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Microstructure of colloidal liquid aphrons (CLAs) by freeze fracture transmission electron microscopy (FF-TEM)
Colloids and Surfaces A: Physicochem. Eng. Aspects 264 (2005) 139–146
Microstructure of colloidal liquid aphrons (CLAs) by freeze fracture transmission electron microscopy (FF-TEM) Yong-li Yan a,∗ , Ning-sheng Zhang b , Cheng-tun Qu b , Li Liu c a
School of Energy and Power Engineering, Xi’an Jiaotong University, Xi’an 710049, China b Xi’an Shiyou University, Xi’an 710065, China c Research Institute of Engineering Technology, Changqing Petroleum Exploration Bureau, Xi’an 710021, China
Received 13 October 2004; received in revised form 29 March 2005; accepted 14 April 2005 Available online 18 August 2005
Abstract Colloidal liquid aphrons (CLAs) composed of triethylene glycol monododecyl ether (C12 E3 )/n-decane/sodium dodecyl sulphate (SDS) or Tween 80/water have been visualized by freeze fracture transmission electron microscopy (FF-TEM) and confirmed by small angle X-ray scattering (SAXS) and polarizing microscopy. The resolution achieved allows detailed inspection of the size, shape of individual micelles, as well as core-shell structure of CLAs. The microstructure of CLAs was compared with that conventional emulsions presented, particularly high internal phase ratio emulsions (HIPREs), from the viewpoint of morphology. The combined results indicate that the CLAs consist of spherical, oil droplets (oil-rich phase) of micron size separated by an aqueous “soapy shell” (water-rich phase) and there exist supramolecular structures such as reversed micelles and O/W micelles or microemulsions in oil-rich and water-rich phases, respectively. The total interfacial area of stable CLAs is consistent with that of colloidal gas aphrons (CGAs) reported previously, with an approximate thickness of 0.3–0.4 m. It has been shown throughout this work that the overall microstructure CLAs presented here is somewhat analogous to that of HIPREs, with biphasic structure and presence of the supramolecular aggregates in these phases, which seems to support the Princen’s opinion. © 2005 Elsevier B.V. All rights reserved. Keywords: Colloidal liquid aphrons (CLAs); Emulsions; High internal phase ratio emulsions (HIPREs); Microstructure; Supramolecular structures; Freeze fracture transmission electron microscopy (FF-TEM)
1. Introduction Colloidal liquid aphrons (CLAs) were first proposed by Sebba [1,2] to consist of a micron-sized solvent droplet encapsulated in a thin aqueous film (“soapy shell”), as shown schematically in Fig. 1. This structure is stabilized by a mixture of non-ionic and ionic surfactants, which forms three distinct interfaces (a monolayer and a separate bilayer) in the soapy shell [3]. Potential uses of these dispersion systems include predispersed solvent extraction such as extraction of antibiotics [4] and organic pollutants [5,6], soil remediation [7], and enzyme immobilization [8,9]. However, despite ∗
Corresponding author. Tel.: +86 29 82674979. E-mail address: [email protected] (Y.-l. Yan).
0927-7757/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2005.04.025
the broad range of potential applications, little work has been carried out to clarify Sebba’s proposed structure for CLAs. There is considerable debate as to whether a CLA should be considered an emulsion [10,11]. Some authors, noticeably Princen [11], refute Sebba’s proposed structures for polyaphron phase and CLAs claiming that they are no different from those of high internal phase emulsions (HIPREs) and the term aphron should be abandoned to avoid confusion. It would thus appear that a definitive structural study is needed before we can rationalize our understanding and nomenclature in this area. Attempts were made at several laboratories to clarify Sebba’s proposed structures for CLAs. Sebba [2] originally used fluorescent dyes to probe the structure of CLAs and
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surfactant layers, and the aggregation behavior in these systems.
2. Experimental section 2.1. Chemicals
Fig. 1. Proposed structure of a single colloidal liquid aphron by Sebba.
presented indirect evidence for the existence of a shell, but the structure of the shell has not yet been determined. Lye et al. [12] have investigated the structure of the CLAs using cryo-transmission electron microscopy (Cryo-TEM), differential scanning calorimetry (DSC) and other light scattering techniques, which seems to confirm the macroscopic structure of these phases as proposed by Sebba [3]. However, these studies did not provide direct evidence for the structure of the surfactant-laden interfaces responsible for the stabilization of CLAs. Electron microscopy techniques are frequently used to directly visualize macromolecule structures. In particular, direct imaging Cryo-TEM [13,14] and freeze fracture transmission electron microscopy (FF-TEM) [15,16] have emerged as artifact-free methods for observing diverse systems such as surfactant aggregates, polymer and polymersurfactant solutions, and microemulsions, as well as biological and biomedical systems. These methods provide the ability to preserve microstructures by rapid vitrification of the solution containing the aggregates. However, little attention has been devoted to the investigation of colloidal liquid/gas aphrons (CLAs/CGAs) using this method [12,17,18]. Only Lye et al. [12] and Jauregi et al [17] have employed the technique of Cryo-TEM or FF-TEM to determine the shape, size and film thickness of CLAs/CGAs, independently. Recently, Srivastava [18] used diverse techniques of FF-TEM, DSC, small angle X-ray scattering (SAXS) and isothermal titration calorimetry (ITC) to provide direct evidence of such a soapy shell. The multilamellar structure of the shell appears to have liquid-crystal-like ordering of the surfactant layers. This ordering has been shown to be a unique property of the surfactant system used to form the aphrons. However, evidence for the structure and location of the three surfactant layers is less obvious. In the present study, we used FF-TEM, SAXS and polarizing microscopy to elucidate the microstructure of the CLA suspensions, focusing on the thickness, number of
For the typical CLAs dispersion formulation, the solvent used was n-decane (CP, Japan); the non-ionic surfactants were triethylene glycol monododecyl ether, C12 E3 (>99% pure, Xingtai Lanxing Reagent Co. China), as well as Tween 80 (AR, Xi’an Chemical Reagent Factory, China); and the anionic surfactant used was sodium dodecyl sulphate, SDS (AR, Xi’an Chemical Reagent Factory, China). All aqueous phases were prepared from freshly deionized water (School of Medicine, Xi’an Jiaotong University, China) with a conductivity of <0.5 mS/cm that has been filtered through a 0.2 mm filter. 2.2. Preparation A serious of four CLAs and one emulsion were prepared using the technique described by Matsushita et al. [19]. CLAs were prepared by the dropwise addition of the organic phase containing non-ionic surfactant (1% (v/v) C12 E3 in n-decane) into a foaming aqueous solution containing ionic or nonionic surfactant (0.5% (w/v) SDS or Tween 80 in deionized water). The initial volume of the aqueous phase was typically 10 ml, which was stirred at approximately 800 rpm, and the organic phase was added at an average flow rate of 0.5 ml/min until the desired phase volume ratio was reached (PVR = Vorg /Vaq ). The resulting CLAs formed in this way were very viscous, having a creamy white appearance, with stable CLAs formulation showing no phase separation over a period of months. Comparative conventional emulsions were made using identical techniques except that the oil phase was free of the C12 E3 surfactant used in the CLAs. 2.3. FF-TEM For this investigation, a small droplet (∼10 l) of each sample was placed on a small holder plate (Φ = 3 mm), and it was frozen in liquid nitrogen at 77 K. The specimens thus prepared were transferred to a freeze-fracture unit (HFZ-1, HITACHI) and fractured by a knife at 123 K with a residual pressure of 1.33 × 10−3 Pa. The fractured faces were etched for 10 min at 173 K prior to shadowing. Subsequently, the etched surfaces were shadowed, with ∼2 nm of Pt at a 45◦ angle to the plane of the sample followed by 10–15 nm of carbon (at = 90◦ angle) by arc evaporation. The resulting replicas were washed in a 10% graded acetone series, rinsed in deionized water, and then collected on 300 mesh grids. Transmission electron microscopy (TEM) was performed on a HITACHI H-600 operated at 80 keV with images recorded on Kodak SO 163 film.
Y.-l. Yan et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 264 (2005) 139–146
2.4. Optical microscopy An Olympus BH-2 microscope, attached to an automatic exposure camera was used for photomicrography in the ordinary and crossed polarizing mode, respectively. 2.5. SAXS Small-angle X-ray scattering experiments were operated at 298 K by means of a Kratky compact small-angle system equipped with a position sensitive detector (OED 50M from Mbraun, Graz, Austria) containing 1024 channels of width 54 mm. The range of scattering angle was chosen from ˚ −1 , where the magnitude of scattering vech = 0.005 to 0.6 A tor h = 2πsin θ/λ, 2θ and λ being, respectively, the scattering ˚ The disangle and incident X-ray wavelength of 1.542 A. tance from sample to detector was 27.7 cm and the exposure time was 600 s for each sample. 2.6. Droplet size The droplet size analysis was performed using a laser diffractometer Mastersizer 2000 (Malvern Instruments, UK). Each sample was measured in triplicate.
3. Results and discussion 3.1. Observations by light microscopy Fig. 2(A) and (B) shows light micrographs of conventional emulsions and CLA dispersions, respectively. The overall form of the CLAs prepared in this study is morphologically similar to the conventional emulsions at the light microscopy level. As seen in Fig. 2(A) and (B), both the CLAs and conventional emulsions at the dispersed state display a core-shell structure. The difference is that the droplet size of CLAs (average about 10 m in diameter) is smaller than that of the conventional emulsions (average about 33 m in diameter). Sebba [2] mentioned the use of differential staining and used light microscopy to characterize the outer shell of the CLAs. However, no comparison with similarly formed conventional emulsions was made. Indeed, the multilamel-
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lar CLAs interfacial region presented is below the resolution limit of conventional light microscopy. In addition, accurate separation of the details of a continuous interface from diffraction artifact at the surface of spherical objects is problematic. TEM allows resolution of macromolecular dimension; however, the sample must be rendered stable at very low pressure. Typically this is accomplished either by keeping the specimen at a temperature below which there is no significant vapor pressure in the microscope column, or “fixing” the specimen chemically to allow the removal or conversion of the volatile phases. In order to determine the fine structure inside CLAs, FF-TEM was conducted for these studies. 3.2. Observations by FF-TEM 3.2.1. Conventional emulsions Fig. 3(A) and (B) shows FF-TEM micrographs of conventional emulsion composed of n-decane/SDS/water at PVR of 4. As shown in Fig. 3(A) and (B), the conventional emulsion prepared in this study consists of two isotropic liquid phases: the dispersed phase (core) is composed of micronsized, oil droplets, and the continuous phase is an aqueous membrane. There is no fine structure presented in the organic phase droplet. In contrast, a lot of small liquid droplets were observed inside the aqueous membrane, which is due to the interaction of surfactant SDS in the aqueous solution. These aggregates show the forms of spherical or close to spherical shape with the size of spanning hundreds of microns. So they fall into the domains of miniemulsions. 3.2.2. CLAs The results presented in Fig. 4(A)–(D) are FF-TEM micrographs of CLAs made of C12 E3 /n-decane/SDS or Tween 80/water at various PVR. From the images of the CLA series, we can see that the CLA emulsions consist of spherical, oil droplets (oil-rich phase) of micron size surrounded by an aqueous soapy shell (water-rich phase). As the PVR equals 2, the freeze fracture of the CLAs yields many spherical or wormlike O/W micelles or microemulsions, but the longrange ordered organizations do not present in the soapy shell as shown in Fig. 4(A). As the PVR equals 3 or 4, the CLAs display spherical or close to spherical micelles or
Fig. 2. Light microscopy micrographs of conventional emulsions and CLAs. 1 bar = 50 m. (A) Conventional emulsions of n-decane/SDS (0.5%, w/v)/water. (B) CLAs of C12 E3 (1%, v/v)/n-decane/SDS (0.5%, w/v)/water.
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Fig. 3. FF-TEM micrographs of conventional emulsions at PVR of 4. (A), (B) emulsions of n-decane/SDS (0.5%, w/v)/water.
O/W microemulsions in aqueous shells (water-rich phase) as depicted in Fig. 4(B) and (C). These micelles (or O/W microemulsions) are closely packed and linked as “necklace” shape which are further packed side by side to be scale-like films, which is similar to the black liquid film and has the approximate thickness of 10 nm. These aggregates could be attributed to the interaction of surfactant SDS and C12 E3 in the aqueous solutions and contribute to the stability for these dispersion systems [20,21]. As for CLAs composed of C12 E3 /n-decane/Tween 80/water at PVR of 4 in Fig. 4(D), the close-packed spherical micelles or O/W microemulsions are presented in the water-rich phase. We could also find some fine structures looking like reverse micelles inside the
oil droplets (oil-rich phase) from the Fig. 4(A1-2, B3, C12, D2). These reverse micelles result from the interaction of C12 E3 and SDS in oil phases. Here, there is partitioning of the two surfactants SDS (Tween 80)/C12 E3 at the water/oil interfaces and into the two bulk phases, which is beyond the scope for this paper. SAXS and polarizing microscopy were performed on a serious of CLAs and emulsions in this study, mainly in order to characterize the isotropic and liquid crystal (lamellar and hexagonal) phase domains when their presence is suspected. When the CLAs or emulsions examined under a polarizing microscope, no birefringence was observed, except for a very small amount of crystalline impurity. Thus, anisotropic
Fig. 4. FF-TEM micrographs of CLAs at different PVR. (A–C) CLAs composed of AEO-3(1%, v/v)/n-decane/SDS (0.5%, w/v)/water, PVR of which are A-2, B-3 and C-4, respectively; (D) CLAs made of AEO-3 (1%, v/v)/n-decane/Tween 80 (0.5%, w/v)/water, PVR of which is 4.
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Fig. 4. (Continued )
structures such as lamellae or hexagonal close-packed cylinders are ruled out. SAXS measurements were undertaken on a serious of CLAs and emulsions as shown in Fig. 5. The small angle X-ray curve of the CLAs and emulsions does not show any small angle X-ray diffraction pattern typical of emulsifier multilayers at oil–water interfaces and therefore SAXS supports the FF-TEM and polarizing microscopy observations. The results of the particle size analysis performed on CLAs and emulsions at various PVR are presented in Fig. 6 for comparison. The findings indicate that the majority of CLAs had diameters in the range between 1 and 50 m for PVR 2, 1 and 40 m for PVR 4, and 1 and 15 m for PVR 8, respectively. The mean diameter, which is calculated from the mean volume of the suspension, was found to be 15.5,
10.7 and 4.6 m, respectively and affected by the different PVR in these dispersions. It was also found that the conventional emulsions at PVR of 4 generated in our laboratory had diameters between 1 and 100 m with a mean diameter of 33.3 m, which is obviously larger than those of all CLAs systems. This is because the organic phase surfactant (C12 E3 ) was found to greatly lower the interfacial tension, which results in smaller average droplet size of the CLAs. This suggests that the formation of CLAs is owing to the interaction between two surfactants, which is consistent with previous reports on the synergistic effect of the different surfactants at the oil/water interface [18,21,22]. Based on these FF-TEM images, one can measure the thickness of the aqueous films. At a PVR 2, the finite interface is about 0.7 m thickness for CLA emulsions; as a
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Fig. 4. (Continued ).
PVR equals or exceeds 3, the film thickness of CLAs ranges between 0.3 and 0.4 m. These measured values are larger than the thickness of 0.15 m for polyaphrons at PVR 4 determined by Lye et al. [12], as well as larger than the soap film thickness of 0.096 m for colloidal gas aphrons (CGAs) studied by Jauregi et al. [17] previously. However, these determined thickness give the best fit with that of CGAs estimated by Amiri et al. [23], that is, 0.75 m, for a cationic surfactant, tetradecyltrimethyl ammonium bromide (TTAB) and Bredwell et al. [24], that is, 0.2–0.3 m for a nonionic surfactant, Tween 20. It should be noted that FF-TEM observation is a direct measurement of the film thickness at the particular time, and therefore it is a reliable measure of the size ranges. It has been shown through discussion above that the CLAs and HIPREs display similar structural model. The structure of
the HIPREs has been elucidated some years ago using a serious techniques of Video-enhanced microscopy (VEM), TEM, SAXS/SANS (small angle neutron scattering) and PGSENMR (pulsed-field-gradient spin-echo NMR) experiments [25–35]. The HIPREs consist of two isotropic liquid phases: the dispersed phase is composed of oil droplets, and the continuous phase is an O/W micelle or microemulsion or a lamellar liquid crystal. In contrast to earlier findings on HIPREs, we could find that both the CLAs and HIPREs present an identical biphasic structure. As for CLAs, the supramolecular structures exist both the oil- and water-rich phases; in contrast, the aggregates occur only at the water-rich region for HIPREs. This difference is the cause of absence of C12 E3 at the oil phase for the latter. These supramolecular structures present various forms including micelles, microemulsions,
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phases, respectively. The total interfacial area of stable CLAs has an approximate thickness of 0.3–0.4 m, which is consistent with that of CGAs reported previously. In addition, it has been shown throughout this work that CLAs and HIPREs display similar characteristics with respect to overall structure, with biphasic structure and presence of the supramolecular structures in these phases, which seems to support Princen’s opinion [11]. There are, however, a number of significant differences in the formulation and manufacture of the two systems [12]. Accordingly, it is too early to make any definite statements. In order to get some conclusive evidence on microstructure of CLAs and HIPREs, we need to employ more techniques such as DSC, FT-IR, PGSE-NMR and SANS in the future. Fig. 5. SAXS curves of various CLAs. CLAs system is composed of AEO3 (1%, v/v)/n-decane/SDS (0.5%, w/v)/water, PVR of which are 2, 3, 4, 8 and 16, respectively; CLAs-Tween system consists of AEO-3 (1%, v/v)/ndecane/Tween 80 (0.5%, w/v)/water, PVR of which are 4 and 8, respectively; Emulsion system is made of n-decane/SDS (0.5%, w/v)/water, PVR of which is 4. The notes for Fig. 6 are same like in (5).
Acknowledgement This project has been financially supported by the National “Shiwu” Key Technologies R&D Programme of China (No.2004BA61010).
References [1] [2] [3] [4] [5] [6] [7] [8] [9] Fig. 6. Typical size distribution of various CLAs.
miniemulsions and lamellar liquid crystals, depending on the nature and composition of the amphiphile in the system, ionic strength, and external conditions such as temperature. The two most frequently encountered structures in thin liquid soap films are repeating units of surfactant bilayers or ordered arrays of micellar domains [20].
4. Conclusions The FF-TEM investigations carried out in this study reveal new important information on the microstructure of CLAs. The micrographs show that the CLAs consist of spherical, oil droplets (oil-rich phase) of micron size separated by an aqueous “soapy shell” (water-rich phase) and there exist supramolecular structures such as inverse micelles and micelles or O/W microemulsions in oil-rich and water-rich
[10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]
F. Sebba, J. Colloid Interface Sci. 40 (1972) 468. F. Sebba, Chem. Ind. 10 (1984) 367. F. Sebba, Foams and Biliquid Foams-Aphrons, Wiley, 1987, p. 103. G.J. Lye, D.C. Stuckey, Proceedings ISEC’ 96, vol. II, University of Melbourne, 1996, pp. 1399–1405. D.L. Michelsen, K.W. Ruettimann, K.R. Hunter, F. Sebba, Chem. Eng. Commun. 48 (1986) 155. C. Zhang, K.T. Valsaraj, W.D. Constant, D. Roy, Sep. Sci. Technol. 31 (1996) 1463. W.K. Vidrine, C.S. Willson, K.T. Valsaraj, Colloid. Surf., APhysicochem. Eng. Asp. 175 (2000) 277. G.J. Lye, O.P. Pavlou, M. Rosjidi, D.C. Stuckey, Biotechnol. Bioeng. 51 (1996) 69. S.B. Lamb, D.C. Lamb, S.L. Kelly, D.C. Stuckey, FEBS Lett. 431 (1998) 343. V. Bergeron, F. Sebba, Langmuir 3 (1987) 857. H.M. Princen, Langmuir 4 (1988) 486. G.J. Lye, D.C. Stuckey, Colloid Surf. A-Physicochem. Eng. Asp. 131 (1998) 119. P.K. Vinson, J.R. Bellare, H.T. Davis, W.G. Miller, L.E. Scriven, J. Colloid Interface Sci. 142 (1991) 74. M. Almgren, K. Edwards, J. Gustafsson, Curr. Opin. Colloid Interf. Sci. 1 (1996) 270. W. Jahn, R. Strey, J. Phys. Chem. 92 (1988) 2294. T. GulikKrzywicki, Curr. Opin. Colloid Interf. Sci. 2 (1997) 137. P. Jauregi, G.R. Mitchell, J. Varley, AIChE J. 46 (2000) 24. P. Srivastava, Ph.D. Dissertation, Michigan State University, 2000. K. Matsushita, A.H. Mollah, D.C. Stuckey, C. del Cerro, A.I. Bailey, Colloids Surfs. 69 (1992) 65. V. Bergeron, J. Phys.: Condens. Matter 11 (1999) R215. E. Staples, J. Penfold, I. Tucker, J. Phys. Chem. B 104 (2000) 606. J.H. Schulman, E.G. Cockbain, Trans. Faraday Soc. 35 (1940) 651. M.C. Amiri, E.T. Woodburn, Trans. Inst. Chem. Eng. 68 (1990) 154. M.D. Bredwell, R.M. Worden, Biotechnol. Progr. 14 (1998) 31. C. Solans, J.G. Dominguez, J.L. Parra, J. Heuser, S.E. Friberg, Colloid Polym. Sci. 266 (1988) 570.
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[26] H. Kunieda, D.F. Evans, C. Solans, M. Yoshida, Colloids Surfs. 47 (1990) 35. [27] R. Pons, J.C. Ravey, S. Sauvage, M.J. St´eb´e, P. Erra, C. Solans, Colloid Surf. A-Physicochem. Eng. Asp. 76 (1993) 171. [28] K. Westesen, T. Wehler, Colloid Surf. A-Physicochem. Eng. Asp. 78 (1993) 115. [29] C. Solans, R. Pons, S. Zhu, H.T. Davis, D.F. Evans, K. Nakamura, H. Kunieda, Langmuir 9 (1993) 1479. [30] J.C. Ravey, M.J. St´eb´e, S. Sauvage, Colloid Surf. A-Physicochem. Eng. Asp. 91 (1994) 237.
[31] M.P. Krafft, J.G. Riess, Angew. Chem. Int. Ed. Engl. 33 (1994) 1100. [32] K. Ozawa, C. Solans, H. Kunieda, J. Colloid Interface. Sci. 188 (1997) 275. [33] S. Rocca, M.J. St´eb´e, J. Phys. Chem. B 104 (2000) 10490. [34] P.A. Reynolds, E.P. Gilbert, J.W. White, J. Phys. Chem. B 104 (2000) 7012. [35] P.A. Reynolds, E.P. Gilbert, J.W. White, J. Phys. Chem. B 105 (2001) 6925.
Microstructure of colloidal liquid aphrons (CLAs) by freeze fracture transmission electron microscopy (FF-TEM) Yong-li Yan a,∗ , Ning-sheng Zhang b , Cheng-tun Qu b , Li Liu c a
School of Energy and Power Engineering, Xi’an Jiaotong University, Xi’an 710049, China b Xi’an Shiyou University, Xi’an 710065, China c Research Institute of Engineering Technology, Changqing Petroleum Exploration Bureau, Xi’an 710021, China
Received 13 October 2004; received in revised form 29 March 2005; accepted 14 April 2005 Available online 18 August 2005
Abstract Colloidal liquid aphrons (CLAs) composed of triethylene glycol monododecyl ether (C12 E3 )/n-decane/sodium dodecyl sulphate (SDS) or Tween 80/water have been visualized by freeze fracture transmission electron microscopy (FF-TEM) and confirmed by small angle X-ray scattering (SAXS) and polarizing microscopy. The resolution achieved allows detailed inspection of the size, shape of individual micelles, as well as core-shell structure of CLAs. The microstructure of CLAs was compared with that conventional emulsions presented, particularly high internal phase ratio emulsions (HIPREs), from the viewpoint of morphology. The combined results indicate that the CLAs consist of spherical, oil droplets (oil-rich phase) of micron size separated by an aqueous “soapy shell” (water-rich phase) and there exist supramolecular structures such as reversed micelles and O/W micelles or microemulsions in oil-rich and water-rich phases, respectively. The total interfacial area of stable CLAs is consistent with that of colloidal gas aphrons (CGAs) reported previously, with an approximate thickness of 0.3–0.4 m. It has been shown throughout this work that the overall microstructure CLAs presented here is somewhat analogous to that of HIPREs, with biphasic structure and presence of the supramolecular aggregates in these phases, which seems to support the Princen’s opinion. © 2005 Elsevier B.V. All rights reserved. Keywords: Colloidal liquid aphrons (CLAs); Emulsions; High internal phase ratio emulsions (HIPREs); Microstructure; Supramolecular structures; Freeze fracture transmission electron microscopy (FF-TEM)
1. Introduction Colloidal liquid aphrons (CLAs) were first proposed by Sebba [1,2] to consist of a micron-sized solvent droplet encapsulated in a thin aqueous film (“soapy shell”), as shown schematically in Fig. 1. This structure is stabilized by a mixture of non-ionic and ionic surfactants, which forms three distinct interfaces (a monolayer and a separate bilayer) in the soapy shell [3]. Potential uses of these dispersion systems include predispersed solvent extraction such as extraction of antibiotics [4] and organic pollutants [5,6], soil remediation [7], and enzyme immobilization [8,9]. However, despite ∗
Corresponding author. Tel.: +86 29 82674979. E-mail address: [email protected] (Y.-l. Yan).
0927-7757/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2005.04.025
the broad range of potential applications, little work has been carried out to clarify Sebba’s proposed structure for CLAs. There is considerable debate as to whether a CLA should be considered an emulsion [10,11]. Some authors, noticeably Princen [11], refute Sebba’s proposed structures for polyaphron phase and CLAs claiming that they are no different from those of high internal phase emulsions (HIPREs) and the term aphron should be abandoned to avoid confusion. It would thus appear that a definitive structural study is needed before we can rationalize our understanding and nomenclature in this area. Attempts were made at several laboratories to clarify Sebba’s proposed structures for CLAs. Sebba [2] originally used fluorescent dyes to probe the structure of CLAs and
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Y.-l. Yan et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 264 (2005) 139–146
surfactant layers, and the aggregation behavior in these systems.
2. Experimental section 2.1. Chemicals
Fig. 1. Proposed structure of a single colloidal liquid aphron by Sebba.
presented indirect evidence for the existence of a shell, but the structure of the shell has not yet been determined. Lye et al. [12] have investigated the structure of the CLAs using cryo-transmission electron microscopy (Cryo-TEM), differential scanning calorimetry (DSC) and other light scattering techniques, which seems to confirm the macroscopic structure of these phases as proposed by Sebba [3]. However, these studies did not provide direct evidence for the structure of the surfactant-laden interfaces responsible for the stabilization of CLAs. Electron microscopy techniques are frequently used to directly visualize macromolecule structures. In particular, direct imaging Cryo-TEM [13,14] and freeze fracture transmission electron microscopy (FF-TEM) [15,16] have emerged as artifact-free methods for observing diverse systems such as surfactant aggregates, polymer and polymersurfactant solutions, and microemulsions, as well as biological and biomedical systems. These methods provide the ability to preserve microstructures by rapid vitrification of the solution containing the aggregates. However, little attention has been devoted to the investigation of colloidal liquid/gas aphrons (CLAs/CGAs) using this method [12,17,18]. Only Lye et al. [12] and Jauregi et al [17] have employed the technique of Cryo-TEM or FF-TEM to determine the shape, size and film thickness of CLAs/CGAs, independently. Recently, Srivastava [18] used diverse techniques of FF-TEM, DSC, small angle X-ray scattering (SAXS) and isothermal titration calorimetry (ITC) to provide direct evidence of such a soapy shell. The multilamellar structure of the shell appears to have liquid-crystal-like ordering of the surfactant layers. This ordering has been shown to be a unique property of the surfactant system used to form the aphrons. However, evidence for the structure and location of the three surfactant layers is less obvious. In the present study, we used FF-TEM, SAXS and polarizing microscopy to elucidate the microstructure of the CLA suspensions, focusing on the thickness, number of
For the typical CLAs dispersion formulation, the solvent used was n-decane (CP, Japan); the non-ionic surfactants were triethylene glycol monododecyl ether, C12 E3 (>99% pure, Xingtai Lanxing Reagent Co. China), as well as Tween 80 (AR, Xi’an Chemical Reagent Factory, China); and the anionic surfactant used was sodium dodecyl sulphate, SDS (AR, Xi’an Chemical Reagent Factory, China). All aqueous phases were prepared from freshly deionized water (School of Medicine, Xi’an Jiaotong University, China) with a conductivity of <0.5 mS/cm that has been filtered through a 0.2 mm filter. 2.2. Preparation A serious of four CLAs and one emulsion were prepared using the technique described by Matsushita et al. [19]. CLAs were prepared by the dropwise addition of the organic phase containing non-ionic surfactant (1% (v/v) C12 E3 in n-decane) into a foaming aqueous solution containing ionic or nonionic surfactant (0.5% (w/v) SDS or Tween 80 in deionized water). The initial volume of the aqueous phase was typically 10 ml, which was stirred at approximately 800 rpm, and the organic phase was added at an average flow rate of 0.5 ml/min until the desired phase volume ratio was reached (PVR = Vorg /Vaq ). The resulting CLAs formed in this way were very viscous, having a creamy white appearance, with stable CLAs formulation showing no phase separation over a period of months. Comparative conventional emulsions were made using identical techniques except that the oil phase was free of the C12 E3 surfactant used in the CLAs. 2.3. FF-TEM For this investigation, a small droplet (∼10 l) of each sample was placed on a small holder plate (Φ = 3 mm), and it was frozen in liquid nitrogen at 77 K. The specimens thus prepared were transferred to a freeze-fracture unit (HFZ-1, HITACHI) and fractured by a knife at 123 K with a residual pressure of 1.33 × 10−3 Pa. The fractured faces were etched for 10 min at 173 K prior to shadowing. Subsequently, the etched surfaces were shadowed, with ∼2 nm of Pt at a 45◦ angle to the plane of the sample followed by 10–15 nm of carbon (at = 90◦ angle) by arc evaporation. The resulting replicas were washed in a 10% graded acetone series, rinsed in deionized water, and then collected on 300 mesh grids. Transmission electron microscopy (TEM) was performed on a HITACHI H-600 operated at 80 keV with images recorded on Kodak SO 163 film.
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2.4. Optical microscopy An Olympus BH-2 microscope, attached to an automatic exposure camera was used for photomicrography in the ordinary and crossed polarizing mode, respectively. 2.5. SAXS Small-angle X-ray scattering experiments were operated at 298 K by means of a Kratky compact small-angle system equipped with a position sensitive detector (OED 50M from Mbraun, Graz, Austria) containing 1024 channels of width 54 mm. The range of scattering angle was chosen from ˚ −1 , where the magnitude of scattering vech = 0.005 to 0.6 A tor h = 2πsin θ/λ, 2θ and λ being, respectively, the scattering ˚ The disangle and incident X-ray wavelength of 1.542 A. tance from sample to detector was 27.7 cm and the exposure time was 600 s for each sample. 2.6. Droplet size The droplet size analysis was performed using a laser diffractometer Mastersizer 2000 (Malvern Instruments, UK). Each sample was measured in triplicate.
3. Results and discussion 3.1. Observations by light microscopy Fig. 2(A) and (B) shows light micrographs of conventional emulsions and CLA dispersions, respectively. The overall form of the CLAs prepared in this study is morphologically similar to the conventional emulsions at the light microscopy level. As seen in Fig. 2(A) and (B), both the CLAs and conventional emulsions at the dispersed state display a core-shell structure. The difference is that the droplet size of CLAs (average about 10 m in diameter) is smaller than that of the conventional emulsions (average about 33 m in diameter). Sebba [2] mentioned the use of differential staining and used light microscopy to characterize the outer shell of the CLAs. However, no comparison with similarly formed conventional emulsions was made. Indeed, the multilamel-
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lar CLAs interfacial region presented is below the resolution limit of conventional light microscopy. In addition, accurate separation of the details of a continuous interface from diffraction artifact at the surface of spherical objects is problematic. TEM allows resolution of macromolecular dimension; however, the sample must be rendered stable at very low pressure. Typically this is accomplished either by keeping the specimen at a temperature below which there is no significant vapor pressure in the microscope column, or “fixing” the specimen chemically to allow the removal or conversion of the volatile phases. In order to determine the fine structure inside CLAs, FF-TEM was conducted for these studies. 3.2. Observations by FF-TEM 3.2.1. Conventional emulsions Fig. 3(A) and (B) shows FF-TEM micrographs of conventional emulsion composed of n-decane/SDS/water at PVR of 4. As shown in Fig. 3(A) and (B), the conventional emulsion prepared in this study consists of two isotropic liquid phases: the dispersed phase (core) is composed of micronsized, oil droplets, and the continuous phase is an aqueous membrane. There is no fine structure presented in the organic phase droplet. In contrast, a lot of small liquid droplets were observed inside the aqueous membrane, which is due to the interaction of surfactant SDS in the aqueous solution. These aggregates show the forms of spherical or close to spherical shape with the size of spanning hundreds of microns. So they fall into the domains of miniemulsions. 3.2.2. CLAs The results presented in Fig. 4(A)–(D) are FF-TEM micrographs of CLAs made of C12 E3 /n-decane/SDS or Tween 80/water at various PVR. From the images of the CLA series, we can see that the CLA emulsions consist of spherical, oil droplets (oil-rich phase) of micron size surrounded by an aqueous soapy shell (water-rich phase). As the PVR equals 2, the freeze fracture of the CLAs yields many spherical or wormlike O/W micelles or microemulsions, but the longrange ordered organizations do not present in the soapy shell as shown in Fig. 4(A). As the PVR equals 3 or 4, the CLAs display spherical or close to spherical micelles or
Fig. 2. Light microscopy micrographs of conventional emulsions and CLAs. 1 bar = 50 m. (A) Conventional emulsions of n-decane/SDS (0.5%, w/v)/water. (B) CLAs of C12 E3 (1%, v/v)/n-decane/SDS (0.5%, w/v)/water.
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Fig. 3. FF-TEM micrographs of conventional emulsions at PVR of 4. (A), (B) emulsions of n-decane/SDS (0.5%, w/v)/water.
O/W microemulsions in aqueous shells (water-rich phase) as depicted in Fig. 4(B) and (C). These micelles (or O/W microemulsions) are closely packed and linked as “necklace” shape which are further packed side by side to be scale-like films, which is similar to the black liquid film and has the approximate thickness of 10 nm. These aggregates could be attributed to the interaction of surfactant SDS and C12 E3 in the aqueous solutions and contribute to the stability for these dispersion systems [20,21]. As for CLAs composed of C12 E3 /n-decane/Tween 80/water at PVR of 4 in Fig. 4(D), the close-packed spherical micelles or O/W microemulsions are presented in the water-rich phase. We could also find some fine structures looking like reverse micelles inside the
oil droplets (oil-rich phase) from the Fig. 4(A1-2, B3, C12, D2). These reverse micelles result from the interaction of C12 E3 and SDS in oil phases. Here, there is partitioning of the two surfactants SDS (Tween 80)/C12 E3 at the water/oil interfaces and into the two bulk phases, which is beyond the scope for this paper. SAXS and polarizing microscopy were performed on a serious of CLAs and emulsions in this study, mainly in order to characterize the isotropic and liquid crystal (lamellar and hexagonal) phase domains when their presence is suspected. When the CLAs or emulsions examined under a polarizing microscope, no birefringence was observed, except for a very small amount of crystalline impurity. Thus, anisotropic
Fig. 4. FF-TEM micrographs of CLAs at different PVR. (A–C) CLAs composed of AEO-3(1%, v/v)/n-decane/SDS (0.5%, w/v)/water, PVR of which are A-2, B-3 and C-4, respectively; (D) CLAs made of AEO-3 (1%, v/v)/n-decane/Tween 80 (0.5%, w/v)/water, PVR of which is 4.
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Fig. 4. (Continued )
structures such as lamellae or hexagonal close-packed cylinders are ruled out. SAXS measurements were undertaken on a serious of CLAs and emulsions as shown in Fig. 5. The small angle X-ray curve of the CLAs and emulsions does not show any small angle X-ray diffraction pattern typical of emulsifier multilayers at oil–water interfaces and therefore SAXS supports the FF-TEM and polarizing microscopy observations. The results of the particle size analysis performed on CLAs and emulsions at various PVR are presented in Fig. 6 for comparison. The findings indicate that the majority of CLAs had diameters in the range between 1 and 50 m for PVR 2, 1 and 40 m for PVR 4, and 1 and 15 m for PVR 8, respectively. The mean diameter, which is calculated from the mean volume of the suspension, was found to be 15.5,
10.7 and 4.6 m, respectively and affected by the different PVR in these dispersions. It was also found that the conventional emulsions at PVR of 4 generated in our laboratory had diameters between 1 and 100 m with a mean diameter of 33.3 m, which is obviously larger than those of all CLAs systems. This is because the organic phase surfactant (C12 E3 ) was found to greatly lower the interfacial tension, which results in smaller average droplet size of the CLAs. This suggests that the formation of CLAs is owing to the interaction between two surfactants, which is consistent with previous reports on the synergistic effect of the different surfactants at the oil/water interface [18,21,22]. Based on these FF-TEM images, one can measure the thickness of the aqueous films. At a PVR 2, the finite interface is about 0.7 m thickness for CLA emulsions; as a
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Fig. 4. (Continued ).
PVR equals or exceeds 3, the film thickness of CLAs ranges between 0.3 and 0.4 m. These measured values are larger than the thickness of 0.15 m for polyaphrons at PVR 4 determined by Lye et al. [12], as well as larger than the soap film thickness of 0.096 m for colloidal gas aphrons (CGAs) studied by Jauregi et al. [17] previously. However, these determined thickness give the best fit with that of CGAs estimated by Amiri et al. [23], that is, 0.75 m, for a cationic surfactant, tetradecyltrimethyl ammonium bromide (TTAB) and Bredwell et al. [24], that is, 0.2–0.3 m for a nonionic surfactant, Tween 20. It should be noted that FF-TEM observation is a direct measurement of the film thickness at the particular time, and therefore it is a reliable measure of the size ranges. It has been shown through discussion above that the CLAs and HIPREs display similar structural model. The structure of
the HIPREs has been elucidated some years ago using a serious techniques of Video-enhanced microscopy (VEM), TEM, SAXS/SANS (small angle neutron scattering) and PGSENMR (pulsed-field-gradient spin-echo NMR) experiments [25–35]. The HIPREs consist of two isotropic liquid phases: the dispersed phase is composed of oil droplets, and the continuous phase is an O/W micelle or microemulsion or a lamellar liquid crystal. In contrast to earlier findings on HIPREs, we could find that both the CLAs and HIPREs present an identical biphasic structure. As for CLAs, the supramolecular structures exist both the oil- and water-rich phases; in contrast, the aggregates occur only at the water-rich region for HIPREs. This difference is the cause of absence of C12 E3 at the oil phase for the latter. These supramolecular structures present various forms including micelles, microemulsions,
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phases, respectively. The total interfacial area of stable CLAs has an approximate thickness of 0.3–0.4 m, which is consistent with that of CGAs reported previously. In addition, it has been shown throughout this work that CLAs and HIPREs display similar characteristics with respect to overall structure, with biphasic structure and presence of the supramolecular structures in these phases, which seems to support Princen’s opinion [11]. There are, however, a number of significant differences in the formulation and manufacture of the two systems [12]. Accordingly, it is too early to make any definite statements. In order to get some conclusive evidence on microstructure of CLAs and HIPREs, we need to employ more techniques such as DSC, FT-IR, PGSE-NMR and SANS in the future. Fig. 5. SAXS curves of various CLAs. CLAs system is composed of AEO3 (1%, v/v)/n-decane/SDS (0.5%, w/v)/water, PVR of which are 2, 3, 4, 8 and 16, respectively; CLAs-Tween system consists of AEO-3 (1%, v/v)/ndecane/Tween 80 (0.5%, w/v)/water, PVR of which are 4 and 8, respectively; Emulsion system is made of n-decane/SDS (0.5%, w/v)/water, PVR of which is 4. The notes for Fig. 6 are same like in (5).
Acknowledgement This project has been financially supported by the National “Shiwu” Key Technologies R&D Programme of China (No.2004BA61010).
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miniemulsions and lamellar liquid crystals, depending on the nature and composition of the amphiphile in the system, ionic strength, and external conditions such as temperature. The two most frequently encountered structures in thin liquid soap films are repeating units of surfactant bilayers or ordered arrays of micellar domains [20].
4. Conclusions The FF-TEM investigations carried out in this study reveal new important information on the microstructure of CLAs. The micrographs show that the CLAs consist of spherical, oil droplets (oil-rich phase) of micron size separated by an aqueous “soapy shell” (water-rich phase) and there exist supramolecular structures such as inverse micelles and micelles or O/W microemulsions in oil-rich and water-rich
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