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Brij30 water-in-oil microemulsions
Journal of Colloid and Interface Science 306 (2007) 143–153 www.elsevier.com/locate/jcis
Unusually large acrylamide induced effect on the droplet size in AOT/Brij30 water-in-oil microemulsions Allan K. Poulsen a,∗ , Lise Arleth b , Kristoffer Almdal c , Anne Marie Scharff-Poulsen d a Celcom, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark b Biophysics, Department of Natural Sciences, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark c Risø National Laboratory, Danish Polymer Centre, P.O. Box 49, Frederiksborgvej 399, DK-4000 Roskilde, Denmark d Risø National Laboratory, Biosystems Department, P.O. Box 49, Frederiksborgvej 399, DK-4000 Roskilde, Denmark
Received 27 June 2006; accepted 21 October 2006 Available online 26 October 2006
Abstract Droplet microemulsions are widely used as templates for controlled synthesis of nanometer sized polymer gel beads for use as, e.g., nanobiosensors. Here we examine water-in-oil microemulsions typically used for preparation of sensors. The cores of the microemulsion droplets are constituted by an aqueous component consisting of water, reagent monomer mixture, buffer salts, and the relevant dyes and/or enzymes. The cores are encapsulated by a mixture of the surfactants Brij30 and AOT and the resulting microemulsion droplets are suspended in a continuous hexane phase. The size of the final polymer particles may be of great importance for the applications of the sensors. Our initial working hypothesis was that the size of the droplet cores and therefore the size of the synthesized polymer gel beads could be controlled by the surfactant-to-water ratio of the template microemulsion. In the present work we have tested this hypothesis and investigated how the monomers and the ratio between the two surfactants affect the size of the microemulsion droplets and the microemulsion domain. We find that the monomers in water have a profound effect on the microemulsion domain as well as on the size of the microemulsion droplets. The relation between microemulsion composition and droplet size is in this case more complicated than assumed in standard descriptions of microemulsions [R. Strey, Colloid Polym. Sci. 272 (1994) 1005–1019; I. Danielsson, B. Lindman, Colloids Surf. 3 (1981) 391–392; Y. Chevalier, T. Zemb, Rep. Progr. Phys. 53 (1990) 279–371]. © 2006 Elsevier Inc. All rights reserved. Keywords: Microemulsion; Acrylamide; Nanobiosensors; AOT; Brij30; Polymer particles; Nanoparticles; Modulation of micelle size
1. Introduction With the increasing desire to control the size and shape of nanoscale structures, microemulsion-templated synthesis have gained increased attention over the last decade. Microemulsions are defined as homogeneous, optically transparent, and thermodynamically stable mixtures of oil and water stabilized by surfactants [2]. They are exploited in polymer chemistry where their well-defined nanoscale structure is used to control the synthesis of materials of certain morphologies and characteristics [4,5]. Microemulsion templated synthesis has a wide range of applications going from preparation of new types of copolymers [6,7], particles for drug delivery [8], preparation of metal * Corresponding author. Fax: +45 6550 2467.
E-mail address: [email protected] (A.K. Poulsen). 0021-9797/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2006.10.048
clusters [9,10], to formation of polymer beads with a narrow polydispersity [11,12]. In particular, microemulsion templated synthesis has been exploited to prepare nanosized sensoring devices for use in, e.g., living cells [13]. Water in oil (w/o) microemulsions are microemulsions that have a microstructure of surfactant encapsulated water droplets suspended in a continuous oil-phase. The radius of the droplets is typically on the order of 5 nm and the size of the droplets is generally controlled by the surfactant-to-water ratio of the microemulsion [1]. The w/o microemulsions are interesting as media for polymer synthesis because hydrophilic reagents may be dissolved and brought to reaction in the aqueous cores of the droplets [14]. The microemulsion droplets can therefore be regarded as nanoreactors with a size that is, in principle, directly controllable. It is well documented in the literature that microemulsion droplets may be used as reaction media and
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templates in polymerization reactions and that they allow for synthesizing well-defined polymer beads with radius less than 50 nm [15], for review read, e.g., Pavel [16]. Polymerization of hydrophilic monomers in confined water droplets makes it possible to incorporate hydrophilic compounds [17] such as compound specific fluorescent dyes [18,19] and proteins [20,21] into the polymer matrix. Provided that the components do not react with the monomer, incorporation happens by physical entrapment in the polymer network, however, covalently attachment of fluorophores to the polyacrylamide matrix is also an option [22]. The technique serves as a basis for a family of optical nanosensors for analysis of single cell metabolic dynamics [19,23–28] and preparation of polyacrylamide beads in microemulsion is applied to embed hydrophilic sensing molecules and enzymes for use as nanobiosensors; for a recent review see Buck et al. [29]. The present study aims at preparing nanosized polyacrylamide beads by polymerizing the hydrophilic monomers acrylamide (AA) and N,N -methylenebisacrylamide (MBA) in the interior of the droplets formed in w/o microemulsions, where the continuous oil-phase is hexane and where a mixture of the surfactants AOT and Brij30 is used to encapsulate the droplets and stabilize the microemulsion. It is generally accepted that the microstructure and phase behavior of microemulsion-systems are controlled by their composition and the curvature free energy of the surfactant layer separating the oil and the water components [1,30]. At fixed temperature and pressure, the phase behavior of a simple surfactant/water/oil microemulsion-system may be represented in a ternary phase diagram [1]. However, addition of polymerizable components to the aqueous component increases the degrees of freedom and therefore the complexity of the system. In the present article we will refer to the mixture of buffer salts, AA, MBA and H2 O as the aqueous component and we will generally consider the pseudo-ternary system consisting of surfactant, the aqueous component and the oil. We will investigate the effects on the ternary phase diagram of the addition of different components to the aqueous component. Initially we had the working hypothesis that the polymer bead size was controlled by the size of the aqueous droplets in the w/o microemulsion [31], which, again was controlled by the ratio of surfactant to aqueous component. Due to the large parameter space given by the number of components forming and affecting the system we decided to focus on the part of the phase diagram where it has been previously shown [15,21] that one-phase w/o microemulsions form and polymer beads can be synthesized. The present study demonstrates that addition of the monomers AA and MBA has a strong effect on the extent of the microemulsion domain and on the microemulsion droplet size. The study also demonstrates that, upon addition of monomers to the aqueous component of the droplets, the assumption that the droplet size can be predicted by the ratio of surfactant-toaqueous component apparently breaks down and particles that are significantly larger than theoretically predicted are formed. Thus apparently, the system no longer exhibits standard microemulsion behavior and radius of micelles in microemulsion
is no longer given by the molar ratio of solubilized component per surfactant [32]. As it will be explained in the following we have searched for an explanation to this observation, however, the question remains open. 2. Experimental 2.1. Materials and methods Brij30 (polyoxyethylene-4-lauryl ether) was obtained from SIGMA. C12 E4 , 99+% were obtained from Nikko Chemicals, Japan. AOT or aerosol OT (sodium bis-2-ethylhexylsulfosuccinate, 98% purity), acrylamide (99+%, electrophoresis grade), hexane (95+%, HPLC-grade), N ,N -methylenebisacrylamide (99+%, electrophoresis grade) were obtained from Aldrich, Germany and used as supplied. 10 mM sodium phosphate buffer, pH 7.2, was prepared in Milli-Q water using a mixture of Na2 HPO4 and NaH2 PO4 to obtain the desired pH. The buffer salts were obtained from Merck, Germany. Sulforhodamine 101 was obtained from Invitrogen, Molecular Probes, Oregon, USA. Brij30 is a mixture of components H(CH2 )n O–(CH2 CH2 O)m –H with significant amount for n = 10, 12, 14, 16 and m = 0, 1, . . . , 9 and stated from the manufacturer to give an average of C12 E4 . The mass fraction of each component has been determined using normal phase HPLC and gas chromatography coupled with mass spectrometry (GC-MS). Once these mass /Mn can be calculated from Mw /Mn = fractions are known Mw 2 M m m /M /( i i i i i i i mi ) , where Mi and mi are the molar mass and mass of component i. For Brij30 we found that Mn = 323.3 g/mol and Mw /Mn = 1.075. For the H(CH2 )n OH fraction Mn = 186.9 g/mol and Mw /Mn = 1.0006. For the –(CH2 CH2 O)m – part of the molecule Mn = 174 g/mol and Mw /Mn = 1.32. A detailed analysis of Brij30 is in preparation and will be published elsewhere. For the microemulsion preparations, we first dissolved the surfactants in hexane. Then the desired amount of aqueous component was added to the solution of hexane and surfactant. The aqueous component consisted of buffer with or without dissolved AA and MBA. Two procedures were used for studying the phase boundaries of the microemulsion region of the phase diagram: Either the aqueous component was titrated with a solution of surfactants in hexane until a homogeneous transparent microemulsion was obtained or an existing microemulsion was titrated with the aqueous component until phase separation occurred. All experiments were carried out at 25 ◦ C. 2.2. Surface tension measurements The Du Nouy-method was used for determination of surface tension measurements using a “Digital Tensiometer” from Krüss (Hamburg, Germany). The critical micelle concentration (cmc) and molecular area per head group for Brij30 was determined from the surface tension measurements on concentration series of the Brij30 in buffer with and without monomers [33].
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2.3. Dynamic light scattering The hydrodynamic radius of w/o microemulsion droplets was determined by dynamic light scattering (DLS) using a BI200SM goniometer from Brookhaven Instruments that incorporates a 632.8 nm He–Ne laser. The instrument was operated at a fixed scattering angle of 90◦ . The temperature was kept constant with a thermostatted water bath operating at 25 ◦ C. The standard analysis software of the instrument was used for analyzing the data. Unimodal size distributions were generally found and the 2 order cumulant analysis method could be applied to analyze the experimentally obtained autocorrelation function and hence determine the hydrodynamic radius and polydispersity of the droplets [34]. The droplets studied in the present project are sufficiently small that the obtained results show no significant dependence on scattering angle.
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the mixture are expressed as the weight fraction of the oil in the mixture of oil and buffer: α ≡ B/(A + B + E). The total weight fraction of the non-ionic plus ionic surfactants in the system: γ ≡ (C + D)/(A + B + C + D + E). The weight fraction of ionic surfactant in the mixture of the two surfactants: δ ≡ D/(C + D). And the weight fraction of buffer salt in the aqueous component (i.e. the mixture of H2 O, including monomers, and buffer-salt): ε ≡ E/(A + E). Phase behavior in three-dimensional space can be obtained by keeping α and δ constant and varying T , ε, and γ and hence determine the extension of the three-phase body of emulsion and microemulsion. We used this approach to study temperature stability of the microemulsion at different δ-values, as well as to investigate the effect of exchanging water with buffer or adding monomers to the aqueous component. 3. Results and discussion
2.4. Ultracentrifugation
3.1. Temperature and salt dependence of the phase behavior
Ultracentrifugation was used to gain information about the continuous phase and cmc of AOT. Runs were made at 20 ◦ C with a Sorvall Discovery 90SE ultracentrifuge and a 70TI rotor at speeds up to 50.000 rpm. When a water-in-oil microemulsion is subjected to a centrifugal field the microemulsion droplets move away from the centre of rotation because their density is larger than the continuous phase. As a result a region near the centre of rotation is cleared of drops and a distinct region forms between the cleared region and the remaining microemulsion. We measured the content of AOT in the cleared region using FTIR spectrometry. As control experiment to ensure that ultracentrifugation left the continuous phase without any watercontaining droplets in the cleared region we centrifuged down microemulsions to which we added the hydrophilic dye sulforhodamine 101 and measured the cleared region with fluorescence [35].
The central motivation for starting our examination of microemulsions was the possibility for controlling the size of the final polyacrylamide beads via the composition and hence the size of the microemulsion droplets. Microemulsion-systems have a rich and complex phase behavior: The sensitivity of microemulsions towards variations of temperature and buffer salt concentration is controlled in a non-trivial fashion by the chemical nature and mixing ratio of the applied surfactants as well as the types and compositions of respectively apolar and polar phases, added salt and other additives [36,39–41]. However, the region in the phase diagram where microemulsions form is important to know for practical applications of droplet microemulsions as nanoreactors. Consequently we performed a preliminary examination of the effect of buffer salts and monomers on the microemulsion region. The temperature and salt dependence of the phase behavior of our pseudo-ternary system was examined by preparing a series of microemulsions in the classical Kahlweit fish-cut [37,40]. Hence, the microemulsions were symmetric in the volume fractions of oil and water, corresponding to α = 50 wt%. We used δ = 66 wt% corresponding to a weight ratio of (μ = AOT/Brij30, w/w) μ = 1.94/1. Several weight fractions of surfactant in the system were investigated and γ ranged from 0.5 to 20 wt%. The concentration of the buffer salts was varied for each γ -value and the salt concentration in terms of ε ranged from 0–7.3 wt%: This corresponds to buffer salt concentrations from 0–300 mM. The phase behavior of each sample was investigated from melting point to boiling point. For all temperatures and sample compositions the samples separated into two phases where the upper phase remained as a translucent mixture of surfactants, aqueous component and hexane in equilibrium with the lower water rich phase that had a clouded appearance. We found that the mixture is quite stable towards variations in temperature and salt, and we did not observe any phase inversion in the whole range of temperatures, buffer salt concentrations, and surfactant contents. We did observe slight changes in the volumes of the two phases as temperature was changed, however, the changes were small and not significant. The same observa-
2.5. FTIR spectrometry Quantification of the amount of AOT in microemulsions and in hexane was performed on a Perkin–Elmer Spectrum One spectrometer. For quantification we used the intensity of the peak at 1737 cm−1 caused by the carbonyl group in the ester bond in AOT. We verified by known concentrations of AOT in hexane, that we were able to recover and quantify AOT in microemulsions prepared of buffer, AOT, Brij30, AA, MBA, and hexane. 2.6. Phase behavior studies and determination of the microemulsion domain Phase behavior of the system; H2 O including AA and MBA (A), hexane (B), Brij30 (C), AOT (D), and buffer salts (E) was examined with regard to temperature and the weight percentage of surfactants in the system at various buffer salt concentrations. In the following we will use the variables and nomenclature as they have been introduced by Kahlweit et al. [36–40] who have also described the method thoroughly. The variables in
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tion was made when the aqueous component was loaded with the monomers AA (3.46 M) and MBA (0.472 M). The separation into two stable phases and the nearly constant ratio between the volumes of the two phases indicate that we never reached phase inversion nor the three-phase body as temperature was increased. AOT is the major component of the two surfactants and the phase behavior of the system is, as in line with those observed by Kahlweit et al. [37], similar to that of a system of pure AOT below phase inversion temperature. Furthermore, temperature and salt insensitivity has been found in similar systems and is most likely a property of the mixture of AOT and Brij30 as well as the ratio μ [32,42]. From this we concluded that the presence of monomers and the concentration of buffer salts did not have a significant effect on phase inversion temperature, but also that the fish-method described by Kahlweit et al. [36,39–41] is not practically applicable for determination of surfactant efficiency in the present system and can therefore not be directly applied to find the relationship between solubilization efficiency and content of monomers or salts in the aqueous component. The central, but indirect, result of this experiment is that the system has a very weak temperature dependence and that temperature effects can be neglected in the temperature range (20–35 ◦ C) where the microemulsion is used for polymerization. 3.2. Monomers affect surfactant solubilization capacity The microemulsion domain of the surfactants/aqueous component/hexane system is indicated in the pseudo-ternary-phase diagram shown in Fig. 1. The phase diagram was determined at room temperature and at the fixed surfactant weight ratio μ = 1.94/1. The aqueous component is a 10 mM 7.2 sodium phosphate buffer in which AA (3.46 M) and MBA (0.472 M)
Fig. 1. Pseudo-ternary-phase diagram for the surfactants (AOT/Brij30, 1.94/1, w/w)/aqueous-component/hexane system showing the microemulsion domain determined at 25 ◦ C. The aqueous component is a sodium phosphate buffer solution (10 mM, pH 7.2) in which AA (3.46 M) and MBA (0.472 M) are dissolved. The boxes indicate the compositions where we observed phase separation. The continuous line is drawn through the experimental results and inserted to guide the eye and delineate the one-phase microemulsion which is the area at right to the line. X refers to the composition often used for polymerization reactions and preparation of nanobiosensors.
are dissolved. The obtained phase diagram is quite similar to the one obtained by Daubresse et al. [21], despite that this group has used a different AOT/Brij30 ratio (3/1, w/w). The observations are also consistent with previous findings that polarity of the continuous phase has a significant effect on the microemulsion domain when monomers are present in the aqueous component [15,43–46]. The interplay between solutes in the aqueous component, surfactants, and the continuous phase is of major importance when it comes to solubilization capacity of the surfactants. The proposed explanation is that the more polar solvent molecules may localize themselves in the surfactant layer separating the w/o interface and thereby act as co-surfactants [15,43,46,47] and help increasing the solubilization capacity of the system. As seen from Fig. 1, the microemulsion domain is characterized by a relatively high ratio of surfactant/aqueous component, i.e. it takes relatively much surfactant to form a microemulsion in this system. The microemulsion composition used for polymerization is indicated in the plot with an x. It is composed by: surfactants (12.7 wt%; AOT/Brij30, 1.94/1, w/w), hexane (81.8 wt%), and aqueous component (5.5 wt%). The composition lies close to the maximum solubilization capacity. This should, in principle, allow for forming the largest possible droplet sizes. We have found that this composition is well suited for encapsulation of proteins and sensoring dyes [22] and it is very similar to the microemulsions previously used for comparable work on polymerization of nanobiosensors [17,48]. The monomer concentration in the aqueous component is important to the properties of the final polymer gel particles. We therefore determined the phase behavior (see Fig. 2) for a series of different weight fractions of the monomers (abscissa-axis) and surfactant/aqueous component weight ratios (ordinate-axis). As it is clearly seen, addition of monomers to the aqueous component also has a major impact on the microemulsion solubilization capacity and hence the microemulsion domain. When the weight fraction of monomers in the aqueous component is increased from 0.0 to 0.45 the solubilization capacity of the surfactants decreases from 0.5 g of surfactant per gram of the aqueous component to 2.5 g of surfactant per gram of the aqueous component. In order to ensure that, e.g., slow equilibrium kinetics did not affect the determination of the phase transition, two different approaches for determining the phase boundary between the translucent homogeneous microemulsion and macroscopic phase separation were utilized. As it may be seen from Fig. 2, it did not have any significant effect on the determination of the phase boundary how it was approached and the two data sets are indistinguishable. It is well documented that Brij30 has a great impact on the solubilization capacity for the monomer-containing microemulsion [21]. In line with this observation it was speculated whether the large decrease of the solubilization capacity of the surfactants could be driven by a change of the surface active properties of one of the surfactants, either AOT or Brij30, upon addition of monomers. This suggested determining a series of phase diagrams similar to the one of Fig. 2, but for variable ratios of AOT to Brij30. We varied the composition of surfactant from 100% pure AOT to mostly Brij30 by titrating from
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ubilization capacity mainly had to do with the surface active properties of the AOT. 3.3. Microemulsion composition versus microemulsion droplet size According to the standard picture of droplet microemulsions, see, e.g., Chevalier and Zemb [3], the radius, r, of the droplets is basically determined by the surface to volume ratio of the droplets. It is assumed that the macroscopic ratio of surfactant to enclosed phase is the same as the ratio in the single droplets. Thus, if the head-group area of the applied surfactants are known, the average radius of the aqueous droplet cores, raq , may be calculated, from [3]: raq = Fig. 2. Phase boundary between one-phase microemulsion and phase separation given for surfactants (AOT + Brij30)/aqueous-component/hexane microemulsion. The solubilization capacity of surfactants is affected by presence of the hydrophilic monomers AA and MBA and decreases as the monomer concentration is increased. This phase diagram was determined by conducting a series of titration experiments at fixed temperature (25 ◦ C) and phase boundary was approached from either side: (1) going from a two-phase system to a one-phase system by titration of hexane containing surfactant into the aqueous component until the mixture turned into a translucent 1-phase system; (!) going from a one-phase system to a two-phase system by titration of the aqueous component into the hexane/surfactant mixture. The ratio μ = mAOT /mBrij30 and the surfactant/hexane ratio were kept constant at μ = 1.94/1, and 0.1086 g surfactant per ml of pure hexane, respectively. The amount of aqueous component and the total concentration of monomers within the aqueous components were varied systematically until the boundary between the 1- and 2-phase regions could be observed. The ratio of AA to MBA monomers was 3.375/1 in these experiments.
the two-phase region and into the one-phase region. The obtained phase diagrams are shown in Figs. 3a–3e and combined in Fig. 3f. It is seen that pure AOT has a higher solubilization capacity with respect to the monomer-free phosphate buffer than the AOT/Brij30 mixture. In the pure AOT microemulsions, 1 g of AOT solubilizes approximately 4 g of the phosphate buffer, but when the concentration of monomers increases to 0.45 (w/w), 1 g of AOT solubilizes only 0.3 g of the aqueous component. The effect of monomer in the aqueous component decreases as the AOT/Brij30 weight ratio μ decreases. However, the four studied AOT/Brij30 ratios have comparable solubilization capacities when it comes to the 0.45 (w/w) monomer loaded aqueous component. When the AOT/Brij30 ratio is decreased to the smallest possible value that still allows for forming microemulsions, that is μ = 0.12/1, the effect of the presence of AA and MBA monomers in the aqueous component nearly vanishes. Unfortunately, the μ = 0.12/1 microemulsions generally have a very low solubilization capacity: 1 g of surfactant only solubilizes 0.4 g of aqueous component, independent of the concentration of monomers. Hence, besides from demonstrating that there is a strong dependence between microemulsion solubilization capacity and the concentration of monomers in the aqueous component, this series of experiments also suggested that the change of sol-
3Vaq , Asurf
(1)
where Vaq is the volume of the enclosed aqueous phase and Asurf is the total surface area of the surfactant layer. Since the head-group areas of surfactants are a weak function of the condition under which they are used, it is generally reasonable to assume a constant head-group area at the water–surfactant interface. Under this assumption, a straightforward geometrical calculation leads to the following expression for the outer radius, router , of the water + surfactant droplet: 1/3 2 n 3raq surf 3 + , router = raq asurf
(2)
where nsurf is the (partial specific) molecular volume of a single surfactant molecule and asurf is the area per head group of a single surfactant molecule. For the mixed AOT/Brij30 system, averaged values for nsurf and asurf are calculated from: nsurf = XAOT νAOT + XBrij30 νBrij30 and asurf = XAOT aAOT + XBrij30 aBrij30 , where XAOT and XBrij30 are the mole fractions of AOT and Brij30, respectively. For our calculations of the droplet radius we used the following parameters: aBrij30 = 45 Å2 , as found by surface tension measurements (Table 1) and in agreement with Kjellin et al. [49] and aAOT = 55 Å2 [50,51]. The molecular volumes for AOT and Brij30 were calculated from their corresponding mass densities. We used νBrij30 = 633 Å3 and νAOT = 649 Å3 per molecule. If it is assumed that the critical micelle concentration (cmc) is equal to the concentration of free surfactant molecules that are not located in the surfactant film, a cmc-effect may be included in the calculations by calculating Asurf in Eq. (1) from Asurf = (csurf − cmc)asurf . It is seen from Eq. (1) and Eq. (2) that for constant volume of the aqueous component and constant amount of surfactant, the microemulsion droplet size is in theory constant. The equations have been verified experimentally for a number of systems, see, e.g., [3] and references therein. Figs. 2 and 3 show that the amount of aqueous component, which can be solubilized by the microemulsions, is substantially reduced when monomer is present. According to Eq. (1) and Eq. (2), this implies that the maximum stable w/o microemulsion droplet size is reduced. Alternatively the surface area is reduced which conceivably can
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(a)
(b)
(c)
(d)
(e)
(f)
Fig. 3. Phase boundary between one-phase microemulsion and phase separation given for surfactants (AOT + Brij30)/aqueous component/hexane microemulsion at various monomer concentrations and for various AOT/Brij30 ratios: μ = mAOT /mBrij30 . (a) μ = 1/0 (pure AOT); (b) μ = 1.94/1; (c) μ = 0.80/1; (d) μ = 0.52/1; (e) μ = 0.12/1, and (f) shows lines fitted to the data points shown in Figs. 3a–3e to summarize the effect of the AOT/Brij30 ratio. The numbers in (f) indicate the ratio μ. Hexane was added to the aqueous component until phase separation. The hexane contained 0.1086 g surfactant per ml and the aqueous component contained AA and MBA in the ratio 3.375/1 (w/w). The measurements were performed at fixed temperature (25 ◦ C).
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Table 1 Critical micellar concentration, cmc and surface head-group area, Ahead , for Brij30 in buffer solution with and without AA and MBA monomers
Brij30 in buffer without monomers Brij30 in buffer with monomers
cmc [mM]
Area head [Å2 /molecule]
0.021 0.716
45 (±10) 53 (±5)
The results are obtained by measurements of surface tension as described in Section 2.2.
only happen if surfactant is removed from the water/oil interface. DLS has often been used for determination of droplet size in microemulsions and results have been verified and discussed in comparison with other methods for droplet size determination and structure studies, e.g., SANS and SAXS, see [52–56] and references therein. Here DLS was used to investigate whether the given system followed Eqs. (1) and (2). The droplet size was measured for a series of samples where the amounts of aqueous component, surfactants, and hexane were kept constant and where the concentration of monomers was gradually increased. The obtained hydrodynamic radii are plotted as a function of the concentration of monomers in Fig. 4. The data indicate that the monomer concentration has a tremendous effect on the experimental droplet sizes. The hydrodynamic radius increases from 4.4 nm in the monomer-free sample to 42 nm in the most monomer-rich sample. It should be emphasized that all samples remained as a homogeneous translucent microemulsion throughout the experiment. The same samples were re-measured at the same instrument after several weeks of storage and no significant changes were observed. The observed non-linearity of the droplet sizes as a function of monomer concentration show that our system does not follow Eqs. (1) and (2). It should be emphasized that the obtained DLS results clearly indicated that the droplets had unimodal size distributions. We hypothesized that the observed non-linearity of the microemulsion droplet sizes was related to a polydispersity of Brij30, see Section 2.1, and prepared a similar series of samples, where Brij30 was replaced by C12 E4 . The results are plotted in Fig. 4. As it may be seen, the observed dependence between hydrodynamic radius and monomer concentration was fully reproduced when Brij30 was replaced by C12 E4 . Hence, a non-linear effect on w/o microemulsion droplet size due to polydispersity of Brij30 can be ruled out. When performing medium or large scale synthesis, the monodisperse C12 E4 is often substituted with the less expensive and polydisperse alternative, Brij30. Fig. 4 suggests that this can be done without any significant effect on the resulting droplet size. The series of samples of Fig. 4 corresponds to moving along a horizontal line in the binary phase diagram of Fig. 3b. When moving along such a line, the distance to the boundary between the one-phase and the two-phase system decreases with the increase of the monomer concentration in the aqueous component. Even though this ought not to have any effect on the droplet size (according to Eqs. (1) and (2)), it was hypothe-
Fig. 4. The hydrodynamic droplet radius of the microemulsion droplets measured at increasing monomer concentration. (1) AOT and Brij30 and (!) AOT and C12 E4 . The overall sample composition used in this experiment was; surfactants (10.6 wt%; AOT/Brij30, 1.94/1, w/w), hexane (83.5 wt%), and aqueous component (5.7 wt%). The aqueous component was a 10 mM pH 7.2 Na-phosphate buffer where the concentration of monomers was varied from 0 M to 3.47 M AA and 0.474 M MBA, the ratio between AA and MBA was kept constant at 3.375/1 w/w.
Table 2 The amount of aqueous component that the microemulsion is able to solubilize as concentration of monomers is varied in the aqueous component Monomer in aq. comp. [wt%]
Aq. comp. in microemulsion [wt%]
0.0 0.1 0.2 0.3 0.4
24.0 10.2 7.3 5.7 5.3
The mass of hexane and surfactants are kept constant. It is seen that solubilization capacity of surfactants decreases dramatically as monomer content is increased. The AOT/Brij30 ratio is 1.94/1. The aqueous component contained AA and MBA in the ratio 3.375/1 (w/w). The results are obtained at 25 ◦ C.
sized whether it could have an effect on the droplet–droplet interactions and therefore give rise to an apparent increase of the droplet radii. In order to test this hypothesis, four series of samples were prepared. In each series the monomer concentration was varied while the distance to the one-phase/twophase boundary was kept constant by loading the microemulsions with 50, 70, 80 and 90% of the amount of aqueous component at the one-phase/two-phase emulsion boundary, respectively, as found in Fig. 2, see Fig. 5b and Table 2. The hydrodynamic radii of the resulting microemulsion droplets were measured by DLS and the results are plotted in Fig. 5a. The volumes of the aqueous component was relatively low in these experiments, see Table 2 and never constituted more than 20 wt% during the experiments in the most extreme case. As it may be seen from Fig. 5a the experimentally observed hydrodynamic radius was basically constant within each of the series. At 50% load an average droplet radius of 6.2 nm was
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The theoretical w/o microemulsion droplet size for the same compositions have been calculated according to Eqs. (1) and (2) and is plotted in Fig. 5b. As the monomer content increases, the solubilization capacity of the microemulsions decreases. This obviously leads to a decrease of the theoretical droplet size. Thus, theoretically, and as shown in Fig. 5b, it is expected that the droplet sizes should decrease when the monomer concentration is increased in each of the series. Clearly, this is not what we observe. Hence, for fixed overall surfactant composition, an increase of the concentration of monomers in the aqueous component induces a strong and systematic increase of the hydrodynamic droplet radius. Furthermore, iso-radius lines, that is, lines of nearly constant hydrodynamic droplet radii, may be observed if the monomer load is varied for constant distance to the boundary between the one-phase and two-phase regions. (a)
3.4. The breakdown of microemulsion behavior A sufficiently large cmc, which furthermore depends on the concentration of monomers in the aqueous component, may provide an explanation of the atypical behavior of both phase diagrams and growth of droplet radii. In order to investigate such a possible effect, the cmc values of the two different surfactants in the different components were studied in more detail. This study was partly based on literature data and partly on our own experimental work.
(b) Fig. 5. (a) Experimental hydrodynamic radius of monomer-containing microemulsion droplets. The effect on the droplet radius of adding monomers is measured for (P) 50%, (1) 70%, (E) 80% and (!) 90% of maximum solubilization capacity of the surfactants in one-phase microemulsion at the given amount monomer. The AOT/Brij30 ratio was 1.94/1, the ratio of AA to MBA was 3.375/1, and the overall volume of hexane and mass of surfactants was constant. (b) The theoretical droplet size calculated at each component and connected with lines to guide the eye.
found, at 70% load an average droplet radius of 11.3 nm was found, at 80% a droplet radius of 17.4 nm was found and at 90% load the droplet radius had increased to 32.4 nm. The droplet polydispersity as determined by DLS remained low (σ/Rav ≈ 10%) and constant throughout the series. Thus the results are in good agreement with the surprising observations of Fig. 4 that the hydrodynamic radius increases significantly as the emulsification one-phase/two-phase emulsion boundary is approached. Another rather surprising observation is that the experimental hydrodynamic radius appears to be determined by the distance to the boundary between the one-phase and two-phase regions.
3.4.1. Brij30 Presently, we have no means for determining the Brij30 concentration in the water cores of the microemulsion droplets. However, as a first approximation it is reasonable to assume that it equals the Brij30 concentration in bulk water (or bulk monomer buffer solution when relevant). The cmc of Brij30 in monomer-free buffer and in buffer containing 4.44 M AA and 0.10 M MBA. (WAA + WMBA = 0.33), respectively, was determined from a series of surface tension measurements. The results are summarized in Table 1. It is seen that the cmc of Brij30 increases strongly (a factor of 34) when AA and MBA is present in the buffer at the given concentration. We do not observe a significant change of the area per head group. For the microemulsion preparations studied so far, we have typically used 400 mg Brij30 per 1 ml of aqueous component. This corresponds to a molarity of Brij30 in aqueous component of ∼0.8 M and implies that even with the large cmc-increase, the effect of the surfactant removed from the surface area when increasing the monomer concentration is negligible. It will only induce a decrease of the surface area to 99.97% of the area in the corresponding monomer-free sample. Hence, with respect to Brij30, a cmc effect is not relevant and not able to explain the decreased solubilization capacity of surfactants. Brij30 does not make any ordered structure as globular micelles, however the solubility of Brij30 in pure hexane is high [57,58]. 3.4.2. AOT An AOT driven cmc effect was also considered. The cmc for AOT is 2.23 mM and 1.09 mM in pure water and hexane, re-
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Table 3 Critical micelle concentration (cmc) in the continuous phase of hexane is determined for AOT by use of ultracentrifugation and FTIR spectrometry in microemulsion composed of surfactants (AOT/Brij30, 1.94/1, w/w)/aqueouscomponent/hexane system Acrylamide [M]
N ,N -methylenebisacrylamide [M]
cmc [M]
3.46 1.73 0.00
0.47 0.24 0.00
0.001 0.001 0.002
The aqueous component is a sodium phosphate buffer solution (10 mM, pH 7.2) in which AA (3.46 M) and MBA (0.472 M) is dissolved.
spectively [59,60]. However, in order to examine the effect of monomers on the free surfactant concentration of AOT in the continuous phase, we studied microemulsions by a combination of ultracentrifugation and FTIR spectrometry (see further details in Sections 2.4 and 2.5). Our examination was based on the microemulsion composition indicated by x in Fig. 1 and composed of surfactants (12.7 wt%; AOT/Brij30, 1.94/1, w/w), hexane (81.8 wt%), and aqueous component (5.5 wt%). We determined the cmc for AOT in the continuous phase for mixtures where the aqueous component contained 32 wt%, 16 wt%, and no monomers, respectively. The results are summarized in Table 3. For the sample with monomer-free aqueous component we found a cmc for AOT in hexane of 0.001 M. This is similar to results found in literature [59,60]. The presence of monomers did not significantly affect the cmc of AOT in the continuous hexane phase of the microemulsion (Table 3). Another AOT driven explanation could be a micro-phase-separation of the droplets into two populations of the w/o microemulsion droplets: A population of large droplets/micelles containing the main part of the aqueous component and a population of small almost empty micelles. This explanation was suggested by a small-angle X-ray scattering (SAXS) based analysis of AOT microemulsion that indicated that not all the AOT participated in the droplet formation, instead some of it self-organized into small “dry” micelles in the alkane solution [61,62]. By dynamic light scattering (DLS) a population of weakly scattering small “dry” micelles would be difficult to detect among a population of larger and more strongly scattering microemulsion droplets. Instead, the hypothesis was tested in the ultracentrifugation experiments. Using the difference in mass density between the water filled micelles and the dry micelles two such populations could be separated by ultracentrifugation: If small dry micelles were present we would find a high AOT concentration in the continuous phase of the centrifuged samples. However, the concentration of AOT in the continuous phase turned out constant and remained at the cmc independent of the concentration of AA and MBA. No indications of a population of the above mentioned small dry micelles that could explain our observations were found. An unusual growth of microemulsion droplet sizes with monomer contents has previously been detected on similar AOT based microemulsion systems by Candau et al. [15] and Munshi et al. [31]. However, the extent of the growth and the effect of a co-surfactant was not studied in detail. In the previous work it was suggested that the increase in radius is due to a
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monomer induced enhancement of attractive forces between the droplets, which makes the w/o microemulsion droplets coalesce and form dynamic clusters of droplets. In more detail, it was proposed that the monomers partly localize themselves in between the hydrocarbon chains of the surfactants and therefore induce a disorganization of the surfactant interface which leads to an effective increase of the “stickyness” of the droplets [51,63,64]. Findings that support this theory is that addition of AA to a microemulsion comparable to ours has shown to increase microemulsion viscosity [65] and it has been found experimentally by Bergenholtz et al. [64] that the increased viscosity seems to be caused by an increased disorganization at the water/surfactant interface and that this results in increased attractions between the droplets as well as a lower percolation threshold. As DLS do not have a resolution that directly allows us to distinguish between a large droplet and a cluster of smaller droplets, the cluster-model may also consistent with our findings. However, it would be most probable that the polydispersity of the clusters was different from that of the single microemulsion droplets, and, as a matter of fact, we observe no significant changes of the polydispersity of the droplets in relation to the growth of the particle radius. Thus the DLS results for the droplet sizes and their polydispersities do not contain any indications in support of the cluster-model and a more detailed structural analysis will be necessary in order to verify or reject the hypothesis. Small-angle scattering is an obvious choice of experimental method for such a study. However, this lies outside the scope of the present work. Hence, the apparent breakdown of the standard microemulsion behavior could be naturally explained by either a large cmc effect, or a microphase separation of the microemulsion droplets into a population of small dry micelles and larger water filled microemulsion droplets. However, our studies show that none of these effects are sufficiently large to explain the observed behavior. Another explanation could be provided by increased attractive interactions between the droplets leading to a cluster formation. However, our DLS results do not contain any indications in favor of this explanation. 4. Conclusion The phase behavior and droplet sizes of a w/o microemulsion composed of hexane, AOT, Brij30, and an aqueous component containing monomers (AA and MBA) and buffer salts were studied. The w/o microemulsion droplets may be applied for polymerization of monomers dissolved in the confined water. Sensoring dyes and functional proteins may be dissolved in the water together with the polymerization monomers and may be incorporated into the synthesized polymer latexes for nanobiosensors [21,48]. In this context modulation of particle size is desirable in order to prepare nanobiosensors for different purposes. The use of w/o microemulsion droplets as nanoreactors is based on the implicit assumption that the system can be understood as a pseudo 3-component system. The 3 components are the oil, the mixture of surfactants and the aqueous component.
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We observed an extremely high sensitivity of the microemulsion towards the presence of the monomers AA and MBA: The solubilization capacity of the surfactants decreases while the apparent size of the w/o microemulsion droplets increase as the concentration of monomers increase. These results were somewhat contradicting and lead us to more systematic studies of the effect of adding monomers to the aqueous component of the microemulsion. Our results show that a mixture of the surfactants AOT and Brij30 has a better solubilization capacity than AOT alone when monomers are present, and the effect of monomers on surfactant solubilization capacity decreases as the AOT/Brij30 ratio decreases. This means that AOT is most strongly affected by the monomers. We found that the monomers did not cause an increase in cmc of either surfactant that is sufficiently large to account for the decreased solubilization capacity and that any effect on w/o microemulsion droplet radius from the polydispersity of Brij30 can be disregarded. It was found by use of DLS that droplet radius increases nonlinearly as monomer content increases, when all ratios between surfactant (AOT/Brij30)/aqueous component/hexane were kept constant and only the content of AA and MBA monomers in the aqueous component was increased. The experimentally found hydrodynamic microemulsion droplet radius is significantly larger than the theoretical droplet radius. The discrepancy between the experimentally found and the calculated w/o microemulsion droplet radii increases with increasing concentration of the monomers in the aqueous component. It has previously been suggested that the apparent increase of the droplet size is due to a monomer induced increase of the attractive forces between w/o microemulsion droplets, so that the droplets get more sticky and form larger aggregates upon addition of AA and MBA [15,31,64,65]. However, our data from DLS studies of microemulsions with varied monomer content show that the polydispersity of the microemulsion droplets remains constant, this makes it less probable that the increase in hydrodynamic radius as monomer content is increased is caused by an aggregation of the droplets. Thus we are presently unable to explain the observed large discrepancy. Ability to modulate the size of functional nanoparticles is a desired feature. If we are able to modulate size of w/o microemulsion droplets it enables us to modulate final polymer particle size [31]. We have shown that the microemulsion is rather temperature stable and that the droplet size is rather temperature independent. However, microemulsion droplet size is highly affected by the monomer concentration in the aqueous component. Unfortunately, monomer concentration is also an important parameter when it comes to pore size and crosslinking in the polymer [66,67]. To nanobiosensors based on encapsulated dyes and enzymes the pore size and the macroporous structure of the final polymer latex may be important to enzyme activity and leaching of the incorporated components [68,69]. The influence of monomers on w/o microemulsion droplets has to be examined further with a medium-to-high resolution technique like small-angle scattering in order to describe the underlying mechanism of the observed unusually large acrylamide induced effect on the droplet size.
Acknowledgments The authors thank Professor Lars Folke Olsen and the Danish Medical Research Council for a fellowship to A.K.P. (Grant no. 22-03-0236) and the Danish Natural Science Research Council for support of the project (Grant Nos. 21-04-054 and 272-05-110). The authors acknowledge support from the Human Frontier Science Program, Grant No. RGP0041/2004C. We thank Professor Reinhard Strey and the staff from his group for useful discussions in the initial phase of the project. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34]
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Unusually large acrylamide induced effect on the droplet size in AOT/Brij30 water-in-oil microemulsions Allan K. Poulsen a,∗ , Lise Arleth b , Kristoffer Almdal c , Anne Marie Scharff-Poulsen d a Celcom, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark b Biophysics, Department of Natural Sciences, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark c Risø National Laboratory, Danish Polymer Centre, P.O. Box 49, Frederiksborgvej 399, DK-4000 Roskilde, Denmark d Risø National Laboratory, Biosystems Department, P.O. Box 49, Frederiksborgvej 399, DK-4000 Roskilde, Denmark
Received 27 June 2006; accepted 21 October 2006 Available online 26 October 2006
Abstract Droplet microemulsions are widely used as templates for controlled synthesis of nanometer sized polymer gel beads for use as, e.g., nanobiosensors. Here we examine water-in-oil microemulsions typically used for preparation of sensors. The cores of the microemulsion droplets are constituted by an aqueous component consisting of water, reagent monomer mixture, buffer salts, and the relevant dyes and/or enzymes. The cores are encapsulated by a mixture of the surfactants Brij30 and AOT and the resulting microemulsion droplets are suspended in a continuous hexane phase. The size of the final polymer particles may be of great importance for the applications of the sensors. Our initial working hypothesis was that the size of the droplet cores and therefore the size of the synthesized polymer gel beads could be controlled by the surfactant-to-water ratio of the template microemulsion. In the present work we have tested this hypothesis and investigated how the monomers and the ratio between the two surfactants affect the size of the microemulsion droplets and the microemulsion domain. We find that the monomers in water have a profound effect on the microemulsion domain as well as on the size of the microemulsion droplets. The relation between microemulsion composition and droplet size is in this case more complicated than assumed in standard descriptions of microemulsions [R. Strey, Colloid Polym. Sci. 272 (1994) 1005–1019; I. Danielsson, B. Lindman, Colloids Surf. 3 (1981) 391–392; Y. Chevalier, T. Zemb, Rep. Progr. Phys. 53 (1990) 279–371]. © 2006 Elsevier Inc. All rights reserved. Keywords: Microemulsion; Acrylamide; Nanobiosensors; AOT; Brij30; Polymer particles; Nanoparticles; Modulation of micelle size
1. Introduction With the increasing desire to control the size and shape of nanoscale structures, microemulsion-templated synthesis have gained increased attention over the last decade. Microemulsions are defined as homogeneous, optically transparent, and thermodynamically stable mixtures of oil and water stabilized by surfactants [2]. They are exploited in polymer chemistry where their well-defined nanoscale structure is used to control the synthesis of materials of certain morphologies and characteristics [4,5]. Microemulsion templated synthesis has a wide range of applications going from preparation of new types of copolymers [6,7], particles for drug delivery [8], preparation of metal * Corresponding author. Fax: +45 6550 2467.
E-mail address: [email protected] (A.K. Poulsen). 0021-9797/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2006.10.048
clusters [9,10], to formation of polymer beads with a narrow polydispersity [11,12]. In particular, microemulsion templated synthesis has been exploited to prepare nanosized sensoring devices for use in, e.g., living cells [13]. Water in oil (w/o) microemulsions are microemulsions that have a microstructure of surfactant encapsulated water droplets suspended in a continuous oil-phase. The radius of the droplets is typically on the order of 5 nm and the size of the droplets is generally controlled by the surfactant-to-water ratio of the microemulsion [1]. The w/o microemulsions are interesting as media for polymer synthesis because hydrophilic reagents may be dissolved and brought to reaction in the aqueous cores of the droplets [14]. The microemulsion droplets can therefore be regarded as nanoreactors with a size that is, in principle, directly controllable. It is well documented in the literature that microemulsion droplets may be used as reaction media and
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templates in polymerization reactions and that they allow for synthesizing well-defined polymer beads with radius less than 50 nm [15], for review read, e.g., Pavel [16]. Polymerization of hydrophilic monomers in confined water droplets makes it possible to incorporate hydrophilic compounds [17] such as compound specific fluorescent dyes [18,19] and proteins [20,21] into the polymer matrix. Provided that the components do not react with the monomer, incorporation happens by physical entrapment in the polymer network, however, covalently attachment of fluorophores to the polyacrylamide matrix is also an option [22]. The technique serves as a basis for a family of optical nanosensors for analysis of single cell metabolic dynamics [19,23–28] and preparation of polyacrylamide beads in microemulsion is applied to embed hydrophilic sensing molecules and enzymes for use as nanobiosensors; for a recent review see Buck et al. [29]. The present study aims at preparing nanosized polyacrylamide beads by polymerizing the hydrophilic monomers acrylamide (AA) and N,N -methylenebisacrylamide (MBA) in the interior of the droplets formed in w/o microemulsions, where the continuous oil-phase is hexane and where a mixture of the surfactants AOT and Brij30 is used to encapsulate the droplets and stabilize the microemulsion. It is generally accepted that the microstructure and phase behavior of microemulsion-systems are controlled by their composition and the curvature free energy of the surfactant layer separating the oil and the water components [1,30]. At fixed temperature and pressure, the phase behavior of a simple surfactant/water/oil microemulsion-system may be represented in a ternary phase diagram [1]. However, addition of polymerizable components to the aqueous component increases the degrees of freedom and therefore the complexity of the system. In the present article we will refer to the mixture of buffer salts, AA, MBA and H2 O as the aqueous component and we will generally consider the pseudo-ternary system consisting of surfactant, the aqueous component and the oil. We will investigate the effects on the ternary phase diagram of the addition of different components to the aqueous component. Initially we had the working hypothesis that the polymer bead size was controlled by the size of the aqueous droplets in the w/o microemulsion [31], which, again was controlled by the ratio of surfactant to aqueous component. Due to the large parameter space given by the number of components forming and affecting the system we decided to focus on the part of the phase diagram where it has been previously shown [15,21] that one-phase w/o microemulsions form and polymer beads can be synthesized. The present study demonstrates that addition of the monomers AA and MBA has a strong effect on the extent of the microemulsion domain and on the microemulsion droplet size. The study also demonstrates that, upon addition of monomers to the aqueous component of the droplets, the assumption that the droplet size can be predicted by the ratio of surfactant-toaqueous component apparently breaks down and particles that are significantly larger than theoretically predicted are formed. Thus apparently, the system no longer exhibits standard microemulsion behavior and radius of micelles in microemulsion
is no longer given by the molar ratio of solubilized component per surfactant [32]. As it will be explained in the following we have searched for an explanation to this observation, however, the question remains open. 2. Experimental 2.1. Materials and methods Brij30 (polyoxyethylene-4-lauryl ether) was obtained from SIGMA. C12 E4 , 99+% were obtained from Nikko Chemicals, Japan. AOT or aerosol OT (sodium bis-2-ethylhexylsulfosuccinate, 98% purity), acrylamide (99+%, electrophoresis grade), hexane (95+%, HPLC-grade), N ,N -methylenebisacrylamide (99+%, electrophoresis grade) were obtained from Aldrich, Germany and used as supplied. 10 mM sodium phosphate buffer, pH 7.2, was prepared in Milli-Q water using a mixture of Na2 HPO4 and NaH2 PO4 to obtain the desired pH. The buffer salts were obtained from Merck, Germany. Sulforhodamine 101 was obtained from Invitrogen, Molecular Probes, Oregon, USA. Brij30 is a mixture of components H(CH2 )n O–(CH2 CH2 O)m –H with significant amount for n = 10, 12, 14, 16 and m = 0, 1, . . . , 9 and stated from the manufacturer to give an average of C12 E4 . The mass fraction of each component has been determined using normal phase HPLC and gas chromatography coupled with mass spectrometry (GC-MS). Once these mass /Mn can be calculated from Mw /Mn = fractions are known Mw 2 M m m /M /( i i i i i i i mi ) , where Mi and mi are the molar mass and mass of component i. For Brij30 we found that Mn = 323.3 g/mol and Mw /Mn = 1.075. For the H(CH2 )n OH fraction Mn = 186.9 g/mol and Mw /Mn = 1.0006. For the –(CH2 CH2 O)m – part of the molecule Mn = 174 g/mol and Mw /Mn = 1.32. A detailed analysis of Brij30 is in preparation and will be published elsewhere. For the microemulsion preparations, we first dissolved the surfactants in hexane. Then the desired amount of aqueous component was added to the solution of hexane and surfactant. The aqueous component consisted of buffer with or without dissolved AA and MBA. Two procedures were used for studying the phase boundaries of the microemulsion region of the phase diagram: Either the aqueous component was titrated with a solution of surfactants in hexane until a homogeneous transparent microemulsion was obtained or an existing microemulsion was titrated with the aqueous component until phase separation occurred. All experiments were carried out at 25 ◦ C. 2.2. Surface tension measurements The Du Nouy-method was used for determination of surface tension measurements using a “Digital Tensiometer” from Krüss (Hamburg, Germany). The critical micelle concentration (cmc) and molecular area per head group for Brij30 was determined from the surface tension measurements on concentration series of the Brij30 in buffer with and without monomers [33].
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2.3. Dynamic light scattering The hydrodynamic radius of w/o microemulsion droplets was determined by dynamic light scattering (DLS) using a BI200SM goniometer from Brookhaven Instruments that incorporates a 632.8 nm He–Ne laser. The instrument was operated at a fixed scattering angle of 90◦ . The temperature was kept constant with a thermostatted water bath operating at 25 ◦ C. The standard analysis software of the instrument was used for analyzing the data. Unimodal size distributions were generally found and the 2 order cumulant analysis method could be applied to analyze the experimentally obtained autocorrelation function and hence determine the hydrodynamic radius and polydispersity of the droplets [34]. The droplets studied in the present project are sufficiently small that the obtained results show no significant dependence on scattering angle.
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the mixture are expressed as the weight fraction of the oil in the mixture of oil and buffer: α ≡ B/(A + B + E). The total weight fraction of the non-ionic plus ionic surfactants in the system: γ ≡ (C + D)/(A + B + C + D + E). The weight fraction of ionic surfactant in the mixture of the two surfactants: δ ≡ D/(C + D). And the weight fraction of buffer salt in the aqueous component (i.e. the mixture of H2 O, including monomers, and buffer-salt): ε ≡ E/(A + E). Phase behavior in three-dimensional space can be obtained by keeping α and δ constant and varying T , ε, and γ and hence determine the extension of the three-phase body of emulsion and microemulsion. We used this approach to study temperature stability of the microemulsion at different δ-values, as well as to investigate the effect of exchanging water with buffer or adding monomers to the aqueous component. 3. Results and discussion
2.4. Ultracentrifugation
3.1. Temperature and salt dependence of the phase behavior
Ultracentrifugation was used to gain information about the continuous phase and cmc of AOT. Runs were made at 20 ◦ C with a Sorvall Discovery 90SE ultracentrifuge and a 70TI rotor at speeds up to 50.000 rpm. When a water-in-oil microemulsion is subjected to a centrifugal field the microemulsion droplets move away from the centre of rotation because their density is larger than the continuous phase. As a result a region near the centre of rotation is cleared of drops and a distinct region forms between the cleared region and the remaining microemulsion. We measured the content of AOT in the cleared region using FTIR spectrometry. As control experiment to ensure that ultracentrifugation left the continuous phase without any watercontaining droplets in the cleared region we centrifuged down microemulsions to which we added the hydrophilic dye sulforhodamine 101 and measured the cleared region with fluorescence [35].
The central motivation for starting our examination of microemulsions was the possibility for controlling the size of the final polyacrylamide beads via the composition and hence the size of the microemulsion droplets. Microemulsion-systems have a rich and complex phase behavior: The sensitivity of microemulsions towards variations of temperature and buffer salt concentration is controlled in a non-trivial fashion by the chemical nature and mixing ratio of the applied surfactants as well as the types and compositions of respectively apolar and polar phases, added salt and other additives [36,39–41]. However, the region in the phase diagram where microemulsions form is important to know for practical applications of droplet microemulsions as nanoreactors. Consequently we performed a preliminary examination of the effect of buffer salts and monomers on the microemulsion region. The temperature and salt dependence of the phase behavior of our pseudo-ternary system was examined by preparing a series of microemulsions in the classical Kahlweit fish-cut [37,40]. Hence, the microemulsions were symmetric in the volume fractions of oil and water, corresponding to α = 50 wt%. We used δ = 66 wt% corresponding to a weight ratio of (μ = AOT/Brij30, w/w) μ = 1.94/1. Several weight fractions of surfactant in the system were investigated and γ ranged from 0.5 to 20 wt%. The concentration of the buffer salts was varied for each γ -value and the salt concentration in terms of ε ranged from 0–7.3 wt%: This corresponds to buffer salt concentrations from 0–300 mM. The phase behavior of each sample was investigated from melting point to boiling point. For all temperatures and sample compositions the samples separated into two phases where the upper phase remained as a translucent mixture of surfactants, aqueous component and hexane in equilibrium with the lower water rich phase that had a clouded appearance. We found that the mixture is quite stable towards variations in temperature and salt, and we did not observe any phase inversion in the whole range of temperatures, buffer salt concentrations, and surfactant contents. We did observe slight changes in the volumes of the two phases as temperature was changed, however, the changes were small and not significant. The same observa-
2.5. FTIR spectrometry Quantification of the amount of AOT in microemulsions and in hexane was performed on a Perkin–Elmer Spectrum One spectrometer. For quantification we used the intensity of the peak at 1737 cm−1 caused by the carbonyl group in the ester bond in AOT. We verified by known concentrations of AOT in hexane, that we were able to recover and quantify AOT in microemulsions prepared of buffer, AOT, Brij30, AA, MBA, and hexane. 2.6. Phase behavior studies and determination of the microemulsion domain Phase behavior of the system; H2 O including AA and MBA (A), hexane (B), Brij30 (C), AOT (D), and buffer salts (E) was examined with regard to temperature and the weight percentage of surfactants in the system at various buffer salt concentrations. In the following we will use the variables and nomenclature as they have been introduced by Kahlweit et al. [36–40] who have also described the method thoroughly. The variables in
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tion was made when the aqueous component was loaded with the monomers AA (3.46 M) and MBA (0.472 M). The separation into two stable phases and the nearly constant ratio between the volumes of the two phases indicate that we never reached phase inversion nor the three-phase body as temperature was increased. AOT is the major component of the two surfactants and the phase behavior of the system is, as in line with those observed by Kahlweit et al. [37], similar to that of a system of pure AOT below phase inversion temperature. Furthermore, temperature and salt insensitivity has been found in similar systems and is most likely a property of the mixture of AOT and Brij30 as well as the ratio μ [32,42]. From this we concluded that the presence of monomers and the concentration of buffer salts did not have a significant effect on phase inversion temperature, but also that the fish-method described by Kahlweit et al. [36,39–41] is not practically applicable for determination of surfactant efficiency in the present system and can therefore not be directly applied to find the relationship between solubilization efficiency and content of monomers or salts in the aqueous component. The central, but indirect, result of this experiment is that the system has a very weak temperature dependence and that temperature effects can be neglected in the temperature range (20–35 ◦ C) where the microemulsion is used for polymerization. 3.2. Monomers affect surfactant solubilization capacity The microemulsion domain of the surfactants/aqueous component/hexane system is indicated in the pseudo-ternary-phase diagram shown in Fig. 1. The phase diagram was determined at room temperature and at the fixed surfactant weight ratio μ = 1.94/1. The aqueous component is a 10 mM 7.2 sodium phosphate buffer in which AA (3.46 M) and MBA (0.472 M)
Fig. 1. Pseudo-ternary-phase diagram for the surfactants (AOT/Brij30, 1.94/1, w/w)/aqueous-component/hexane system showing the microemulsion domain determined at 25 ◦ C. The aqueous component is a sodium phosphate buffer solution (10 mM, pH 7.2) in which AA (3.46 M) and MBA (0.472 M) are dissolved. The boxes indicate the compositions where we observed phase separation. The continuous line is drawn through the experimental results and inserted to guide the eye and delineate the one-phase microemulsion which is the area at right to the line. X refers to the composition often used for polymerization reactions and preparation of nanobiosensors.
are dissolved. The obtained phase diagram is quite similar to the one obtained by Daubresse et al. [21], despite that this group has used a different AOT/Brij30 ratio (3/1, w/w). The observations are also consistent with previous findings that polarity of the continuous phase has a significant effect on the microemulsion domain when monomers are present in the aqueous component [15,43–46]. The interplay between solutes in the aqueous component, surfactants, and the continuous phase is of major importance when it comes to solubilization capacity of the surfactants. The proposed explanation is that the more polar solvent molecules may localize themselves in the surfactant layer separating the w/o interface and thereby act as co-surfactants [15,43,46,47] and help increasing the solubilization capacity of the system. As seen from Fig. 1, the microemulsion domain is characterized by a relatively high ratio of surfactant/aqueous component, i.e. it takes relatively much surfactant to form a microemulsion in this system. The microemulsion composition used for polymerization is indicated in the plot with an x. It is composed by: surfactants (12.7 wt%; AOT/Brij30, 1.94/1, w/w), hexane (81.8 wt%), and aqueous component (5.5 wt%). The composition lies close to the maximum solubilization capacity. This should, in principle, allow for forming the largest possible droplet sizes. We have found that this composition is well suited for encapsulation of proteins and sensoring dyes [22] and it is very similar to the microemulsions previously used for comparable work on polymerization of nanobiosensors [17,48]. The monomer concentration in the aqueous component is important to the properties of the final polymer gel particles. We therefore determined the phase behavior (see Fig. 2) for a series of different weight fractions of the monomers (abscissa-axis) and surfactant/aqueous component weight ratios (ordinate-axis). As it is clearly seen, addition of monomers to the aqueous component also has a major impact on the microemulsion solubilization capacity and hence the microemulsion domain. When the weight fraction of monomers in the aqueous component is increased from 0.0 to 0.45 the solubilization capacity of the surfactants decreases from 0.5 g of surfactant per gram of the aqueous component to 2.5 g of surfactant per gram of the aqueous component. In order to ensure that, e.g., slow equilibrium kinetics did not affect the determination of the phase transition, two different approaches for determining the phase boundary between the translucent homogeneous microemulsion and macroscopic phase separation were utilized. As it may be seen from Fig. 2, it did not have any significant effect on the determination of the phase boundary how it was approached and the two data sets are indistinguishable. It is well documented that Brij30 has a great impact on the solubilization capacity for the monomer-containing microemulsion [21]. In line with this observation it was speculated whether the large decrease of the solubilization capacity of the surfactants could be driven by a change of the surface active properties of one of the surfactants, either AOT or Brij30, upon addition of monomers. This suggested determining a series of phase diagrams similar to the one of Fig. 2, but for variable ratios of AOT to Brij30. We varied the composition of surfactant from 100% pure AOT to mostly Brij30 by titrating from
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ubilization capacity mainly had to do with the surface active properties of the AOT. 3.3. Microemulsion composition versus microemulsion droplet size According to the standard picture of droplet microemulsions, see, e.g., Chevalier and Zemb [3], the radius, r, of the droplets is basically determined by the surface to volume ratio of the droplets. It is assumed that the macroscopic ratio of surfactant to enclosed phase is the same as the ratio in the single droplets. Thus, if the head-group area of the applied surfactants are known, the average radius of the aqueous droplet cores, raq , may be calculated, from [3]: raq = Fig. 2. Phase boundary between one-phase microemulsion and phase separation given for surfactants (AOT + Brij30)/aqueous-component/hexane microemulsion. The solubilization capacity of surfactants is affected by presence of the hydrophilic monomers AA and MBA and decreases as the monomer concentration is increased. This phase diagram was determined by conducting a series of titration experiments at fixed temperature (25 ◦ C) and phase boundary was approached from either side: (1) going from a two-phase system to a one-phase system by titration of hexane containing surfactant into the aqueous component until the mixture turned into a translucent 1-phase system; (!) going from a one-phase system to a two-phase system by titration of the aqueous component into the hexane/surfactant mixture. The ratio μ = mAOT /mBrij30 and the surfactant/hexane ratio were kept constant at μ = 1.94/1, and 0.1086 g surfactant per ml of pure hexane, respectively. The amount of aqueous component and the total concentration of monomers within the aqueous components were varied systematically until the boundary between the 1- and 2-phase regions could be observed. The ratio of AA to MBA monomers was 3.375/1 in these experiments.
the two-phase region and into the one-phase region. The obtained phase diagrams are shown in Figs. 3a–3e and combined in Fig. 3f. It is seen that pure AOT has a higher solubilization capacity with respect to the monomer-free phosphate buffer than the AOT/Brij30 mixture. In the pure AOT microemulsions, 1 g of AOT solubilizes approximately 4 g of the phosphate buffer, but when the concentration of monomers increases to 0.45 (w/w), 1 g of AOT solubilizes only 0.3 g of the aqueous component. The effect of monomer in the aqueous component decreases as the AOT/Brij30 weight ratio μ decreases. However, the four studied AOT/Brij30 ratios have comparable solubilization capacities when it comes to the 0.45 (w/w) monomer loaded aqueous component. When the AOT/Brij30 ratio is decreased to the smallest possible value that still allows for forming microemulsions, that is μ = 0.12/1, the effect of the presence of AA and MBA monomers in the aqueous component nearly vanishes. Unfortunately, the μ = 0.12/1 microemulsions generally have a very low solubilization capacity: 1 g of surfactant only solubilizes 0.4 g of aqueous component, independent of the concentration of monomers. Hence, besides from demonstrating that there is a strong dependence between microemulsion solubilization capacity and the concentration of monomers in the aqueous component, this series of experiments also suggested that the change of sol-
3Vaq , Asurf
(1)
where Vaq is the volume of the enclosed aqueous phase and Asurf is the total surface area of the surfactant layer. Since the head-group areas of surfactants are a weak function of the condition under which they are used, it is generally reasonable to assume a constant head-group area at the water–surfactant interface. Under this assumption, a straightforward geometrical calculation leads to the following expression for the outer radius, router , of the water + surfactant droplet: 1/3 2 n 3raq surf 3 + , router = raq asurf
(2)
where nsurf is the (partial specific) molecular volume of a single surfactant molecule and asurf is the area per head group of a single surfactant molecule. For the mixed AOT/Brij30 system, averaged values for nsurf and asurf are calculated from: nsurf = XAOT νAOT + XBrij30 νBrij30 and asurf = XAOT aAOT + XBrij30 aBrij30 , where XAOT and XBrij30 are the mole fractions of AOT and Brij30, respectively. For our calculations of the droplet radius we used the following parameters: aBrij30 = 45 Å2 , as found by surface tension measurements (Table 1) and in agreement with Kjellin et al. [49] and aAOT = 55 Å2 [50,51]. The molecular volumes for AOT and Brij30 were calculated from their corresponding mass densities. We used νBrij30 = 633 Å3 and νAOT = 649 Å3 per molecule. If it is assumed that the critical micelle concentration (cmc) is equal to the concentration of free surfactant molecules that are not located in the surfactant film, a cmc-effect may be included in the calculations by calculating Asurf in Eq. (1) from Asurf = (csurf − cmc)asurf . It is seen from Eq. (1) and Eq. (2) that for constant volume of the aqueous component and constant amount of surfactant, the microemulsion droplet size is in theory constant. The equations have been verified experimentally for a number of systems, see, e.g., [3] and references therein. Figs. 2 and 3 show that the amount of aqueous component, which can be solubilized by the microemulsions, is substantially reduced when monomer is present. According to Eq. (1) and Eq. (2), this implies that the maximum stable w/o microemulsion droplet size is reduced. Alternatively the surface area is reduced which conceivably can
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(a)
(b)
(c)
(d)
(e)
(f)
Fig. 3. Phase boundary between one-phase microemulsion and phase separation given for surfactants (AOT + Brij30)/aqueous component/hexane microemulsion at various monomer concentrations and for various AOT/Brij30 ratios: μ = mAOT /mBrij30 . (a) μ = 1/0 (pure AOT); (b) μ = 1.94/1; (c) μ = 0.80/1; (d) μ = 0.52/1; (e) μ = 0.12/1, and (f) shows lines fitted to the data points shown in Figs. 3a–3e to summarize the effect of the AOT/Brij30 ratio. The numbers in (f) indicate the ratio μ. Hexane was added to the aqueous component until phase separation. The hexane contained 0.1086 g surfactant per ml and the aqueous component contained AA and MBA in the ratio 3.375/1 (w/w). The measurements were performed at fixed temperature (25 ◦ C).
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Table 1 Critical micellar concentration, cmc and surface head-group area, Ahead , for Brij30 in buffer solution with and without AA and MBA monomers
Brij30 in buffer without monomers Brij30 in buffer with monomers
cmc [mM]
Area head [Å2 /molecule]
0.021 0.716
45 (±10) 53 (±5)
The results are obtained by measurements of surface tension as described in Section 2.2.
only happen if surfactant is removed from the water/oil interface. DLS has often been used for determination of droplet size in microemulsions and results have been verified and discussed in comparison with other methods for droplet size determination and structure studies, e.g., SANS and SAXS, see [52–56] and references therein. Here DLS was used to investigate whether the given system followed Eqs. (1) and (2). The droplet size was measured for a series of samples where the amounts of aqueous component, surfactants, and hexane were kept constant and where the concentration of monomers was gradually increased. The obtained hydrodynamic radii are plotted as a function of the concentration of monomers in Fig. 4. The data indicate that the monomer concentration has a tremendous effect on the experimental droplet sizes. The hydrodynamic radius increases from 4.4 nm in the monomer-free sample to 42 nm in the most monomer-rich sample. It should be emphasized that all samples remained as a homogeneous translucent microemulsion throughout the experiment. The same samples were re-measured at the same instrument after several weeks of storage and no significant changes were observed. The observed non-linearity of the droplet sizes as a function of monomer concentration show that our system does not follow Eqs. (1) and (2). It should be emphasized that the obtained DLS results clearly indicated that the droplets had unimodal size distributions. We hypothesized that the observed non-linearity of the microemulsion droplet sizes was related to a polydispersity of Brij30, see Section 2.1, and prepared a similar series of samples, where Brij30 was replaced by C12 E4 . The results are plotted in Fig. 4. As it may be seen, the observed dependence between hydrodynamic radius and monomer concentration was fully reproduced when Brij30 was replaced by C12 E4 . Hence, a non-linear effect on w/o microemulsion droplet size due to polydispersity of Brij30 can be ruled out. When performing medium or large scale synthesis, the monodisperse C12 E4 is often substituted with the less expensive and polydisperse alternative, Brij30. Fig. 4 suggests that this can be done without any significant effect on the resulting droplet size. The series of samples of Fig. 4 corresponds to moving along a horizontal line in the binary phase diagram of Fig. 3b. When moving along such a line, the distance to the boundary between the one-phase and the two-phase system decreases with the increase of the monomer concentration in the aqueous component. Even though this ought not to have any effect on the droplet size (according to Eqs. (1) and (2)), it was hypothe-
Fig. 4. The hydrodynamic droplet radius of the microemulsion droplets measured at increasing monomer concentration. (1) AOT and Brij30 and (!) AOT and C12 E4 . The overall sample composition used in this experiment was; surfactants (10.6 wt%; AOT/Brij30, 1.94/1, w/w), hexane (83.5 wt%), and aqueous component (5.7 wt%). The aqueous component was a 10 mM pH 7.2 Na-phosphate buffer where the concentration of monomers was varied from 0 M to 3.47 M AA and 0.474 M MBA, the ratio between AA and MBA was kept constant at 3.375/1 w/w.
Table 2 The amount of aqueous component that the microemulsion is able to solubilize as concentration of monomers is varied in the aqueous component Monomer in aq. comp. [wt%]
Aq. comp. in microemulsion [wt%]
0.0 0.1 0.2 0.3 0.4
24.0 10.2 7.3 5.7 5.3
The mass of hexane and surfactants are kept constant. It is seen that solubilization capacity of surfactants decreases dramatically as monomer content is increased. The AOT/Brij30 ratio is 1.94/1. The aqueous component contained AA and MBA in the ratio 3.375/1 (w/w). The results are obtained at 25 ◦ C.
sized whether it could have an effect on the droplet–droplet interactions and therefore give rise to an apparent increase of the droplet radii. In order to test this hypothesis, four series of samples were prepared. In each series the monomer concentration was varied while the distance to the one-phase/twophase boundary was kept constant by loading the microemulsions with 50, 70, 80 and 90% of the amount of aqueous component at the one-phase/two-phase emulsion boundary, respectively, as found in Fig. 2, see Fig. 5b and Table 2. The hydrodynamic radii of the resulting microemulsion droplets were measured by DLS and the results are plotted in Fig. 5a. The volumes of the aqueous component was relatively low in these experiments, see Table 2 and never constituted more than 20 wt% during the experiments in the most extreme case. As it may be seen from Fig. 5a the experimentally observed hydrodynamic radius was basically constant within each of the series. At 50% load an average droplet radius of 6.2 nm was
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The theoretical w/o microemulsion droplet size for the same compositions have been calculated according to Eqs. (1) and (2) and is plotted in Fig. 5b. As the monomer content increases, the solubilization capacity of the microemulsions decreases. This obviously leads to a decrease of the theoretical droplet size. Thus, theoretically, and as shown in Fig. 5b, it is expected that the droplet sizes should decrease when the monomer concentration is increased in each of the series. Clearly, this is not what we observe. Hence, for fixed overall surfactant composition, an increase of the concentration of monomers in the aqueous component induces a strong and systematic increase of the hydrodynamic droplet radius. Furthermore, iso-radius lines, that is, lines of nearly constant hydrodynamic droplet radii, may be observed if the monomer load is varied for constant distance to the boundary between the one-phase and two-phase regions. (a)
3.4. The breakdown of microemulsion behavior A sufficiently large cmc, which furthermore depends on the concentration of monomers in the aqueous component, may provide an explanation of the atypical behavior of both phase diagrams and growth of droplet radii. In order to investigate such a possible effect, the cmc values of the two different surfactants in the different components were studied in more detail. This study was partly based on literature data and partly on our own experimental work.
(b) Fig. 5. (a) Experimental hydrodynamic radius of monomer-containing microemulsion droplets. The effect on the droplet radius of adding monomers is measured for (P) 50%, (1) 70%, (E) 80% and (!) 90% of maximum solubilization capacity of the surfactants in one-phase microemulsion at the given amount monomer. The AOT/Brij30 ratio was 1.94/1, the ratio of AA to MBA was 3.375/1, and the overall volume of hexane and mass of surfactants was constant. (b) The theoretical droplet size calculated at each component and connected with lines to guide the eye.
found, at 70% load an average droplet radius of 11.3 nm was found, at 80% a droplet radius of 17.4 nm was found and at 90% load the droplet radius had increased to 32.4 nm. The droplet polydispersity as determined by DLS remained low (σ/Rav ≈ 10%) and constant throughout the series. Thus the results are in good agreement with the surprising observations of Fig. 4 that the hydrodynamic radius increases significantly as the emulsification one-phase/two-phase emulsion boundary is approached. Another rather surprising observation is that the experimental hydrodynamic radius appears to be determined by the distance to the boundary between the one-phase and two-phase regions.
3.4.1. Brij30 Presently, we have no means for determining the Brij30 concentration in the water cores of the microemulsion droplets. However, as a first approximation it is reasonable to assume that it equals the Brij30 concentration in bulk water (or bulk monomer buffer solution when relevant). The cmc of Brij30 in monomer-free buffer and in buffer containing 4.44 M AA and 0.10 M MBA. (WAA + WMBA = 0.33), respectively, was determined from a series of surface tension measurements. The results are summarized in Table 1. It is seen that the cmc of Brij30 increases strongly (a factor of 34) when AA and MBA is present in the buffer at the given concentration. We do not observe a significant change of the area per head group. For the microemulsion preparations studied so far, we have typically used 400 mg Brij30 per 1 ml of aqueous component. This corresponds to a molarity of Brij30 in aqueous component of ∼0.8 M and implies that even with the large cmc-increase, the effect of the surfactant removed from the surface area when increasing the monomer concentration is negligible. It will only induce a decrease of the surface area to 99.97% of the area in the corresponding monomer-free sample. Hence, with respect to Brij30, a cmc effect is not relevant and not able to explain the decreased solubilization capacity of surfactants. Brij30 does not make any ordered structure as globular micelles, however the solubility of Brij30 in pure hexane is high [57,58]. 3.4.2. AOT An AOT driven cmc effect was also considered. The cmc for AOT is 2.23 mM and 1.09 mM in pure water and hexane, re-
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Table 3 Critical micelle concentration (cmc) in the continuous phase of hexane is determined for AOT by use of ultracentrifugation and FTIR spectrometry in microemulsion composed of surfactants (AOT/Brij30, 1.94/1, w/w)/aqueouscomponent/hexane system Acrylamide [M]
N ,N -methylenebisacrylamide [M]
cmc [M]
3.46 1.73 0.00
0.47 0.24 0.00
0.001 0.001 0.002
The aqueous component is a sodium phosphate buffer solution (10 mM, pH 7.2) in which AA (3.46 M) and MBA (0.472 M) is dissolved.
spectively [59,60]. However, in order to examine the effect of monomers on the free surfactant concentration of AOT in the continuous phase, we studied microemulsions by a combination of ultracentrifugation and FTIR spectrometry (see further details in Sections 2.4 and 2.5). Our examination was based on the microemulsion composition indicated by x in Fig. 1 and composed of surfactants (12.7 wt%; AOT/Brij30, 1.94/1, w/w), hexane (81.8 wt%), and aqueous component (5.5 wt%). We determined the cmc for AOT in the continuous phase for mixtures where the aqueous component contained 32 wt%, 16 wt%, and no monomers, respectively. The results are summarized in Table 3. For the sample with monomer-free aqueous component we found a cmc for AOT in hexane of 0.001 M. This is similar to results found in literature [59,60]. The presence of monomers did not significantly affect the cmc of AOT in the continuous hexane phase of the microemulsion (Table 3). Another AOT driven explanation could be a micro-phase-separation of the droplets into two populations of the w/o microemulsion droplets: A population of large droplets/micelles containing the main part of the aqueous component and a population of small almost empty micelles. This explanation was suggested by a small-angle X-ray scattering (SAXS) based analysis of AOT microemulsion that indicated that not all the AOT participated in the droplet formation, instead some of it self-organized into small “dry” micelles in the alkane solution [61,62]. By dynamic light scattering (DLS) a population of weakly scattering small “dry” micelles would be difficult to detect among a population of larger and more strongly scattering microemulsion droplets. Instead, the hypothesis was tested in the ultracentrifugation experiments. Using the difference in mass density between the water filled micelles and the dry micelles two such populations could be separated by ultracentrifugation: If small dry micelles were present we would find a high AOT concentration in the continuous phase of the centrifuged samples. However, the concentration of AOT in the continuous phase turned out constant and remained at the cmc independent of the concentration of AA and MBA. No indications of a population of the above mentioned small dry micelles that could explain our observations were found. An unusual growth of microemulsion droplet sizes with monomer contents has previously been detected on similar AOT based microemulsion systems by Candau et al. [15] and Munshi et al. [31]. However, the extent of the growth and the effect of a co-surfactant was not studied in detail. In the previous work it was suggested that the increase in radius is due to a
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monomer induced enhancement of attractive forces between the droplets, which makes the w/o microemulsion droplets coalesce and form dynamic clusters of droplets. In more detail, it was proposed that the monomers partly localize themselves in between the hydrocarbon chains of the surfactants and therefore induce a disorganization of the surfactant interface which leads to an effective increase of the “stickyness” of the droplets [51,63,64]. Findings that support this theory is that addition of AA to a microemulsion comparable to ours has shown to increase microemulsion viscosity [65] and it has been found experimentally by Bergenholtz et al. [64] that the increased viscosity seems to be caused by an increased disorganization at the water/surfactant interface and that this results in increased attractions between the droplets as well as a lower percolation threshold. As DLS do not have a resolution that directly allows us to distinguish between a large droplet and a cluster of smaller droplets, the cluster-model may also consistent with our findings. However, it would be most probable that the polydispersity of the clusters was different from that of the single microemulsion droplets, and, as a matter of fact, we observe no significant changes of the polydispersity of the droplets in relation to the growth of the particle radius. Thus the DLS results for the droplet sizes and their polydispersities do not contain any indications in support of the cluster-model and a more detailed structural analysis will be necessary in order to verify or reject the hypothesis. Small-angle scattering is an obvious choice of experimental method for such a study. However, this lies outside the scope of the present work. Hence, the apparent breakdown of the standard microemulsion behavior could be naturally explained by either a large cmc effect, or a microphase separation of the microemulsion droplets into a population of small dry micelles and larger water filled microemulsion droplets. However, our studies show that none of these effects are sufficiently large to explain the observed behavior. Another explanation could be provided by increased attractive interactions between the droplets leading to a cluster formation. However, our DLS results do not contain any indications in favor of this explanation. 4. Conclusion The phase behavior and droplet sizes of a w/o microemulsion composed of hexane, AOT, Brij30, and an aqueous component containing monomers (AA and MBA) and buffer salts were studied. The w/o microemulsion droplets may be applied for polymerization of monomers dissolved in the confined water. Sensoring dyes and functional proteins may be dissolved in the water together with the polymerization monomers and may be incorporated into the synthesized polymer latexes for nanobiosensors [21,48]. In this context modulation of particle size is desirable in order to prepare nanobiosensors for different purposes. The use of w/o microemulsion droplets as nanoreactors is based on the implicit assumption that the system can be understood as a pseudo 3-component system. The 3 components are the oil, the mixture of surfactants and the aqueous component.
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We observed an extremely high sensitivity of the microemulsion towards the presence of the monomers AA and MBA: The solubilization capacity of the surfactants decreases while the apparent size of the w/o microemulsion droplets increase as the concentration of monomers increase. These results were somewhat contradicting and lead us to more systematic studies of the effect of adding monomers to the aqueous component of the microemulsion. Our results show that a mixture of the surfactants AOT and Brij30 has a better solubilization capacity than AOT alone when monomers are present, and the effect of monomers on surfactant solubilization capacity decreases as the AOT/Brij30 ratio decreases. This means that AOT is most strongly affected by the monomers. We found that the monomers did not cause an increase in cmc of either surfactant that is sufficiently large to account for the decreased solubilization capacity and that any effect on w/o microemulsion droplet radius from the polydispersity of Brij30 can be disregarded. It was found by use of DLS that droplet radius increases nonlinearly as monomer content increases, when all ratios between surfactant (AOT/Brij30)/aqueous component/hexane were kept constant and only the content of AA and MBA monomers in the aqueous component was increased. The experimentally found hydrodynamic microemulsion droplet radius is significantly larger than the theoretical droplet radius. The discrepancy between the experimentally found and the calculated w/o microemulsion droplet radii increases with increasing concentration of the monomers in the aqueous component. It has previously been suggested that the apparent increase of the droplet size is due to a monomer induced increase of the attractive forces between w/o microemulsion droplets, so that the droplets get more sticky and form larger aggregates upon addition of AA and MBA [15,31,64,65]. However, our data from DLS studies of microemulsions with varied monomer content show that the polydispersity of the microemulsion droplets remains constant, this makes it less probable that the increase in hydrodynamic radius as monomer content is increased is caused by an aggregation of the droplets. Thus we are presently unable to explain the observed large discrepancy. Ability to modulate the size of functional nanoparticles is a desired feature. If we are able to modulate size of w/o microemulsion droplets it enables us to modulate final polymer particle size [31]. We have shown that the microemulsion is rather temperature stable and that the droplet size is rather temperature independent. However, microemulsion droplet size is highly affected by the monomer concentration in the aqueous component. Unfortunately, monomer concentration is also an important parameter when it comes to pore size and crosslinking in the polymer [66,67]. To nanobiosensors based on encapsulated dyes and enzymes the pore size and the macroporous structure of the final polymer latex may be important to enzyme activity and leaching of the incorporated components [68,69]. The influence of monomers on w/o microemulsion droplets has to be examined further with a medium-to-high resolution technique like small-angle scattering in order to describe the underlying mechanism of the observed unusually large acrylamide induced effect on the droplet size.
Acknowledgments The authors thank Professor Lars Folke Olsen and the Danish Medical Research Council for a fellowship to A.K.P. (Grant no. 22-03-0236) and the Danish Natural Science Research Council for support of the project (Grant Nos. 21-04-054 and 272-05-110). The authors acknowledge support from the Human Frontier Science Program, Grant No. RGP0041/2004C. We thank Professor Reinhard Strey and the staff from his group for useful discussions in the initial phase of the project. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34]
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