volume (10 mL) of emulsion was prepared in glass tubes with a height of 10 cm, an internal diameter of 2.2 cm and a flat bottom by sonication with a 3.2 mm diameter probe (Sonicator 4000, 20 kHz, 600 W, Misonix, Newtown, USA). The energy of sonication is given by the apparatus, and was recorded when the hexadecane phase was completely incorporated in the oleic acid–oleate mixture. States of droplet flocculation were assessed by examining emulsions by phase contrast microscopy.

Results and discussion

Phase behavior

In water, the formation of vesicles in the oleic acid system has been shown to occur by varying the pH of the solution.¹¹Sodium oleate forms micelles, and decreasing the pH yields vesicles at a pH from about 10.5 to 8.5, at which carboxylic and carboxylate groups coexist. In glycerol, rather than varying the pH to disperse HCl or NaOH, we used mixtures of sodium oleate and oleic acid. Both components were dispersed at different molar ratios in glycerol. For comparison, we also studied the same system in water, and the dispersions were produced in a similar fashion, i.e., by using sodium oleate and oleic acid. The molar ratio of oleic acid to sodium oleate was varied from 0.1 to 2.5.

In water, at low molar ratio (up to 0.5), the solutions were transparent, probably made of micelles. Then, upon increasing the molar ratio until 2.0, the turbidity increased. Above this value, the oleic acid formed droplets in water. These findings are in agreement with previous observations made by varying the pH.¹¹ By contrast, in the case of glycerol, solutions appeared non-turbid whatever the molar ratio between sodium oleate and oleic acid. This visual difference is expected to be due to the difference between the refractive index of the solvent (water or glycerol) and the fatty acid. Indeed, the difference between the refractive index of water (n²⁰_D = 1.33) and that of oleic acid (n²⁰_D = 1.46) is 0.13. By contrast, in the case of glycerol, which has a refractive index of 1.47, that difference is only 0.01. As in the case of water, a suspension of oleic droplets was also observed for a molar ratio above 2.0 by phase contrast microscopy (not shown).

In water, at a molar ratio of 1.0, micrometer-length polydispersed vesicles were observed by phase contrast microscopy (Fig. 1). In order to have more information on the size of these vesicles, the DLS technique was used. A very broad spectrum was obtained (not shown), confirming the large polydispersity observed by microscopy. By contrast, in glycerol an average size of 71 nm was found with a polydispersity index of 0.6. Such a small size also explains why the samples are not turbid in glycerol. Note that such small assemblies cannot be observed by phase contrast microscopy, and the use of glycerol does not allow one to perform transmission electron microscopy (TEM) or even cryo-TEM. The formation of vesicles in glycerol was further demonstrated by SANS (see below). The smaller size of the self-assembled structure observed in glycerol is a general feature in that solvent. Previous studies have focused on the self-assembly of surfactants in mixtures of glycerol and water. For instance, for surfactant-forming micelles, these assemblies have been shown to be smaller.^34,35

Fig. 1 Oleic acid–sodium oleate vesicles at a molar ratio of 1 in water observed by phase contrast microscopy.

Determination of the cvc

Oleic acid–sodium oleate vesicles are thus present both in water and in glycerol. The subsequent experiments with these systems were only done at a molar ratio of 1.0. In order to determine the cvc, we used the fluorescence of pyrene, which has already been successfully employed for determining the critical micelle concentration (cmc) of surfactants in water.^36,37 Below the cmc, the pyrene remains in water, and is incorporated into micelles when they form by concentrating the surfactant. This is accompanied by a change of the polarity of its environment, and then by a variation of its fluorescence. The same principle was used in the current work, except that we focus on the cvc and not the cmc. The fluorescence spectrum of pyrene exihibits five predominant peaks. It has been shown that the ratio of intensity of the first peak (I₁ at 375 nm) and the third one (I₃ at 394 nm) is a sensitive parameter characterizing the polarity of the probe's environment.^38,39 The cvc was determined for the oleic acid–sodium oleate system at a molar ratio of 1.0 in both water and glycerol. For this, the ratio I₁/I₃ was plotted as a function of the concentration (Fig. 2). In both cases I₁/I₃ first decreased sharply upon increasing the concentration. This effect was more pronounced in the case of water. Above a given concentration (which corresponds to a breaking point), the I₁/I₃ still decreased. Therefore, the concentration at which the breaking point occurs corresponds to the critical vesicle concentration (cvc), which was 0.10 mM in water and 27.1 mM in glycerol.

Fig. 2 Variation of the I₁/I₃ ratio as a function of the concentration for the oleic acid–sodium oleate system at 25 °C: (a) in water, (b) in glycerol.

Therefore, the cvc in glycerol is much higher than in water. Once again, this finding is in agreement with the literature reporting on the self-assembly of micelles in glycerol. The cmc is also known to be higher in the presence of glycerol.^34,35

In addition, we also determined the cvc for the similar palmitoleic acid–sodium palmitoleate system at a molar ratio of 1.0. For this, the fluorescence of pyrene was also used. Fig. 3 shows the ratio I₁/I₃vs. the palmitoleic acid–sodium palmitoleate concentration in glycerol. The variation was similar to that for the previous system, with a breaking point at a concentration of 35 mM. As expected, this value is higher than for the oleic acid system – it is already known that in water that the higher the alkyl chain length, the lower the cmc.⁴⁰

Fig. 3 Determination of the cvc in the palmitoleic acid–sodium palmitoleate system in glycerol at 25 °C.

SANS

To obtain additional information on the vesicles formed in both glycerol and water, we performed SANS experiments. To carry out these experiments, oleic acid and sodium oleate mixtures were prepared in deuterated water or d₅-glycerol at 10 mg mL⁻¹, i.e., at a concentration above the cvc. The azimuthally averaged spectra (Fig. 4) exhibit a small-angle scattering signal in both cases. In water, two peaks are located at q₀ = 0.009 and 0.018 Å⁻¹ (i.e., in an exact ratio of 1 : 2) and can be readily identified as Bragg peaks. This ratio indicates that bilayers of oleic acid–sodium oleate in water are stacked within the vesicular assembly (multilayer structure). The fact that the peaks are relatively flat indicates that either the bilayers exhibit undulations,^41,42 or that there is a low number of bilayers in the multilayer structure. A lamellar spacing (i.e., the repeat distance corresponding to one lipid layer and a water layer) can be determined according to 2π/q₀, and was found to be 70 nm in water.

Fig. 4 SANS intensity profile for the oleic acid–sodium oleate system at a concentration of 10 mg mL⁻¹: (a) in water, and (b) in glycerol. The arrows indicate the Bragg peaks (see text).

The signal of the system in glycerol also undergoes a strong small-angle scattering, but only displays one sharp peak, located at q₀ = 0.017 Å⁻¹. The fact that only one flat peak is observed suggests that the bilayers exhibit higher undulations than in water, or that the multilamellarity is weaker. The latter hypothesis is the most probable, since the high viscosity of glycerol should lower the bilayer undulations. In any case, this confirms that bilayers are formed and that self-assembly of vesicles also occurs in glycerol. Even without observable higher-order peaks, one can determine from the SANS data a repeat distance of 37 nm. Then, compared to water, the lamellar spacing is two times lower than in glycerol. Once again, this is in agreement with the fact that glycerol has a higher viscosity than water. Indeed, the bilayer undulations are weaker in that solvent, which lowers the repulsive interactions between two adjacent layers.⁴² Interestingly, since a diameter of only 71 nm was measured by light scattering, this means that the vesicles are formed by a maximum of two fatty acid bilayers.

In the so-called Porod representation, where q⁴ times the scattering intensity is displayed as a function of q, a fit to the form factor of the main oscillation yielded a value of the bilayer thickness δ equal to 3 ± 0.1 and 2.8 ± 0.1 nm in water and in glycerol, respectively (not shown). It must be noted that the oleic chain in its extended conformation has a length of about 2.0 nm. The experimental values measured by SANS are markedly lower than twice the oleic chain length in its extended conformation. This means that for both systems, the alkyl chains are in a disordered state, i.e., in a fluid Lα phase.

In addition, a SANS experiment was performed for that system in glycerol below the cvc at 3.5 mg mL⁻¹ (not shown). No scattering was observed for this sample, confirming our previous finding by fluorescence, i.e., only free fatty acids are present at low concentration, below the cmc.

Emulsions

In the last part of this study, we used these dispersions in water and in glycerol to produce emulsions with hexadecane as the oil component. Emulsions were produced by ultrasonication of the solutions. In water, after one cycle of 15 s, all the hexadecane could be fully dispersed in the oleic acid dispersion, yielding a white solution of emulsion droplets. This procedure corresponds to an energy of sonication of 418 J. In glycerol, after only one cycle of 15 s, complete demixing of the oil and glycerol phases was observed. This means that the emulsion is not formed or stable under these conditions. This is probably because the high viscosity of the glycerol hampering the migration of the fatty acids at the interface. Four cycles of 15 s were necessary to produce an emulsion in which no hexadecane was released at the surface of the solution. In that case, an energy of sonication of 2044 J was measured. Both the emulsions were observed by phase contrast microscopy (Fig. 5). The pictures showed individual oil droplets dispersed in water or glycerol, which were then stabilized by the fatty acids. Interestingly, the size of those droplets was similar in both solvents. Emulsions were stable, and after 1 month, no release of hexadecane could be observed by visual inspection of the sample tubes. However, coalescence of oil droplets was observed microscopically, since larger droplets were formed (Fig. 5c and d). Interestingly, larger droplets were observed in the case of water. Once again, this is probably because of the higher viscosity of glycerol, which decreases the Ostwald ripening.⁴³

Fig. 5 Emulsion droplets observed by phase contrast microscopy at 25 °C: after emulsification in (a) water, (b) glycerol; after 28 days in (c) water, (d) glycerol. Scale bar: 5 μm.

Conclusions

In this paper, we have shown that oleic acid–sodium oleate mixtures can form vesicles in pure glycerol, and can be emulsified in this solvent. The most striking finding is that these vesicles have considerably lower diameters in glycerol. This could be of interest for many applications in the field of materials or organic chemistry. For example, surfactant assemblies are widely used as templates for the preparation of mesoporous silica⁴⁴ or silica spheres,⁴⁵ so the use of glycerol could allow the preparation of spheres with a lower diameter. In addition, stable emulsions can also be produced in glycerol, and could be used in cosmetics or again in materials chemistry for the synthesis of hollow materials and nanomaterials. Finally, studying the formation and stability of emulsions in glycerol should contribute to improvement of the experimental conditions required to perform organic reactions in this solvent.^29–31

Acknowledgements

MD is grateful to the CNRS and INRA for the allocation of his PhD grant. The program CPDD of the CNRS (RDR-1 action) is also gratefully acknowledged for financial support. We also thank the Laboratoire Léon-Brillouin for the allocation of neutron beam time on the spectrometer PAXY. Several experiments were done during the annual training period ‘les fans du LLB’. We gratefully acknowledge the assistance of our local contact, Fabrice Cousin, during the neutron scattering run.

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