water microemulsions

Colloids and Surfaces A: Physicochem. Eng. Aspects 281 (2006) 230–236

Formation and characterization of soy bean oil/surfactant/water microemulsions Marcos Alexandre Polizelli a , Vˆania Regina Nicoletti Telis b , Lia Q. Amaral c , Eloi Feitosa a,∗ a

IBILCE/UNESP, Departamento de F´ısica, Rua Cristovao Colombo, 2265, S˜ao Jos´e do Rio Preto, SP 15054-000, Brazil b UNESP, Departamento de Engenharia e Tecnologia de Alimentos, Campus de S˜ao Jos´e do Rio Preto, SP, Brazil c USP, Instituto de F´ısica, S˜ ao Paulo, SP, Brazil

Received 17 October 2005; received in revised form 22 December 2005; accepted 21 February 2006 Available online 3 April 2006

Abstract Pseudoternary phase diagrams, at 25 ◦ C, were constructed for the systems soy bean oil (SBO)/surfactant/water, with single anionic sodium bis(2ethylhexyl)sulfosuccinate (AOT), nonionic monoolein (MO) and mixtures of these surfactants, showing the isotropic phase of W/O microemulsions (MEs). The area of ME formation in the phase diagrams was shown to be dependent of the relative amount of surfactants, being larger for MO:AOT equals to 2:1. Rheological and dynamic light scattering (DLS) studies indicated that the viscosity of the isotropic ME phase exhibited two different behaviors depending on composition. The viscosity of “dry” MEs initially decreased with increasing amount of water following a dilution line in the phase diagram, i.e., a constant surfactant:SBO percentage ratio. As the water content increased the relative viscosity attained a minimum and then increased. This minimum could be related to the transition between two ME regions, L2 and L2 , having different characteristics. DLS measurements confirm the existence of ordinary W/O ME droplets in the L2 region and suggest the existence of another structure in the L2 region. The size of the MEs droplets in L2 phase ranges from 3.6 to 16.5 nm, depending on composition of SBO, surfactant and water. Small angle X-ray scattering (SAXS) also indicates the existence of structures with different characteristics, for the SAXS curves exhibit a typical micelle asymmetrical peak at low scattering vector q for MEs in L2 but a symmetrical correlation peak at higher q vector in L2 . © 2006 Elsevier B.V. All rights reserved. Keywords: Microemulsions; Reverse micelles; Dry micelles; Phase diagram; Soy bean oil; Surfactant; Rheology; Scattering techniques

1. Introduction Microemulsions (MEs) are homogeneous dispersions of water-in-oil (W/O) or oil-in-water (O/W) droplets stabilized by surfactants (emulsifiers). The MEs are formed spontaneously and exhibit typical physicochemical properties, such as optical transparency and isotropy, low viscosity and thermodynamic stability [1]. The transparency of the MEs is due to the fact that the droplet size ranges from 5 to 100 nm, which is smaller than the wavelength of the visible light, ca 150 nm, and the polydispersity increases with the droplet size [2]. In the last years MEs have received increasing interest in cosmetic and pharmaceuti-

∗

Corresponding author. Tel.: +55 17 3221 22 00; fax: +55 17 3221 22 47. E-mail address: [email protected] (E. Feitosa).

0927-7757/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2006.02.043

cal formulations, as well as in food industries, as systems for drug encapsulation and transport, and allow solubility of high polar molecules. As drug vehicle, MEs have the advantage of being stable and of easy preparation. Owing to the nanometric size, MEs improve transdermal diffusivity of drugs in cutaneous application [3,4]. Owing to the high capability to solubilize a great amount of lipophilic or hydrophilic compounds, together with the huge interfacial area, MEs function as catalysts, increasing the efficacy of chemical reactions. The hydrolysis catalyzed by lipases is an example, where the substrate is lipophilic and the enzyme hydrophilic, and the MEs provide a huge interfacial area to promote the substrate–enzyme contact [1,5,6]. MEs have also attracted the attention because of its capability for selective extraction of biomolecules and metal ions in liquid-liquid systems [7–9] and DNA condensation [10].

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Nomenclature ρ φ ηr a and k AOT DL DLS L2 L2 ME MO O/W SAXS SBO TG V W/O X

density of the compounds volume fraction of the disperse phase relative viscosity parameters of Mooney equation (Eq. (1)) bis(2-etilexil)sodium sulfosuccianate lipodynamic diameter dynamic light scattering isotropic regions of W/O microemulsions isotropic regions of “dry” microemulsions microemulsion monoolein oil-in-water small angle X-ray scattering soybean oil triglyceride volume water-in-oil mass fraction

The capability of enhancing solubility of hydrophilic vitamins, flavors and other additives in W/O MEs is of great interest, since they can provide stabilized moieties for the incorporation of these ingredients and protect them from degradation reactions [11,12]. So far, little effort has been done on the formation of MEs using only food grade materials, where these ME droplets could be used, for example, to solubilize polar additives in typical hydrophobic food products. Garti et al. [13] found a ME phase region in a pseudoternary phase diagram of food compounds, using R-limonene as the oil phase. The commonly used oil in food application is saturated or unsaturated long-chain fatty acid triglycerides (TGs), such as soy bean, corn, cotton, or sunflower oil. However, the bulky TGs are not appropriate for ME formation since they are not good solvent for food grade surfactants [13]. This work aims to find a W/O ME single phase region in pseudoternary phase diagrams of systems containing soy bean oil (SBO) and water, as well as the anionic and nonionic surfactants AOT and MO with potential application in food industry, for example to solubilize and protect (against degradation) hydrophilic food additives (ascorbic acid, thiamin and riboflavin) in a hydrophobic matrix, as well as food microreactor [5]. Another potential application of this ME system may be in cosmetic or pharmaceutical industry [1]. Rheology, dynamic light scattering (DLS) and low angle X-ray scattering (SAXS) were used in order to characterize the obtained W/O MEs.

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Aldrich and Fluka, respectively, and used as received. The used SBO (Cargill) was commercial grade oil. High pure deionized water of Milli-Q quality was used in sample preparations. It is worth of noticing that the used MO contained a 60% impurity fraction composed mainly by di- and triglycerides, similar constituents of SBO. Thus the MO:AOT ratios referred to in this communication in fact are the apparent ratios. The use of such an impure MO has to do with the fact that in food application the low cost of the compounds is a point of major importance. The MO used in this work is solid at the low (fridge) temperature of storage, ca. 5 ◦ C, but liquid at room temperature. 2.2. Phase diagram The phase diagrams were constructed by diluting a mixture of x (percent weight of surfactant) with y (percent weight of SBO), so that x + y = 100 wt%. Microvolumes of water were then added to surfactant:SBO mixtures following the x:y dilution lines in the ternary phase diagrams, and the samples observed by eyes and cross polaroids. The x:y “dry” samples contained no water, y% SBO and x% single AOT or MO, or a mixture of these surfactants. Such a dilution method is convenient for liquid systems [13]. The surfactant:SBO (x:y) mixtures were prepared in culture glass tubes sealed with caps. The x:y samples, for x and y varying from 0 to 100 wt%, were titrated with water and, after each water fraction addition, samples were vigorously shaken by vortexing and left at rest for 24 h to attain equilibrium before analyses. Samples observation and experimental measurements were performed at room temperature (ca. 25 ◦ C). 2.3. Rheology Rheological measurements were made using a Brookfield rotational rheometer, equipped with a cone and plate probe (Brookfield Engineering Laboratories, RVIII model, Stoughton, MA, USA). The instrument was operated with the cone speed ranging from 10 to 250 rpm, at increasing steps of 10 rpm per 10 s. Acquisition and analyses of shear stress and shear rate data were made using the Rheocalc software (Brookfield Engineering Laboratories). A thermostatic bath was used to keep the working temperature at 25 ◦ C. The dependence of the relative viscosity (ηr ) on volume fraction (φ) of the dispersed phase in non-diluted solutions could be described by the Mooney equation (Eq. (1)), which relates ηr with φ [2]: 
aφ (1) ηr = exp 1 − kφ where a and k are constants related, respectively, with the intrinsic viscosity and the interparticle interactions.

2. Materials and methods 2.4. Dynamic light scattering (DLS) 2.1. Materials Sodium bis(2-ethylhexyl)sulfosuccinate (AOT) 99% purity and monoolein (MO) 40% purity were purchased from Sigma-

DLS experiments were performed to obtain the “lipodynamic” diameter, DL , of the W/O ME droplets. The measurements were made using a BI-200SM goniometer (Brookhaven

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Instruments Corporation, Holtsville, NY) and a BI-9000AT digital correlator coupled with a 300 mW laser. The diode-pumped, solid-state laser operating at 532 nm was purchased from Coherent Laser Group (Santa Clara, CA). All measurements were performed at the sample temperature of 25 ◦ C. 2.5. Low angle X-ray scattering (SAXS) The SAXS measurements were made in a conventional Rigaku Denki equipment, with Cu K␣ target in a rotating anode, ˚ wavelength λ, line graphite monochromator defining 1.54 A focus, and image plate detector at 48.5 cm from the sample, conditioned in 1.5 mm glass capillary. Images were analyzed in a scanner STORM 820 from Molecular Dynamics, with the Imagequant software, giving curves corresponding to SAXS profiles as a function of the momentum transfer q = 4π sin θ/λ (2θ being the scattering angle).

Fig. 1. Ternary and pseudoternary phase diagrams for the systems SBO/MO/water and SBO/MO:AOT/water for MO:AOT ratios of 1:1, 2:1 and 3:1. The hachured areas represent the ME monophase.

2.6. Volume fraction The volume fraction φ of the disperse micelle phase is given by Eq. (2): φ=

Vmicellle Vsystem

(2)

where V is the volume of the system. The volume of the continuous phase is the volume of the SBO and TG present as impurity in the MO sample. In terms of density (ρ) and mass fraction (X),
 ρsystem ρsystem φ = 1 − XSBO + XMO 0.6 . (3) ρSBO ρtriglicerdes The densities of MEs and SBO were determined by picnometry, at 25 ◦ C. 3. Results and discussion

indicating that the mixtures of surfactants are more efficient in reducing the interfacial tension of water/SBO than the single surfactants themselves. Fig. 1 also shows the phase diagrams for the MO:AOT composition ratios of 1:1, 2:1 and 3:1. In these cases, the area for the ME monophase are, respectively, 30.2, 31.6 and 24.4% of the whole phase diagram area. Note that the surfactant corner of the phase diagrams represents a ME since the MO used is in the liquid state at room temperature. 3.2. Rheology Rheological investigation was made for the ME isotropic phase of the SBO/MO:AOT/water system, for MO:AOT = 2:1 that exhibits the largest area in the phase diagram. The measurements were made by diluting a “dry” (waterless) ME, following a dilution line (referred to as x:y line). It was observed that all samples exhibit a newtonian behavior. Figs. 2 and 3 show the

3.1. Phase diagrams Samples containing SBO/surfactant/water were prepared in the presence of the anionic AOT or the nonionic MO, or mixtures of these surfactants, and the regions of W/O ME phase were indicated in ternary or pseudoternary phase diagrams. Mixtures of SBO/AOT/water give no ME monophase, most probably due to the quite low surfactant solubility in SBO, which does not reduce sufficiently the water/oil interfacial tension. Precipitates were found in the complete binary AOT/SBO phase diagram. AOT, on the other hand, can form large W/O ME regions with other apolar solvents, such as iso-octane and p-xilene [14,15]. Unlike AOT, MO is completely soluble in SBO, thus being a good emulsifier for this system since one of the main compounds of SBO is the oleic acid (23 wt%) [16]. The SBO/MO/water system exhibits a small region of ME monophase (6.5% the whole phase diagram area), as shown in Fig. 1, which is important to food industry since all components of the MEs are food grade and are not purified reagents, yielding to low cost preparation of the MEs. This area is extended in the presence of AOT,

Fig. 2. Relative viscosity vs. relative water mass for dilution lines 50:50, 60:40 and 70:30 of the phase diagram for ratio MO:AOT = 2:1.

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Fig. 3. Relative viscosity vs. relative water mass for dilution lines 75:25, 85:25, 90:10 and 100:0 of the phase diagram for ratio MO:AOT = 2:1.

effect of the water mass fraction on the relative viscosity ␩r of the MEs for the following surfactant:SBO dilution lines x:y = 50:50, 60:40, 70:30, 75:25, 85:25, 90:10 and 100:0. For every surfactant:SBO ratio, ηr initially decreases to a minimum, and then it increases as the water content increases. The minimum in the viscosity plots suggests that the MEs have different properties in different regions of the phase diagram labeled as L2 and L2 in Fig. 1. Fourth-order polynomials were fitted to the viscosity data and the minima in the plots were asserted to the border between L2 and L2 , as indicated in Fig. 1. A simple calculation based on the composition of the surfactant (AOT plus MO) and water molecules present around the L2 –L2 border in the phase diagram, reveals that the transition from L2 to L2 occurs when there is about 4.7 ± 0.9 water molecules per surfactant molecule. This value is in very good agreement with those reported in the literature for the maximum bound water [17] per surfactant molecule. In L2 , the increase in viscosity is related to the increase in the volume fraction of the reverse micelles (dispersed phase) that favors the contact and friction between micelles during the flow. Figs. 2 and 3 also show that in the L2 region and in the absence of water, ηr increases with the surfactant:SBO ratio. An opposite behavior is observed for the L2 region due to a decrease in the micelle size, as confirmed by DLS data shown below. For the L2 region the viscosity was plotted as a function of the micelle volume fraction, and the data are shown in Fig. 4 for the dilution lines x:y = 80:20, 85:15 and 90:10. The solid lines in Fig. 4 indicate the fitting curves according to the Mooney equation (Eq. (1)) to the experimental data. The fitting parameters are summarized in Table 1. The intermicellar interaction parameter k increases with the surfactant:SBO ratio. That is, the intermicellar interaction increases with the volume fraction of the micelles. The parameter a, which is related to intrinsic viscosity, decreases with the surfactant:SBO ratio due to a decrease in the micelle size. It can be due to an increase in the surfactant content that raises the water/SBO interfacial area per volume, yielding to the formation of smaller micelles with larger specific interfacial

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Fig. 4. Relative viscosity as function of micelle volume fraction for the SBO/MO:AOT (2:1)/water system, and dilution line 80:20, 85:15 and 90:10, as shown.

area. The fitting parameter r2 is close to unit, indicating a good fitting curve to the experimental data. The viscosity in the L2 region exhibits an opposite behavior to that predicted by the Mooney equation, possibly due to changes in the aggregate structure, as discussed below. 3.3. Dynamic light scattering (DLS) Dynamic light scattering measurements for selected samples from the L2 and L2 regions were performed. Fig. 5 shows a typical DLS curve for the SBO/MO:AOT (2:1)/water system, dilution line x:y = 85:15, and mass fraction of water Xw = 0.19. The curve is single modal and the peak is rather narrow, indicating that the sample has low polydispersity. Table 2 summarizes the effect of sample composition on the “lipodynamic” diameter DL and polidispersity of the reverse micelles in L2 . Following a dilution line x:y, the micelle DL increases with the water content, indicating that the MEs are of W/O type. It also can be seen that the micelle size decreases when the surfactant:SBO ratio increases. These results are in agreement with the reological results, indicating that the intrinsic viscosity of the micelles decrease when the surfactant:SBO ratio increases. For samples in the L2 region the correlation function does not decay to zero and DL could not be estimated, since it could not assume any stabilized value within the micelle range of size. Table 1 Parameters obtained from the fitting curves for the the viscosity vs. micelle volume fraction data using the Mooney relation (Eq. (1)) MO:AOT

k

a

r2

80:20 85:15 90:10

0.60 0.88 0.90

1.85 1.31 1.09

0.999 0.998 0.999

r2 equals unity indicate very good fitting curves.

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Fig. 5. Typical DLS curve for MEs in the SBO/MO:AOT (2:1)/water system, and dilution line 85:15 and Xw = 0.19. Table 2 Mean lipodynamic diameter of W/O ME droplets in the L2 phase for the SBO/MO:AOT (2:1)/water system MO:AOT

Water content (wt%)

Mean diameter (nm)

Polidispersity

75:25

15 20 25

4.5 7.4 16.5

0.70 0.47 0.70

85:15

19 22 26

3.6 3.7 5.4

0.58 0.47 0.34

20:80

2

13.9

0.28

3.4. Small angle X-ray scattering (SAXS) Figs. 6 and 7 show the SAXS curves for the surfactant:SBO percent ratios (dilution lines x:y) 75:25 and 85:15, for the surfac-

Fig. 7. SAXS curves for the SBO/MO:AOT (2:1)/water system and samples in the L2 (c) and L2 (b) phases, prepared for fixed 85:15 dilution lines and increating amount of water, as shown. As reference it is shown the curve for pure SBO.

tant fraction MO:AOT equals 2:1 and selected amount of water, so that the samples lies either in the L2 or L2 phase, as shown in the Figure legends. For comparison the curve for pure SBO is also shown. The curves exhibit a single broad band centered at a q* value, but with different behavior in the L2 and L2 regions. ˚ −1 are asymmetric, with higher In L2 the peaks at q* ≈ 0.1 A intensities on the lower q side of the curves, both for 20% water on the 75:25 surfactant:SBO percent ratio (Fig. 6) and for 22% water on the 85:15 ratio (Fig. 7). Such curves display the typical asymmetric profile expected for spherical micelle droplets, with a peak due to contrast between the polar heads and both water and paraffin, and an increase in the smaller q region related to the micelle form [18]. In L2 , the curves instead, are symmetric around the peak q values and can be interpreted in terms of a characteristic correlation distance d = 2␲q*−1 in the range of ˚ A possible origin for this peak is the dry ME structure, 29–36 A. as discussed below. 3.5. The region L2 of dry MEs

Fig. 6. SAXS curves for the SBO/MO:AOT (2:1)/water system and samples in the L2 (d) and L2 (b and c) phases, prepared for fixed 75:25 dilution lines and increasing amount of water, as shown. The curve for pure SBO (a) is shown for comparison.

The possible aggregate structures present in the L2 region will be discussed based on the results here obtained as well as those available in the literature for similar systems. It has been reported that for the binary AOT/water system, in presence of reduced water content, the following phase sequence is observed: lamellar, cubic, inverted hexagonal [1,19]. Cylindrical (hexagonal) structures are therefore formed by dry (waterless) AOT. Pure MO, however, forms lamellar crystals in up to 4 wt% ˚ and hydration water, with a bilayer repetition distance of 49.3 A at higher water content and temperatures, a lamellar liquid crystalline phase is formed [20]. It has been reported that the system AOT/ciclohexane/water also forms dry cylindrical reverse micelles with radius of ˚ [21]. The cylinders length however decreases when ∼7.5 A water is added, and the structure becomes W/O droplets when there are ∼5 water molecules per surfactant molecule, in good

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Fig. 8. Schematic models for a dry (a) and a hydrated (b) AOT–MO aggregate having SBO as solvent.

agreement with the value (4.7) here reported for the system SBO/AOT:MO/water. The spherical droplets reported by Steytler et al. [21] resemble those in the L2 phase of the SBO/AOT:MO/water system, but that system does not present the viscosity minimum, which characterizes the L2 region reported here. The insolubility of AOT in SBO evidences that the long chain MO molecules are crucial to mediate the packing of AOT with TG molecules. The repetition distance seen ˚ is too large to be assoby SAXS in the L2 region (29–36 A) ciated to the distance between dry AOT polar head groups in a cylinder, and too small to be associated to the lamellar repetition distance with a bilayer of long chain molecules as MO or TG. The SAXS results were obtained for samples with approximately 1:1:1 AOT/MO/TG molar ratio (by including the MO impurities of SBO). It seems reasonable to propose from the SAXS results a “local lamellar” structure, but with interdigitated monolayers. Fig. 8a shows a sketch of a model suggested for the dry lamellar structure in L2 , in which MO places its head group near the AOT polar head group and chains from all the molecules are inside the bilayer. Compatibility with the SAXS repetition distance is achieved with MO molecules of the two monolayers in mirror position, as displayed in Fig. 8a; this might represent a high entropic cost and the reason for the “local” character of such structure. Alternatively, if MO chains exhibit aleatory distribution, the more flexible TG molecules could fill the empty spaces. It should be stressed that such a “locally lamellar” structure must be distorted, since it does not form a liquid crystalline lamellar phase with long range order. The polar heads of AOT and MO may be connected trough a net of dry hydrogen bonds.

Such a dry structure remains as long as added water molecules are bound to the surfactant polar head groups to form quasidry MEs with “inner bound water”, and as a consequence the relative viscosity is reduced. The minimum in viscosity might correspond to the transition from bound water to free water in the interior of the MEs to start formation of water droplets or ordinary W/O (Fig. 8b). The model we propose for MEs in L2 is compatible with our experimental data reported here, but a better understanding of the structure present in the L2 region requires a more detailed investigation, that is under way, but beyond the scope of this communication. 4. Concluding remarks The formation of MEs in mixtures of SBO and water requires the use of one or more surfactant molecules (emulsifier). A mixture of surfactants may be required to form ME monophases in the SBO–surfactant–water system. In order to obtain ME monophases in this system we have tried the well-known food grade surfactants AOT and MO, with MO being widely used in food industry while the AOT usage being restricted to some types of food and amount of the surfactant. Thus AOT does not form single ME phase whereas MO forms a rather small ME monophase that corresponds to only 6.5% the area of the whole phase diagram. This ME area is augmented when appropriate mixtures of MO/AOT is used. For MO:AOT equals 1:1, 2:1 and 3:1, the ME areas are respectively 30.2, 31.6 and 24.4% of the total area of the phase diagram. Thus the

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system containing ratio of 2:1 MO:AOT exhibits the largest ME area. An important aspect of this investigation is that the ME monophase in the SBO/MO:AOT/water system consists of two ME regions (we referred to as L2 and L2 ) having different rheological and structural characteristics (see Fig. 1 for MO:AOT equals 2:1). The difference in these ME characteristics may be related to the effect of water on the ME structures. In L2 , there is only water bound to the surfactant polar head groups in a “locally lamellar” structure, whereas in L2 spherical droplets of water occupy the inner core of the reverse micelles, with the L2 –L2 transition corresponding to the viscosity minima where this structural transition occurs. Acknowledgments FAPESP foundation gave financial support through grant 03/04478-0 (PhD of MAP) and grant 01/11721-3 (Tematic Project). We also thank Dr. Ana L´ucia Barreto Penna (UNESP) and Dr. Maria Elisabete Darbello Zaniquelli (USP) for kindly supplying the rheometer and scattering equipments, respectively. A useful remark of Dr. W. Loh regarding details of the possible dry structure is also acknowledged. References [1] B. J¨onsson, B. Lindman, K. Holmberg, B. Kronberg, Surfactants and Polymers in Aqueous Solution, John Wiley & Sons, Chichester, 1998. [2] S.P. Moulik, B.K. Paul, Structure, dynamics and transport properties of microemulsions, Adv. Colloid Interf. Sci. 78 (1998) 99–195. [3] L. Djordjevic, M. Primorac, M. Stupar, D. Krajisnik, Characterization of caprylocaproyl macrogolglycerides based microemulsion drug delivery vehicles for an amphiphilic drug, Int. J. Pharm. 271 (2004) 11–19. [4] A.C. Sintov, L. Shapiro, New microemulsion vehicle facilitates percutaneous penetration in vitro and cutaneous drug bioavailability in vivo, J. Control. Rel. 95 (2004) 173–183. [5] N. Garti, Microemulsions as microreactors for food applications, Curr. Opin. Colloid Interf. Sci. 8 (2003) 197–211. [6] A. Lif, K. Holmberg, Chemical and enzymatic ester hydrolysis in a Winsor I system, Colloids Surf. A: Physicochem. Eng. Aspects 129–130 (1997) 273–277.

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