Nanoemulsions Prepared by a Two-Step Low-Energy Process

May 20, 2008 - Ian A. Fallis , Peter C. Griffiths , Terence Cosgrove , Cecile A. Dreiss , Norman Govan , Richard K. Heenan , Ian Holden , Robert L. Je...
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Langmuir 2008, 24, 6092-6099

Nanoemulsions Prepared by a Two-Step Low-Energy Process Lijuan Wang,† Kevin J. Mutch,‡ Julian Eastoe,‡ Richard K. Heenan,§ and Jinfeng Dong*,† College of Chemistry and Molecular Science, Wuhan UniVersity, Wuhan 430072, China, School of Chemistry, UniVersity of Bristol, Bristol BS8 1TS, U.K., and ISIS-STFC, Rutherford Appleton Laboratory, Chilton, Oxon OX11 0QX, U.K. ReceiVed February 27, 2008. ReVised Manuscript ReceiVed March 20, 2008 A simple low-energy two-step dilution process has been applied in oil/surfactant/water systems with pentaoxyethylene lauryl ether (C12E5), dodecyldimethylammonium bromide, sodium bis(2-ethylhexyl)sulfosuccinate, sodium n-dodecyl sulfate-pentanol, and hexadecyltrimethylammonium bromide-pentanol. Appropriate formulations were chosen for the concentrate to be diluted with water to generate oil-in-water (O/W) emulsions or nanoemulsions. For the system of decane/C12E5/water, bluish, transparent nanoemulsions having droplet radii of the order of 15 nm were formed, only when the initial concentrate was a bicontinuous microemulsion, whereas opaque emulsions were generated if the concentrate began in an emulsion-phase region. Nanoemulsions generated in the system decane/C12E5/water have been investigated both by dynamic light scattering (DLS) and contrast-variation small-angle neutron scattering (SANS). The SANS profiles show that nanodroplets exist as spherical core-shell (decane-C12E5) particles, which suffer essentially no structural change on dilution with water, at least for volume fractions φ down to 0.060. These results suggest that the nanoemulsion droplet structure is mainly controlled by the phase behavior of the initial concentrate and is largely independent of dilution. A discrepancy between apparent nanoemulsion droplet sizes was observed by comparing DLS and SANS data, which is consistent with long-range droplet interactions occurring outside of the SANS sensitivity range. These combined phase behavior, SANS, and DLS results suggest a different reason for the stability/instability of nanoemulsions compared with earlier studies, and here it is proposed that a general mechanism for nanoemulsion formation is homogeneous nucleation of oil droplets during the emulsification.

1. Introduction Nanoemulsions1 consist of small quite monodisperse droplets, typically in the 20-200 nm size range. Although they are of similar size to microemulsion droplets (∼1-100 nm), and appear transparent or translucent, they are in fact distinctly quite different from true microemulsions. Nanoemulsions are thermodynamically unstable, and their formation generally requires energy input. The properties of nanoemulsions depend not only on thermodynamic conditions (i.e., composition, temperature, and pressure) but also on the preparation method, and, crucially, on the order of component addition.2 However, the kinetic stability of nanoemulsions, and their transparent or translucent appearance due to the presence of nanometer-sized droplets, makes nanoemulsions of interest for fundamental studies and practical applications (e.g., in chemical, pharmaceutical,3 and cosmetic4 fields). To obtain nanodroplet emulsions, significant amounts of mechanical energy are needed, making high-energy preparation methods unfavorable for industrial applications.5,6 Therefore, the preparation of nanoemulsions with reproducible properties * To whom correspondence should be addressed. E-mail: colloid@ whu.edu.cn. † Wuhan University. ‡ University of Bristol. § Rutherford Appleton Laboratory. (1) Forgiarini, A.; Esquena, J.; Gonza´lez, C.; Solans, C. Langmuir 2002, 17, 2076–2083. (2) Esquena, J.; Solans, C. Prog. Colloid Polym. Sci. 1998, 110, 235–239. (3) Nicolaos, G.; Crauste-Manciet, S.; Farinotti, R.; Brossard, D. Int. J. Pharm. 2003, 263, 165–171. (4) Sonneville-Aubrun, O.; Simonnet, J.-T.; L’Alloret, F AdV. Colloid Interface Sci. 2004, 108-109, 145–149. (5) Liedtke, S.; Wissing, S.; Muller, R. H.; Mader, K. Int. J. Pharm. 2000, 196, 183–185. (6) Shi, R.; Hong, L.; Wu, D.; Ning, X.; Chen, Y.; Lin, T.; Fan, D.; Wu, K. Cancer Biol. Ther. 2005, 4, 218–224. (7) Solans, C.; Izquierdo, P.; Nolla, J.; Azemar, N.; Garcia-Celma, M. J. Curr. Opin. Colloid Interface Sci. 2005, 10, 102–110. (8) Pons, R.; Carrera, I.; Caelles, J.; Rouch, J; Panizza, P. AdV. Colloid Interface Sci. 2003, 106, 129–146.

and small droplet sizes using low-energy methods has been a field of growing interest.7,8 Low-energy methods7 make use of accessible phase transitions occurring during the emulsification process as a result of changes in surfactant film spontaneous curvature. This curvature transition has been achieved through different routes: (a) partitioning of alcohol from the oil to the aqueous phase or diffusion of water into the initial droplet, both producing a shift from lipophilic to hydrophilic conditions;9,10 (b) chemical reactions which convert lipophilic surfactants to hydrophilic surfactants;11,12 (c) a sudden decrease of ionic strength with ionic surfactant systems;13,14 and (d) an increased hydration of poly(oxyethylene) chains of PEOtype nonionic surfactants.15–22 The reversal of spontaneous curvature from water-in-oil (W/O) to an oil-in-water configuration (O/W) reduces the solubilization capacity for oil to such an extent that supersaturation may occur, leading to oil droplet nucleation. (9) Rang, M. J.; Miller, C. A. J. Colloid Interface Sci. 1999, 209, 179–192. (10) Rang, M. J.; Miller, C. A. Prog. Colloid Polym. Sci. 1998, 109, 101–117. (11) Nishimi, T.; Miller, C. A. J. Colloid Interface Sci. 2001, 237, 259–266. (12) Sole`, I.; Maestro, A.; Pey, C. M.; Gonza´lez, C.; Solans, C.; Gutie´rrez, J. M. Colloids Surf., A 2006, 288, 138–143. (13) Nishimi, T.; Miller, C. A. Langmuir 2000, 16, 9233–9241. (14) (a) Bataller, H.; Lamaallam, S.; Lachaise, J.; Graciaa, A.; Dicharry, C. J. Mater. Process Tech. 2004, 152, 215–220. (b) Lamaallam, S.; Bataller, H.; Dicharry, C.; Lachaise, J. Colloids Surf., A 2005, 270-271, 44–51. (15) Leaver, M. S.; Olsson, U.; Wennerstro¨m, H. J. Chem. Soc., Faraday Trans. 1995, 91, 4269–4274. (16) Izquierdo, P.; Esquena, J.; Tadros, Th. F.; Dederen, C; Garcia, M. J.; Azemar, N.; Solans, C. Langmuir 2002, 18, 26–30. (17) Morales, D.; Gutie´rrez, J. M.; Garcı´a-Celma, M. J.; Solans, C. Langmuir 2003, 19, 7196–7200. (18) Izquierdo, P.; Esquena, J.; Tadros, Th. F.; Dederen, J. C.; Feng, J.; GarciaCelma, M. J.; Azemar, N.; Solans, C. Langmuir 2004, 20, 6594–6598. (19) Izquierdo, P.; Feng, J.; Esquena, J.; Tadros, Th. F.; Dederen, J. C.; GarciaCelma, M. J.; Azemar, N.; Solans, C J. Colloid Interface Sci. 2005, 285, 388–394. (20) Morales, D.; Solans, C.; Gutie´rrez, J. M.; Garcı´a-Celma, M. J.; Olsoson., U. Langmuir 2006, 22, 3014–3020. (21) Forgiarini, A.; Esquena, J.; Gonza´lez, C.; Solans, C. Prog. Colloid Polym. Sci. 2000, 115, 36–39. (22) Forgiarini, A.; Esquena, J.; Gonza´lez, C.; Solans, C. Prog. Colloid Polym. Sci. 2001, 118, 184–189.

10.1021/la800624z CCC: $40.75  2008 American Chemical Society Published on Web 05/20/2008

Nanoemulsions Prepared by a Two-Step Process

In these cases, emulsions can be generated spontaneously. Therefore, the low-energy methods can be considered to generate emulsions with small droplet sizes through this “spontaneous emulsification”. Particular attention has been directed to nonionic surfactants, for which PEO hydration can be changed both by temperature (phase inversion temperature (PIT) method)16–20 and composition (emulsion inversion point (EIP) method).1,21,22 With the PIT method, emulsions stabilized by appropriate nonionic surfactants are obtained by rapid temperature changes passing through the hydrophile-lipophile balance (HLB) temperature. Nanoemulsions can be obtained only when the oil is completely dissolved in a single phase prior to the nanoemulsification.16–20 The formation mechanism has been investigated by 1H-pulsed-fieldgradient-spin-echo NMR (PFGSE-NMR), indicating that a thermally induced disruption of a bicontinuous microemulsion is necessary to generate nanoemulsion droplets.19 In the EIP method, water is added dropwise to a mixture of surfactant and oil at constant temperature.1,12,21,22 The formation of nanoemulsions is generally attributed to phase instabilities during emulsification, where the presence of lamellar crystallites and/or bicontinuous microemulsions are thought to play critical roles.1,21,22 However, since the systems may pass through various different phases on the route to nanoemulsion formation, it is far from clear which phase (if any) is key. Nanoemulsions (so-called miniemulsions) stabilized by ionic surfactants have also been prepared by dilution of microemulsions with excess water.8,14,23,24 In the work reported by Pons et al.8, nanoemulsions can be obtained either by dilution of a bicontinuous, a O/W microemulsion, or multiphase mixtures with water contents intermediate between the two microemulsion regions. The flexibility of the surfactant film, its affinity for water, and the interfacial tension were suggested as important factors in the formation of nanoemulsions in two complex systems studied by Bataller et al.14 However, no systematic study into the mechanism of nanoemulsion formation has been conducted for ionic surfactant systems. Therefore, further effort is required in order to better understand the mechanisms of nanoemulsion formation under isothermal conditions, with both nonionic and ionic surfactants, this will help in optimizing nanoemulsification processes for industrial and research applications. An isothermal two-step process, which requires no heat or energy input, has recently been demonstrated to generate nanoemulsions with an industrially relevant system composed of methyl decanoate/technical grade surfactant poly(oxyethylene) 7-lauryl ether AEO-7/water.25 In this system, initial concentrates with different water weight fractions were rapidly diluted with water to reach the same final composition. Although the oil and surfactant are industrial grade, these previous results25 suggest a relationship between the formation of nanoemulsions and the initial concentrate equilibrium-phase behavior. Bluish transparent O/W nanoemulsions with a narrow size distribution were formed only when the concentrate was located in a microemulsion (bicontinuous and/or oil-in-water) region. The formation of O/W nanoemulsions was attributed to homogeneous nucleation of oil from the microemulsion phase upon dilution. The use of this two-step process allow not only generation of nanoemulsions, but also improved understanding of the mechanism of nanoemulsion formation. Therefore, this low-energy two-step process was employed in the present work for several academically relevant systems, (23) Taylor, P.; Ottewill, R. H. Colloids Surf., A 1994, 88, 303–316. (24) Taylor, P.; Ottewill, R. H. Prog. Colloid Polym. Sci. 1994, 97, 199–203. (25) Wang, L. J.; Li, X. F.; Zhang, G. Y.; Dong, J. F.; Eastoe, J. J. Colloid Interface Sci. 2007, 314, 230–235.

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formulated from research grade components and well-known surfactants: decane/pentaoxyethylene lauryl ether (C12E5)/water, decane/dodecyldimethylammonium bromide (DDAB)/water, decane/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/water (with 100 mM NaCl), dodecane/sodium n-dodecyl sulfate (SDS)/ pentanol/water, and dodecane/hexadecyltrimethylammonium bromide (CTAB)/pentanol/water. In this work, both contrast variation small-angle neutron scattering (SANS) and dynamic light scattering (DLS) were employed to gain insight into the structure and stability mechanisms of model nanoemulsions formulated from decane/C12E5/water mixtures at 25 °C. SANS has been used previously to study silicone oil-in-water nanoemulsions, which were generated by a high-energy extreme shear method and stabilized by the anionic surfactant SDS, over a range of nanoemulsion volume fractions (0.008 < φ < 0.6).26 As φ increases, the primary structure factor peak increases in size, indicating stronger interactions and deformation of the droplets at high φ above the jamming point. The systems studied here contrast with those published previously19,26 in three essential ways: (a) they are formed by a low-energy dilution method, rather than PIT method19 or high-energy shear;26 (b) a direct method SANS was used to gain insight into the equilibrium structure and stability mechanisms of the resultant nanoemulsions, rather than the “dynamic” 1H-PFGSE-NMR technique;19 and (c) nonionic, rather than ionic, surfactants have been used,26 minimizing electrostatic structure factor effects which could complicate the detailed analyses of SANS data. Therefore this new work contributes to the studies on nanoemulsions in three important ways: (1) the generality of the low-energy dilution method has been explored for systems with ionic, as well as nonionic surfactants; (2) internal droplet structures of nanoemulsions can be assigned with better confidence, based on model fitting of contrast variation SANS data; and (3) studies with complementary scattering techniques SANS and DLS, both sensitive to different relevant length scales, provide new insight into the instability mechanism, suggesting it is flocculation dominated, at least up to 4 h after the initial nanoemulsion preparation.

2. Experimental Section 2.1. Materials. C12E5 (>98% purity), AOT (>98% purity), and DDAB (98% purity) were purchased from Sigma-Aldrich, and SDS (99% purity) and CTAB (98% purity) from Alfa Aesar. n-Decane (Alfa Aesar, >99% purity), n-decane-d22 (Fluka, >99% D atom), n-dodecane (Aldrich, >99% purity), 1-pentanol (Aldrich, >99% purity), and D2O (Aldrich, 99.9% D atom) were used as received. Milli-Q ultrapure water with resistivity no less than 18.4 MΩ cm was used. 2.2. Phase Diagrams. All components were weighed, sealed in ampules, and homogenized with a vibromixer. The samples were equilibrated at 25 °C. Optically anisotropic liquid crystalline phases were identified by using polarizing light microscopy (OptipHot-2, Nikon, Japan). The boundary lines were found by consecutive addition of one component to mixtures of the other components. 2.3. Preparation Method. Emulsions were prepared for several different systems. For the decane/C12E5/water system, the partial ternary phase diagram is shown in Figure 1, and sample preparation pathways, outlined below. The nanoemulsion volume fraction, given by the oil volume fraction plus the surfactant volume fraction (φ ) φdecane + φC12E5) was varied from 0.006 to 0.120. Two formulations A (φdecane ) 0.020, φC12E5 ) 0.010) and B (φdecane ) 0.017, φC12E5 ) 0.013) were chosen as the final systems. There are two important composition parameters in the work, the overall droplet volume (26) (a) Graves, S.; Meleson, K.; Wiliking, J.; Lin, M. Y.; Mason, T. G J. Chem. Phys. 2005, 122, 134703/1–134703/6. (b) Mason, T. G.; Graves, S.; Wiliking, J. N.; Lin, M. Y. J. Phys. Chem. B 2006, 110, 22097–22102.

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Figure 1. Partial ternary phase diagram of the decane/C12E5/water system at 25 °C. Samples A and B, generated from A5 and B5, respectively, are shown in the inset photographs. The preparation protocol II is outlined in section 2.3. (Om, inverse micellar solution or W/O microemulsion; Lc, optically anisotropic phase; MLc, multiphase region including liquid crystal phase; ME, bicontinuous microemulsion or O/W microemulsion; Em, multiphase region).

fraction φ (defined above) and the weight fraction of water in the initial concentrate w. Note, with reference to Figure 1, sample A ends up in an emulsion region, whereas sample B, at slightly higher surfactant concentration, is in a microemulsion region. The following low-energy dilution pathways were followed to arrive at a suite of samples A and B: (I) the one-step process, where components needed to generate final compositions A and B were mixed by gentle magnetic stirring at 25 ( 0.1 °C (LTD 6G, Grant, England); (II) the two-step process, where, first, oil, surfactant, and an appropriate amount of water were mixed to generate a sequence of concentrates containing water weight fractions w ) 0, 0.1, 0.2, . . . , 0.9 (these concentrates are marked in Figure 1 and denoted as A0, A1, A2, . . . , A9 and B0, B1, B2, . . . , B9, respectively), and then, a certain amount of one of these concentrates was injected into an appropriate amount of water under gentle stirring to achieve the final composition, either A or B. As indicated in Figure 1 the dilution steps were arranged so that A0 w A, A1 w A, A2 w A, . . . , A9 w A and B0 w B, B1 w B, B2 w B, . . . , B9 w B. The temperature was kept constant at 25 ( 0.1 °C. Other systems with different ionic surfactants, decane/DDAB/ water,27 decane/AOT/water (100 mM NaCl),28 dodecane/SDS/ pentanol/water,29 and dodecane/CTAB/pentanol/water,29 were formulated to prepare emulsions by the two-step dilution process as described above. The appropriate formulations were chosen as concentrates from literature27–29 and then injected into water to yield emulsions with final volume fraction of φ ) 0.030. 2.4. Dynamic Light Scattering. DLS (λ ∼ 500 nm) is commonly employed to study droplet size distributions in emulsions and nanoemulsions.1,12–25 Analysis of the autocorrelation function decay obtained by DLS30 from a dilution series of nanodroplets yields information on the effective diffusion coefficient D. In the limit of infinite dilution, the apparent hydrodynamic radius rh can be estimated from

rh )

kT 6πηD

(1)

where η is the viscosity of the medium. The apparent hydrodynamic radius includes solvation effects. DLS experiments were carried out using a Malvern 4800 Autosizer (Malvern Instruments, England). In self-assembling systems such as emulsions, nanoemulsions, and microemulsions, DLS is particularly sensitive to colloidal (27) Blum, F. D.; Pickup, S.; Chen, S. J.; Evans, D. F. J. Phys. Chem. 1985, 89, 711–713. (28) Chen, C. H.; Chang, S. L.; Strey, R.; Samseth, J.; Mortensen, K. J. Phys. Chem. 1991, 95, 7427–7432. (29) Sripriya, R.; Muthu Raja, K.; Santhosh, G.; Chandrasekaran, M; Noel, M. J. Colloid Interface Sci. 2007, 314, 712–717.

interactions on a length scale of 2-1000 nm; hence, the apparent “size” can become obscured if the samples are highly polydisperse, insufficiently dilute, or experiencing attractive/repulsive interactions. Hence, there are potential limitations to structural studies of nanoemulsions by DLS alone. 2.5. Small-Angle Neutron Scattering Experiments. In comparison to DLS, SANS covers length scales which are associated with the internal structure of nanoemulsion droplets (∼1-10 nm).31 An additional advantage of SANS over DLS is that “contrast variation” can be used to obtain detailed structural information on the droplets by selective deuteration of the various components. The time-of-flight LOQ instrument at ISIS, U.K., was used; the data acquisition, treatment, and normalization procedures have been outlined elsewhere.32–34 Samples were held in Hellma quartz cells and thermostated at 25 °C. Following standard procedures, raw data were treated to yield normalized scattering intensities I(Q) in cm-1, where the momentum transfer is related to the scattering angle, θ, by Q ) (4π/λ) sin(θ/2). The observed Q range was 0.007-0.23 Å-1. Scattering data were also corrected for wavelength-dependent transmission factors, as well as cell, background, and any incoherent scattering. The overall nanoemulsion “drop” contrast was generated with decane-h/C12E5-h/D2O, and the external surfactant “shell” contrast was highlighted with decane-d/C12E5-h/D2O. Data were analyzed using standard Guinier limiting laws, Porod analysis, and the multimodel FISH fitting program.35 Details of these analyses can be found in the Supporting Information.

3. Results and Discussion 3.1. Samples Prepared by Methods I and II for the Decane/ C12E5/Water System. The partial ternary-phase diagram of the system decane/C12E5/water at 25 °C is shown in Figure 1. As can be seen from the figure, samples A and B with volume fraction φ ) 0.030 are located in the emulsion (A) and microemulsion regions (B) of the phase diagram, respectively. As the actual phase behavior is quite complicated,15 the exact (30) Pecora, R. Dynamic Light Scattering; Plenum: New York, 1985. (31) Eastoe, J. Surfactants; Wuhan University Press: Wuhan, China, 2005; pp 96-134. (32) Dupont, A.; Eastoe, J.; Murray, M.; Martin, L.; Guittard, F.; Givenchy, E. T.; Heenan, R. K. Langmuir 2004, 20, 9953–9959. (33) Eastoe, J.; Dominguez, M. S.; Wyatt, P.; Orr-Ewing, A. J.; Heenan, R. K. Langmuir 2004, 20, 6120–6126. (34) Summers, M.; Eastoe, J.; Davis, S.; Du, Z.; Richardson, R.; Heenan, R. K.; Steytler, D. C.; Grillo, I. Langmuir 2001, 17, 5388–5397. (35) Heenan, R. K. Fish Data Analysis Program; Report RAL-89-129, Rutherford Appleton Laboratory, CCLRC: Didcot, U.K., 1989.

Nanoemulsions Prepared by a Two-Step Process

Figure 2. Appearance of A and B prepared by method I at different times.

Figure 3. Droplet radii for samples A (0) and B ()) with volume fraction φ of 0.030 at 25 °C as a function of w, the weight fraction of water in the initial concentrate. Inset: appearance of emulsion diluted from different concentrates.

microstructures of the liquid crystal phases have not been fully identified. The appearance of samples A and B prepared by the gentle stirring method I, at different times, are shown in Figure 2. It can be seen that a coarse, white emulsion was obtained by gentle stirring for sample A (emulsion region). On the other hand sample B became transparent over time, which is consistent with eventual microemulsion formation, as expected on the basis of the phase diagram shown in Figure 1. Samples A and B prepared by method I were studied by DLS 24 h after preparation. System A is a white coarse emulsion with radii ∼ 290 nm, whereas the transparent system B gave rh ) 9.3 nm, exactly the same as for an equivalent B sample but formulated by the dilution method II (see below). The apparent droplet size for the presumed microemulsion sample B was constant over 6 months. Samples A and B prepared by method II were investigated by DLS. The relationship between the final droplet radius and the water weight fraction w in the concentrate is shown in Figure 3. It can be seen that, with system A, the droplet radii vary with water level w in the concentrates. Bluish transparent nanoemulsions with droplet radii as low as about 15 nm were obtained on dilution of concentrates with w ) 0.4 and 0.5 (A4 and A5; see Figure 1). For concentrates with w less than 0.3 (A0-A3; refer to Figure 1) or more than 0.6 (A6-A9; refer to Figure 1), white coarse emulsions with radii larger than 100 nm were obtained. On the other hand, the droplet radii for systems B remain essentially constant at about 9 nm, regardless of water level in the concentrate. It can be seen that microemulsions form spontaneously, and the scattering and visual properties are independent of the preparation pathway. However, appearance and the droplet radii of the emulsions from pathway A are clearly

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dependent on the preparation procedure, and crucially the water level in the concentrate. Finally, comparing the DLS results for system A, prepared by the two separate routes I and II (rh ) 290 and 15 nm if generated from A5), shows that different states are achieved. This indicates that the formulation pathway to “nanoemulsion” A is an important factor. 3.2. Relationship between the Formation of Nanoemulsions and Equilibrium Phase Behavior in the Decane/C12E5/Water System. Electrical conductivity measurements on concentrates of systems A with initial water weight fractions w in the range 0.34-0.57 are consistent with the presence of bicontinuous microemulsions (see Supporting Information). It can be seen from Figure 1 that concentrates A4 and A5 are located in the bicontinuous microemulsion region, while other concentrates are located in different regions of the phase diagram. Nanoemulsions with small droplet radii are formed only when the concentrate starts as a bicontinuous microemulsion, with w of 0.4 and 0.5 (samples A4 and A5) (Figure 3). This suggests a close relationship between the equilibrium phase behavior of the initial concentrate and the final droplet radius of the resulting emulsions after they have been diluted to achieve A. These different phases are consistent with changes in the surfactant hydration and solubility of decane as a function of dilution. It is well-known that the preferred curvature of nonionic surfactant layers depends on hydration of the PEO, which with surfactant-rich concentrates can be increased by dilution with water15 (EIP method). At low water contents the preferred curvature may be around water, generating reverse W/O structures. With increased water dilution, hydration of the PEO headgroups may drive the curvature about oil to favor O/W phases. The solubilization capacity for oil is decreased during the phase transition, leading to local supersaturation and then oil droplet nucleation: therefore, spontaneous emulsification occurs. For low water level systems (A0-A3), oil is the continuous phase in these initial concentrates. The existing oil droplets may act as nuclei and trigger heterogeneous nucleation,36 which result in droplets with larger sizes and polydispersity. On the other hand in concentrates A4 and A5, the oil was completely solubilized in a bicontinuous microemulsion phase (see Supporting Information). The dilution of these concentrates with excess water converts them to oil-in-water systems and decreases the concentration of surfactants, which also inevitably leads to a decrease in oil solubilization. Similar to concentrates A0-A3, these systems became supersaturated in oil, leading to nucleation of oil. The point is that supersaturation in the single phase results in homogeneous nucleation, which favors the formation of smallsized oil droplets with narrow polydispersity.36,37 Considering concentrates located in a region with higher water, the surfactant layer is saturated with water and the mean curvature can be considered as water concentration independent.15 Concentrates A6-A9 are located in the emulsion (Em) region, in which the oil droplets coexist with other phase(s). Highly polydisperse emulsions were obtained by dilution of water because no curvature (structural) change is induced by added water; the emulsion just become less concentrated. Therefore, nanoemulsions were obtained by dilution of bicontinuous microemulsion, as a result of homogeneous nucleation during a spontaneous emulsification. These results are consistent with previous reports that nanoemulsions were (36) Vincent, B.; Kiraly, Z.; Obey, T. M. In Modern Aspects of Emulsion Science; Binks, B. P., Ed.; The Royal Society of Chemistry: Cambridge, U.K., 1998; pp 100-114. (37) Morris, J.; Olsson, U.; Wennerstro¨m, H. Langmuir 1997, 13, 606–608.

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obtained only when the oil was completely dissolved in a single phase prior to the nanoemulsification through the PIT method.16–20 Furthermore, similar results have also obtained by an isothermal two-step process in a system with technical grade oil and surfactant.25 3.3. Emulsions Prepared by Method II in Systems with Ionic Surfactants. To explore the generality of the mechanism of nanoemulsion formation, a range of concentrates were formulated with different surfactants. Literature was used to guide the formulation of appropriate concentrates with DDAB,27 AOT,28 and CTAB-pentanol and SDS-pentanol.29 For systems with DDAB, all concentrates including bicontinuous microemulsions do not disperse in water at all; coarsely dispersed oil droplets were observed during stirring which phase-separated as soon as stirring was stopped. This is consistent with the published phase behavior of the decane/ DDAB/water system:27 addition of an appropriate amount of water transforms the bicontinuous concentrate into a discontinuous discrete droplet water-in-oil microemulsion, and then further dilution with water places the samples in an undefined (emulsion) phase region. There is no spontaneous curvature transition to an oil-in-water structure in this system. Therefore, it may be reasonable to assume that spontaneous emulsification is also required for nanoemulsion formation as well as the complete solubilization of oil in a bicontinuous microemulsion. Crash dilution of concentrates with AOT, CTAB-pentanol, and SDS-pentanol in water, to yield final volume fractions of φ ) 0.030, resulted in emulsions having droplet radii of the order 70 nm, when the concentrates were located in a W/O or bicontinuous microemulsion phase region (see the Supporting Information, Table S1). Phase separation was observed in these systems after 3 days. This means nanoemulsions can be prepared by dilution of concentrates even when they are not bicontinuous microemulsions. Due to the decrease of ionic strength13,14 and partitioning of alcohol,9,10 as reported elsewhere, dilution of these concentrates with water allows a change of spontaneous curvature from water-in-oil to a bicontinuous microemulsion and then to an oil-in-water configuration. 3.4. Nanoemulsion Droplet Structure and Stability Determined by SANS. 3.4.1. Nanoemulsion Droplet Structure 30 min after Preparation. Because the nanoemulsions prepared with decane/C12E5/water have droplet sizes ideally matched to SANS (up to 60 nm), with the additional benefit of an absence of interdroplet electrostatic interactions, this neutron scattering method can be used to generate detailed structural information. Figure S2 in the Supporting Information shows the SANS profile from concentrate A5, exhibiting a correlation peak at Q ∼ 0.03 Å-1, consistent with a “bicontinuous” microemulsion at φ ) 0.50. Nanoemulsions (sample A prepared by dilution from A5), at drop contrast decane-h/C12E5-h/D2O, and shell contrast decane-d/C12E5-h/D2O were studied by SANS 30 min after preparation. These two contrasts can be clearly distinguished on a log-log plot, as can be seen in Figure 4, and there is an absence of any S(Q)-type correlation peak. The drop contrast data were initially analyzed by Guinier and Porod approximations and then finally fitted using the multimodel FISH program (details given in the Supporting Information). The Guinier plots show significant linearity at low Q for spheres (ln I(Q) vs Q2, Figure S3 in the Supporting Information); no linearity in an appropriate region of Q space was discerned for cylinders ln [I(Q)Q] vs Q2 or discs (ln [I(Q)Q2] vs Q2). Therefore, a Guinier analysis suggests the nanoemulsions exist in the form of spherical (globular) droplets. Estimates for the nanoemulsion

Wang et al.

Figure 4. SANS profiles of the drop and shell contrasts of the nanoemulsion (sample A, at φ ) 0.030) at 30 min and 25 °C. Error bars are shown. Lines are fits to a polydisperse sphere model described in the Supporting Information. Inset: Schematic outlines of the contrast arrangements. Table 1. Values Obtained from the SANS Data from Nanoemulsions at Different Times (Sample A at O ) 0.030)a sample A, 30 min A shell, 30 min A, 120 min A, 240 min

RGuinier/nm RPorod/nm Cdrop/(µmol dm-3) RFit/nm ts/nm 12.7

11.9

15

12.7 12.3

11.9 11.6

15 15

10.2 10.1 10.0 9.9

1.1

a σ/Rc ) 0.23 ( 0.03, φhs ) φdecane + φsurfactant, Rhs ) RPorod ( 0.8 nm. Parameters: RGuinier and RPorod are radii estimated by the Guinier and Porod approximations; Cdrop is the calculated nanoemulsion droplet concentration as described in the Supporting Information; RFit is the average radius given by Schulz polydisperse spheres fitting routine; ts is the apparent shell thickness given by the core-shell model; σ/Rc is the width of the Schultz distribution function.

radii R were obtained from the Guinier plot, which is valid only for very low Q (QR < 1); at high Q, the SANS intensity is sensitive to scattering from local interfaces. In this regime, the Porod approximation was used to estimate particle radii and the droplet concentration Cdrop, via the total specific area (see the Supporting Information). These values were used as starting points for a more detailed analysis, using a model of Schulz polydisperse spheres.38,39 The data are described very well by this model, with radii quite close to those obtained by the Guinier and Porod analyses (see Table 1). These analyses were consistent with the view that the droplets exist in the form of oil spheres. A core-shell model35 was employed to fit the shell contrast decane-d/C12E5-h/D2O. This model described the profile very well, which suggests that the nanoemulsion with an oil core is surrounded by a surfactant shell of apparent film thickness, ts. The respective model fits, and associated parameters, are shown in Figure 4 and Table 1. It is shown that the overall radius of the nanoemulsion (sample A) is 10.2 nm and the thickness of the surfactant layer is 1.1 nm. This outer layer thickness reflects reasonably well the dimension of a C12H25-h chain (1.7 nm) and would be consistent with minimal contrast difference between the outer hydrated EO-5 groups and the external D2O solvent, as would be expected under highly solvated conditions. The (38) Eastoe, J.; Hetherington, K. J.; Sharpe, D.; Dong, J.; Heenan, R. K.; Steytler, D. C. Langmuir 1996, 12, 3876–3880. (39) Nave, S.; Eastoe, J.; Heenan, R. K.; Steytler, D.; Grillo, I. Langmuir 2000, 16, 8741–8748.

Nanoemulsions Prepared by a Two-Step Process

Figure 5. SANS profiles for nanoemulsions at φ ) 0.030 at different times t ) 30 (0), 120 ()), and 240 min (∆). Error bars are shown. Lines are model fits to a polydisperse sphere model described in the Supporting Information. Inset: shown is the corresponding Porod plot.

overall droplet radius determined by SANS matches quite the value determined by DLS (15 nm) considering the need to include solvation of the surfactant shell. The polydispersity value is about 0.23, which reflects a narrow distribution of nanoemulsion droplet radii, and typical of microemulsion droplets using this model.38,39 Because of preexisting literature,40 the structure of dilute microemulsion B was not investigated by SANS here. Comparison of the composition for system B [φ ) 0.030, and R ) 0.50 ) (mass fraction wtdecane)/(wtdecane + wtC12E5)]40c) with those investigated previously40a,b clearly suggests spherical decane-in-water microemulsion droplets of radius ∼ 9 nm. Therefore, the DLS radii for the microemulsion system B shown in Figure 3 are consistent with previously published data.40 Note the microemulsion droplet radius is consistently lower than for the nanoemulsion A (Figure 3 and ref 40), suggesting different underlying structures for the two systems A and B. 3.4.2. Effect of Time on Nanoemulsion Droplets. The evolution of droplet size is an important aspect of emulsion stability. Nanoemulsions were characterized by SANS, at different times after the initial dilution to generate system A. No obvious changes in the shape or intensity of the SANS were observed. Similar detailed analyses as outlined above in section 3.4.1 were performed, and the resulting values are shown in Table 1 while the fitting lines are displayed in Figure 5. It can be seen that no notable changes in the structure or radius of the droplets was observed over the test time of 4 h after the initial dilution. This is most obviously seen in the inset to Figure 5, which magnifies the supersensitive high Q Porod region: increases in droplet size would result in shifts of the maxima/minima to lower Q values. The constant size demonstrates that the nanoemulsion droplets in this system are quite stable during the test period against coalescence41 and Ostwald ripening,8,42 both of which would be expected to give rise to increases in droplet size. 3.4.3. Structure of Nanoemulsions as a Function of Dilution. Systems were prepared by dilution with different water contents (40) (a) Menge, U.; Lang, P.; Findenegg, G. H.; Strunz, P. J. Phys. Chem. B 2003, 107, 1316–1320. (b) Menge, U.; Lang, P.; Findenegg, G. H. J. Phys. Chem. B 1999, 103, 5768–5774. (c) The composition parameter used in refs 40a and 40b is R is an oil mass fraction ) wt decane/(wt decane + wtC12E5). Translating now to total droplet volume fraction φ used in this paper, spherical droplets were observed in ref 40b for φ ) 0.0141-0.141 and above R ) 0.25: the composition of system B studied here is φ ) 0.030 and R ) 0.5.

Langmuir, Vol. 24, No. 12, 2008 6097

Figure 6. SANS profiles for nanoemulsions at various volume fractions, φ ) 0.120 (0), 0.060 ()), 0.030 (∆), 0.015 (O), and 0.006 (b). Error bars are shown. Lines are model fits to a polydisperse sphere model described in Supporting Information. Inset: shown is the Porod plot corresponding to the SANS profiles. Table 2. Values Obtained from the SANS Data from Nanoemulsions at Various Volume Fractions Oa φ

RGuinier/nm

RPorod/nm

Cdrop/(µmol dm-3)

RFit/nm

0.120 0.060 0.030 0.015 0.006

7.3 11.2 12.7 13.0 12.5

11.3 12.2 11.9 11.3 11.3

69 28 15 7 2

9.4 9.6 10.2 10.3 9.7

a σ/Rc ) 0.23 ( 0.03, φHS ) φdecane + φsurfactant, RHS ) RPorod ( 0.8 nm. Parameters: RGuinier and RPorod are radii estimated by the Guinier and Porod approximations; Cdrop is the calculated nanoemulsion droplet concentration as described in the Supporting Information; RFit is average radius given by Schulz polydisperse spheres fitting routine; σ/Rc is the width of the Schultz distribution function.

from concentrate A5, located in the microemulsion region of the phase diagram. Figure 6 shows SANS data for the nanoemulsions as a function of volume fraction φ, and it can be seen that the shape of the SANS profile changes little over this range. The increase in intensity with φ is an indication of an increase in the nanoemulsion droplet number density. This can be seen clearly from the nearlinear relationship between the droplet concentration Cdrop (calculated from the limiting high-Q intensity on the Porod plot) and φ (details can be seen in Supporting Information, Figure S7) and further verified by the linear relationship between the fitted scale factor (fitted as polydisperse spheres) and φ (can be seen in Figure S8 in the Supporting Information). Fitted and derived parameters are summarized in Table 2 along with the results obtained from the Guinier and Porod approximations. As is shown in Table 2, there is no significant change in the droplet size with φ (except for the R obtained from the Guinier approximation at φ ) 0.120, which may be attributed to the more prominent S(Q), which is exhibited by the downward slope at low Q). Nanoemulsions with radius of the order of 10 nm were obtained, independent of φ, and polydispersity indices were about 0.23, indicating a narrow size distribution. It can then be said that nanoemulsions may be obtained when the initial concentrates are located in a (41) Deminiere, B.; Colin, A.; Calderon, F. L.; Bibette, J. In Modern Aspects of Emulsion Science; Binks, B. P., Ed.; The Royal Society of Chemistry: Cambridge, U.K., 1998; pp 261-291. (42) Tadros, T.; Izquierdo, P.; Esquena, J.; Solans, C. AdV. Colloid Interface Sci. 2004, 108-110, 303–318.

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1 8π ωt ) r20 r 3

( )

(2)

where r is the average droplet radius after time t, r0 is the droplet radius at t ) 0, and ω is the frequency of rupture per unit surface of the film. If the mechanism is Ostwald ripening,43 the droplet radius should vary with time as

ω)

Figure 7. Dependence of apparent droplet radii of nanoemulsions against time, determined by DLS, at various volume fractions, φ ) 0.120 (0), 0.060 ()), 0.030 (∆), 0.015 (O), and 0.006 (b). Inset: the observed eventual phase separation of a nanoemulsion (sample A) after 3 days.

microemulsion region, regardless of the water content used for dilution. Eventual droplet sizes are mainly controlled by the structure of the concentrate and are independent of dilution. This is different from the results reported by Mason et al.26 in that the structure of nanoemulsions deforms from hard sphere to a glassy structure at high volume fraction (with φ larger than 0.3) through screened surface charge repulsions, perhaps because those systems were stabilized by anionic SDS. Due to the lower volume fraction and zero effective surface charge in the system investigated here, the droplets of nanoemulsions are discrete spheres at all test volume fractions, and excess water appears to act only as a dilution medium without having any effect on the structure. 3.5. Stability of Nanoemulsions Determined by DLS. Phase separation of nanoemulsion sample A (prepared from A5) was observed after 3 days (see inset to Figure 7), whereas the microemulsion samples B showed no phase change when left for up to 10 weeks. These results suggest that nanoemulsions are actually thermodynamically unstable. To follow the breakdown process, nanoemulsion droplet sizes were determined by DLS as a function of φ and time; these results are shown in Figure 7. When measured 15 min after preparation, similar droplet radii were obtained for the different volume fractions, agreeing with the SANS results described above. However, the DLS-determined droplet sizes show a marked increase with time; the higher φ is, the faster the droplet size increased. These results are contradictory to those obtained by SANS which suggest no change in the underlying droplet structure, at least 4 h after preparation from the stock concentrates (see Figure 5). These discrepancies are at least consistent with the length scale resolutions of the two scattering techniques: SANS is ideally matched to examine the discrete droplet structure for dimensions up to 30 nm or so; DLS being particularly sensitive to collective motions resulting from attractive interactions of particles, certainly with dimensions 30 nm or so and above. To explore the mechanism of instability with nanoemulsions, the results of Figure 7 were replotted as shown in Figure 8. The change in droplet size with time may follow eq 2 if the instability mechanism is coalescence:41

dr3 8 C(∞)γVmD ) ) dt 9 FRT

(3)

where r is the average droplet size after time t, C(∞) is the bulk phase solubility (the solubility of the oil in an infinitely large droplet), γ is the interfacial tension, Vm is the molar volume of the oil, D is the diffusion coefficient of the oil in the continuous phase, F is the oil density, and R is the gas constant. Figure 8 suggests that there is no linear variation upon plotting 1/r2 or r3 as a function of time. These results indicate that neither coalescence nor Ostwald ripening are the underlying mechanisms for the nanoemulsion instability, which are consistent with the results obtained from SANS in section 3.4.2. Comparing the DLS and SANS results, it is reasonable to assume that droplet-droplet hydrodynamic interactions exist without droplet deformation or rupture of the surfactant film during the test period. Therefore, the mechanism for the instability in this system may be attributed to flocculation,43 where drops cluster without significant rupture of the stabilizing interfacial layer. The droplet radius determined by DLS may be attributed to an effective cluster radius, which increases as more droplets aggregate with time through flocculation. These aggregates rise under gravity because of the density difference between the dispersed oil and the continuous phase, resulting in creaming and eventually phase separation, presumably with accompanying coalescence. At least over the time scale studied here, up to 4 h, it can be said that the individual droplet radius remains constant: SANS data are inconsistent with growth of individual droplets, which would be an inevitable consequence of both coalescence and/or Ostwald ripening. The rate of flocculation depends on the product of a frequency factor (how often drops encounter each other) and a probability factor (how long they stay in contact).43 Thus it is easy to understand that the higher volume fraction φ (in which drops have a higher frequency of encounters), the faster flocculation occurs, which results in the faster increase in droplet radius as determined by DLS (as can be seen in Figure 7). Tests were performed by DLS, swapping H2O for the higher density D2O to assess if this would affect the flocculation (Figure S10), but no significant differences were observed using this technique.

4. Conclusions An isothermal two-step process (method II) has been applied to oil/surfactant/water systems with C12E5, DDAB, AOT, SDS-pentanol, and CTAB-pentanol. Oil-in-water nanoemulsions have been obtained in the decane/C12E5/water system, by crash-dilution of a bicontinuous microemulsion into a large volume of water. The structure of nanoemulsions obtained this way is mainly controlled by the structure of the initial concentrate, being apparently independent of the dilution factor with water. Coupled with the results gained with systems stabilized by DDAB, AOT, SDS-pentanol, and CTAB-pentanol, a general mechanism for nanoemulsion formation may be postulated as homogeneous nucleation of oil droplets during spontaneous emulsification. (43) Binks, B. P. In Modern Aspects of Emulsion Science; Binks, B. P., Ed.; The Royal Society of Chemistry: Cambridge, U.K., 1998; pp 17-38.

Nanoemulsions Prepared by a Two-Step Process

Langmuir, Vol. 24, No. 12, 2008 6099

Figure 8. DLS derived radii, 1/r2 as a function of time (a) and r3 as a function of time (b) for nanoemulsions at various volume fractions, φ ) 0.120 (-0-), 0.060 (-)-), 0.030 (-∆-), 0.015 (-O-), and 0.006 (-b-).

The droplet sizes of nanoemulsions have been determined both by DLS and SANS. The two techniques show no striking differences in the nanoemulsion radii if newly prepared. However, droplet structure determined by SANS shows no change over 4 h, while the apparent DLS droplet size increases with time. This discrepancy suggests flocculation may be responsible for the nanoemulsion instability (at least up to emulsion ages of 4 h), which is different from the mechanisms widely reported before, such as coalescence and Ostwald ripening.8,41,42 This work gains insight into the general mechanism of nanoemulsion formation, which can be used to guide the preparation of nanoemulsions in a wide range of systems. This two-step process is easy to scale up and has low energy consumption, which is of great interest for practical applications. The findings verify that the two-step process can be used for the preparation of nanoemulsions, which are stable without an

increase in the radii of individual droplets over several hours. This new nanoemulsification route paves the way to new potential applications of these easy-to-prepare systems in fields such as foods, pharmaceuticals, and agrochemicals. Acknowledgment. We acknowledge the National Natural Science Foundation of China (Grant NSFC 20573079) and the Ministry of Science and Technology (Grant 2006 BAE01A075) for financial support. STFC (U.K.) is thanked for provision of beam time at ISIS and for financial support for travel and consumables. Supporting Information Available: Details of the conductivity data, SANS analysis, and theoretical background (pdf). This material is available free of charge via the Internet at http://pubs.acs.org. LA800624Z