oil interfaces

Journal of Colloid and Interface Science 298 (2006) 920–934 www.elsevier.com/locate/jcis

Surfactant-mediated water transport at gelatin gel/oil interfaces V.I. Uricanu, M.H.G. Duits ∗ , D. Filip, R.M.F. Nelissen, W.G.M. Agterof Physics of Complex Fluids Group, University of Twente, Faculty of Science and Technology, associated with the J.M. Burgerscentrum for Fluid Mechanics, and Institute of Mechanics, Processes and Control—Twente (IMPACT), P.O. Box 217, 7500 AE Enschede, The Netherlands Received 4 November 2005; accepted 13 January 2006 Available online 3 February 2006

Abstract We studied spontaneous emulsification (SE) at Water/Oil (W/O) interfaces, using several types of aqueous reservoirs immersed in dodecane plus Span80 surfactant. Above a threshold surfactant concentration CSE , aqueous satellite droplets are formed at the W/O interface. Varying the aqueous reservoir size, from below 100 µm (droplets) to centimeters (macroscopic phases), allowed investigating SE with complementary techniques. Release (rates) and size distributions for SE droplets were measured with microscopy. For gelled aqueous phases, water expulsion due to SE was quantified. Values for CSE were measured and were found to be higher for aqueous phases containing gelatin and/or NaCl. We also studied water exudation during network building and syneresis in aqueous gelatin gels immersed in dodecane/Span80. Below CSE (i.e., in the absence of SE) this process is still responsible for significant physico-chemical changes at the W/O interface. To study these in more detail, we performed atomic force microscopy experiments (in force–distance mode) on macroscopic gels. Both changes in the local elastic response and in the wettability of the AFM tip were detected. Together they suggest the formation of “water pockets” after prolonged (gel) setting times, along with a densification of the interfacial gelatin network. © 2006 Elsevier Inc. All rights reserved. Keywords: Spontaneous emulsification; Gelatin gel; Water exudation; Atomic force microscopy; Elasticity; Surfactant

1. Introduction The use of surfactants in liquid/liquid dispersions is intimately related to the structure/texture and other desired properties of the end-product. For emulsions, surfactant layers adsorbed at the interface between the dispersed (droplet) liquid and the continuum medium prevent droplets’ coalescence. On the long term, the overall stability is preserved and no macroscopic phase separation occurs. The proper choice of the surfactant for a given application must take into account all the other ingredients that will be included in the product formulation. For simple emulsions of two (otherwise) immiscible liquids, the chemical structure of the surfactant and its HLB value will dictate whether an O/W emulsion or an inverse (i.e., W/O = water-in-oil) emulsion will be the stable result. Regardless hereof, external energy must be provided to the system as to achieve emulsification. The formation of large * Corresponding author. Fax: +31 53 4891096.

E-mail address: [email protected] (M.H.G. Duits). 0021-9797/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2006.01.030

droplets (few micrometers) is fairly easy and high-speed stirrers such as the Ultraturrax or Silverson Mixer, or sometimes even magnetic stirring, supply enough mechanical energy to produce the emulsion. In contrast, the formation of small (submicronic in size) droplets is difficult and requires a large amount of surfactant and/or energy. Adding surfactant lowers the interfacial tension; hence, the Laplace pressure (the difference in pressure between inside and outside of the droplet) is reduced and the stress needed to break up the large initial drop(s) is lower. In most cases, emulsification is not a spontaneous process. However, given favorable conditions, a spontaneous formation of droplets may take place at the interface between two liquids. This phenomenon is known as “spontaneous emulsification” (SE). The surfactant presence at the interface can induce local disturbances and stimulate mass transport processes. Different mechanisms are responsible for the so-generated “thermodynamic non-equilibrium.” Maragoni effects [1,2], chemical reactions at the interface [3–5], local nucleation in suprasaturation regions resulting from mass diffusion across the interface [6], bursting of swollen bilayers [7–9], or mechanically

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induced non-equilibrium through rapid area contraction [10] were reported as potential SE mechanisms. Despite its observation in various systems [1–18] and even its use in applications [19–28], the (theoretical) understanding of SE is far from being complete [29–33]. Theissen and Gompper [31] concluded that the speed of the SE process is controlled mostly by surfactant diffusion. A review on similar issues was published by Miller [32]. Also on the experimental side, there are still open questions. A precise quantification of the dynamics involved in the spontaneous emulsification process, based on in situ experiments, is rather difficult to achieve. An effective approach was suggested by Lopez-Montilla and co-workers [34]. They used a Coulter Counter Sizer, to quantify not only the SE-rate but also the volume fraction of the final dispersed phase and the SE-droplet size distribution. However, whether such measurements could be extended to systems which are labile under stirring (i.e., prone to disruption of the dispersed phase droplets, or coalescence of the small droplets formed via SE) is questionable. Video-microscopy has also been used as an experimental tool [12,13]. This technique has so far been limited to qualitative/phenomenological observations. The oil-soluble Span80 has been reported in a number of papers [12–16] as one of the SE-inducing surfactants, implicated in water transport in double (W/O/W) emulsions. However, in none of these the extent and the limits of the SE process were considered for cases where the aqueous phase contains a polymer. In this paper we want to investigate the influence of gelatin, a biopolymer which is commonly used in industrial applications and has the ability to gel. Gelatin is also interesting for (potential) use in drug release applications, since a common method to prepare (bio)polymer particles containing an encapsulated active drug is solvent evaporation from a W/O emulsion [35–37]. There are different ways via which the presence of gelatin inside the water droplets may change the SE process. At room temperature, above a certain polymer concentration, gelatin solutions start to build up a network (gel) via entanglements and formation of triple helixes between molecules. Due to its molecular structure (which is a combination of hydrophilic and hydrophobic groups), gelatin molecules may stick through the interface, from the gel into the oil phase. Therefore, increase of the gelatin concentration could influence the adsorption of the Span 80 molecules at the oil/water interface. Another possible influence of gelatin over the water transport processes may come from the biopolymer ability to bind (via hydrogen bonding) the water molecules to the gel network, leaving less free water at the interface that can come into contact with the surfactant and oil. This ‘free water’ may, for gelatin gels, even take the form of water layers formed by a process known as syneresis. Since such a syneresis could provide a different pathway for spontaneous emulsification, we have also studied this process. To contribute to a better understanding of the problems posed, we have performed an experimental study on the SE phenomenon, using several complementary techniques. Direct observations of the satellite droplets formation during SE were done with optical microscopy. Atomic force microscopy turned out to be a very suitable technique to study syneresis, since

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with it one can detect both the expelled water (via its adhesive interaction with the AFM tip) and the local densification of the polymer network under it (via its increased elastic modulus). Gelatin-containing aqueous phases were prepared at two polymer concentrations: 10 and 30%. As oil we used dodecane. Surfactant concentrations were varied from a very low level (0.001%) up to 20%. Experiments with pure dodecane were done as control. To have a good overview of the ongoing processes and a better base to explain the results, besides gelled water droplets, we also investigated systems prepared only with pure or salted water. 2. Experimental 2.1. Materials The oil (O) phase was dodecane (Aldrich) with different surfactant (Span 80, Fluka) concentrations: varied from 0 to 20% w/w. The initial aqueous (A) phase was: (a) pure water (pH = 5.7), (b) salted (0.3 M NaCl) water or (c) solutions of gelatin in the molten state. Acid treated pig gelatin with an isoelectric point: 8–9 (provided by Unilever Netherlands as dry powder, commercial name: Geltec) was used. To prevent bacterial growth, sodium azide (0.2%) was added in all preparations with gelatin. Hot gelatin solutions were prepared according to the procedure described in Ref. [38]. While hot (60 ◦ C), the gelatin solutions are liquid. As the temperature drops below 30–35 ◦ C, a gel starts forming. Its strength grows in time and depends on both polymer concentration and aging (time/temperature) of the network. 2.2. Sample preparation In one type of experiment, an aqueous ‘single mother-drop’ was deposited on the bottom of a glass container already filled with the oil phase. When gelatin was used, a hot (polymercontaining) drop was brought into contact with the glass by allowing it to settle through the oil, which itself was preequilibrated at room temperature (20 ◦ C). The dimensions of the mother drops were controlled by the volume ((i) 2–4 µl or (ii) 30–50 µl) of the aqueous phase injected with the help of a syringe. The droplet diameters corresponding to these volumes are 1.6–4.6 mm. In another type of experiment, much smaller reservoir droplets were prepared. Here, whole emulsions (to be termed: ‘macroemulsion’ later on) were prepared by mixing fixed amounts of (dodecane +1% Span) and aqueous phase in hot conditions (60 ◦ C): first 5 min magnetic stirring, followed by reduction of the droplet size using an Ultraturrax (5 min, 1.7 × 104 rpm). The diameter of the thus obtained emulsion droplets was always below 100 µm; hence, we will consider them as ‘small aqueous reservoirs’. Immediately after the Ultrathurrax step, a small volume of the hot emulsion was injected into an oil phase equilibrated at room temperature. We also prepared ‘macroscopic gels’ (i.e. having a thickness of several mm and a weight ∼2.5 grams), with 10 or 30% gelatin solutions. These gels had an average area of 170 mm2

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Table 1 Summary of samples investigated and found to be affected (X) or not (O) by spontaneous emulsification. The indices are related to the type of reservoir used Span conc. (%)

Water

Water + 0.3 M NaCl

Water + gelatin Water + 10% gelatin + 0.3 M NaCl 10% 30%

0.001 0.002 0.004 0.006 0.008 0.01 0.02 0.04 0.06 0.08 0.1 0.2 0.4 0.5 0.6 0.8 1 5 10 15 20

Oa Oa Oa Xa Xa Xa Xa Xa Xa Xa Xa Xa Xa Xa Xa Xa Xa Xa Xa Xa Xa

– – – – – – – – – – – – – – – – Xa Xa Xa Xa Xa

– – – Oa Oa Oa,c Oa,c Oa Oa Xa Xa Xa Xa Xa Xa Xa Xa,b Xa,b,c Xa,b,c Xa,b,c Xa,b,c

– – – – – Oa Oa Oa Oa Oa Oa Oa Oa Xa Xa Xa Xa Xa,b,c Xa,b,c Xa,b,c Xa,b,c

– – – – – Oa,c Oa,c – – – – – – – – – Xa Xa,c Xa,c Xa,c Xa,c

a Single mother-drop. b Macroemulsion. c Macroscopic.

exposed to the surfactant solutions. Samples covered with small Span concentration: 0.01 or 0.02% were used to study the ageing in the absence of SE. To measure the amount of SE-expelled water, oil phases with relatively high Span80 concentrations (from 5 to 20%) were poured on top of the gelling network. After gelation, the material remained stuck to the bottom of the container, thus allowing easy removal of the top liquid. Small quantities of oil were wiped off the gel surface using the capillary action of a dry tissue. The subsequent percentual weight loss due to water expulsion was then measured gravimetrically after allowing the gel to set at 20 ◦ C for 24 h, 48 h and 2 weeks. A summary of the samples prepared for studying the SE occurrence can be found in Table 1. We remark here that the above procedures cannot preclude some differences in the quenching rates for different-sized gelatin reservoirs. 2.3. Techniques The temporal behaviors at the droplet/oil interfaces were visualized with optical microscopy, using a Nikon Diaphot inverted microscope with a 100× objective and CCD camera attached, as well as with an Olympus microscope with a lateral view and a 20× magnification. For surfactant concentrations high enough to stimulate spontaneous emulsification, size distributions of the SE droplets could be measured. This was done either in situ, i.e., by inspecting the vicinity of the mother droplet surface with the microscope, or by carefully taking aliquots of satellite droplets with a pipette and transferring them to another microscope cell. Both methods gave the

same results. Optical microscopy also allows to determine the conditions when the SE droplets were unstable towards coalescence or flocculation. Image analysis for the size distribution was done with Optimas software. Atomic force microscopy (AFM) was performed on the mmthick gel samples, at given time intervals (measured from the moment of coverage with oil/surfactant mixtures). The forceversus distance-mode was used to measure both the network elasticity and the tip–sample adhesion. The indentor used was a blunt-cone tip supported on a silicon nitride cantilever (Park Scientific Instruments, nominal spring constant 0.1 Nm−1 ). The up and down movement of the indenter was achieved by driving the piezo tube at a constant frequency of 1 Hz. Prior to the force/displacement measurements, the sensitivity of the laser-detection system on the used cantilever was determined via calibration scans on a hard substrate, under oil. A more elaborate description of these experiments can be found in [38]. 3. Results and discussion 3.1. Water-transport regimes 3.1.1. General observations at water/dodecane interfaces Video-microscopy (even in brightfield mode) is a very suitable technique to measure the approximate magnitude of the surfactant concentration necessary to enter the SE-controlled regime. Our first experiments were done with pure water and a very high (15% w/w) surfactant concentration dissolved in the oil. The results are presented in Fig. 1. To allow recording of the SE process from the start, the water reservoir (2.0 µl) was deposited initially under pure dodecane. After adjusting the focus to the water/dodecane interface, a concentrated surfactant solution (20%) was added to the oil phase as to have roughly 15% Span80 in the final state. In the initial state without surfactant, the interface between the “mother drop” and the dodecane is sharp and clearly visible. After adding surfactant, sparse small droplets produced via SE become already visible after 1 min, as distinct dispersed entities in the oil phase. The number of droplets increases fast. After 3 min, the bottom-glass is completely covered with them (on the dodecane side, in the observation range under the microscope). The average SE droplet diameter is around 600 nm. In time, the SE droplets start to fill up the space in three dimensions, and the interface water/oil (located in the middle of the observation field for the image taken after 10 min) becomes obscured by coverage with SE droplets. For similar high surfactant concentrations (in the range between 5 and 20%), time evolution was also monitored via observations on an enhanced (dimensional and temporal) scale, performed using a smaller magnification and a laterally oriented objective (Figs. 2–3). Fig. 2 shows the pictures captured at various times for a (pure water) “mother-drop” placed on the bottom of a container filled with 5% Span80 oil phase. While at t = 0, the glass/dodecane interface was clearly delimited as a sharp line, after 10 min, the spontaneous emulsification creates already enough small droplets dispersed around

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Fig. 1. Time evolution of spontaneous emulsification at the interface of water (right) and dodecane (left) in the presence of 15% Span80. Images were taken with an inverted microscope and correspond to horizontal sections close to the glass substrate.

the water-reservoir that a whitish layer forms on the glass bottom. This layer grows in time. After 60 min, the full contour of the ‘mother-drop’ is completely covered with the small SE droplets. After 24 h, the process is so extended that the initial ‘mother-drop’ is almost completely embedded in a layer of the SE droplets. The last image in Fig. 2 (after 48 h) suggests that the whole process slows down in time and minor differences are noticeable between 24 and 48 h. It is already known that salt can influence the SE process, by creating an osmotic pressure in the water phase [39]. Another known effect of salt—for our system—is that also the interaction of Span80 molecules with the water phase and in between themselves changes and the strength of the surfactant film at the oil/water interface decreases [40]. Our observations confirm that SE is strongly suppressed in the presence of salt. For 0.3 M NaCl (images in Fig. 3), a barely visible white SE layer covered the “mother-drop” and the bottom of the container after 10 min. For the salty case, it took 24 h to obtain a similar coverage with SE as reached for the pure water droplet after 30–60 min.

Between 24 and 48 h, no significant modifications were observed. Long-term observations on the SE evolution in salted conditions revealed an interesting behavior. The SE droplets form a dense layer, which slides on the curved surface of the big aqueous reservoir, accumulating more on the bottom-glass and leaving the top uncovered. Water expulsion (via SE) seems to have stopped and no additional SE droplets are formed (see Fig. 3, image after 9 days). These observations suggest that the whole system has achieved a quasi-equilibrium. The size distribution of the SE droplets was investigated after 10 min, 24 h and 48 h for a number of samples, with practically the same results for samples having Span80 concentrations of 5% or higher. At these high surfactant concentrations, also the salt concentration had no apparent influence on the size distribution. After 10 min, the measured diameters for the small SE droplets are in the 550–700 nm range. While these measures were mostly obtained from pipetted sample volumes, rather similar dimensions were measured from in situ observations (like Fig. 1). Image processing and statistical analysis on a large number of acquired images allowed extracting the av-

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(a)

(b)

(c)

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(e) Fig. 2. Time evolution of spontaneous emulsification for a pure water/dodecane system with 5% Span80. The dimensions of the lateral observation field are 7 × 7 mm2 .

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Fig. 3. Added salt (0.3 M NaCl) changes the time scale of the process. The other parameters are identical with those in the sample for Fig. 2. Note the small whitish layer in the front of the mother-drop.

erage diameter by fitting the data with a Gaussian distribution (Figs. 4a–4c). As becomes evident from Figs. 4 and 5, the size of the SE droplets clearly increases with time. Besides a very large number of small, liberated SE droplets, also much bigger droplets were observed. Probably the latter ones were formed via coalescence. (Note: for SE droplets generated from pure water reservoirs, the coalescence proved to be more advanced. In this study, we could only study size distributions taken from the whole oil phase and, hence, were not able to tell via which mechanism(s) the droplet sizes changed.) After 48 h, droplets such as initially formed, are not found anymore and the entire size distribution is shifted towards larger diameters (see Fig. 4c). These observations clearly indicate a metastable nature for the initially formed SE droplets. Looking more closely into the temporal evolution of the systems affected by SE, over very long time intervals (i.e.,

months), further changes are observed. In time, the SE droplets are not stable but show (further) coalescence. In addition, the mother droplet can no longer be distinguished. The result after 3 months is a coexistence of two clearly separated (aqueous and oil) phases, with no SE droplets present. For all surfactant concentrations higher than 5%, such an ‘equilibrium state’ is reached after a comparable waiting time. Waiting longer (up to 6 months) does not bring any other modifications. The long-term (order of weeks and months) evolution in size distribution for the SE satellite droplets, ending up into phase separation: oil, water (and eventually a dried gel—see Section 3.1.2) has a direct correspondence with the results of Dukhin and Goetz [41]. In their study, authors used the same surfactant as we, with another oil: kerosene. Another important difference is the fact that stirring was applied to maintain the emulsion droplets in suspension. We also expect that, due to this continuous mixing, the transition (between the initial

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(a)

Fig. 5. SE droplets after 24 h, for the same system as depicted in Fig. 1. Observation field: 40 × 40 µm2 . The diameter of the smallest SE droplets is around 600 nm.

(b)

(c) Fig. 4. Typical droplet size distribution for the main population of SE droplets released from a big water reservoir in the oil phase containing Span, after (a) 10 min, (b) 24 h and (c) 48 h. The continuous lines represent fits with Gaussian distributions.

emulsion and the thermodynamically unstable mini-emulsion, with 25 nm droplets) is much faster compared with the unstirred samples. Their conclusion that: “[..] we are dealing with an unstable mini-emulsion, which undergoes coalescence after stirring is turned off,” suggests that smaller (dimensional) species will disappear in time and this is also what we observed in our undisturbed systems. Determination of the precise SE mechanism for the investigated recipes was beyond the scope of the present work. It would also not be an easy task, since Span80 is not a singlecomponent material [42,43], but a mixture of different sorbitan

esters (monooleate—the major component, dioleate, trioleate and tetraoleate) and other start and byproduct (after contact with water) materials like: oleic acid, the very hydrophilic sorbitol and sorbitol isosorbide. In principle, different constituents of the Span80 could be involved in different SE mechanisms (as was mentioned in Section 1). Before presenting the observed behaviors for polymercontaining systems, we would like to elaborate shortly on two issues. The first one concerns the potential influence of SE effects when measuring interfacial/surface properties of Span80 added to an oil/water mixture. We refer in particular to measurements of the cmc (= critical micellar concentration) and Γ (= interfacial tension) values. Peltonen and co-workers [44] showed that change of the oil phase has only a minor influence on the cmc and Γ . However, a survey of the data reported in the literature [40,44–47] brings an astonishing picture; it seems that the Γ values at (and above) cmc are scattered over almost one order of magnitude, between: 22.4 mN/m [44] and 3.5 mN/m [46]. Studying the kinetics of Span80-film formation at a mineral oil/aqueous interface, Opawale and Burgess [40] noted that the temporal changes of the film elasticity indicate: “that either an interfacial association of inverse micelles and/or surfactant multilayer formation has taken place.” Due to these above-mentioned observations, performing experiments with formulations for which SE could play a role is not trivial. The second issue is related to the question: Is there a threshold value of the surfactant concentration (noted in what follows with CSE ) below which the SE is inhibited or absent? For our experiments with pure water reservoirs it was possible to quantify precisely the limit of the two regimes. Below CSE w = 0.006% Span80, no SE droplet formation was seen. This threshold value will change if the aqueous phase is enriched with other additives like salt or gelatin (see Section 3.1.2).

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Fig. 6. Time evolution of the spontaneous emulsification for freshly prepared gelatin-containing droplets with (20%) extra Span80. The images were taken at (a) 0, (b) 20 or (c, d) 60 min after adding the extra surfactant. The (a–c) snapshots refer to the same reservoir. The (d) image shows another large G-drop (in focus) affected by SE and surrounded by other, slightly smaller gelled drops. For advanced stages of SE (like in image d), the large number of SE droplets attached at the surface create the impression that the mother reservoir is ‘filled’ with extremely small spheres.

3.1.2. Gelatin gels/dodecane interfaces Besides pure water droplets, also gelatin (G) containing reservoirs were studied. First, small G-drops from a macroemulsion (see Section 2 for preparative details) were studied either fresh (within 2 h after preparation), or after a few days of aging at 20 ◦ C. For the fresh macroemulsion droplets, the rate of spontaneous emulsification was found to significantly increase after the addition of surfactant to a high concentration (i.e., 1%). The corresponding images of ‘mother’ (diameter >10 µm) and ‘satellite’ (SE, sub-micronic) droplets are shown in Fig. 6. During evolution towards equilibrium, an intermediary stage was observed, in which the SE droplets are ‘arrested’ on the surface of the gelled reservoirs (Figs. 6c and 6d). Meanwhile, the gelled reservoirs do not change significantly their volume. This apparent volume conservation tells us

that only a small fraction of the total water was released as SE drops. It is interesting to note that the SE drops are not able to liberate themselves from the gelling reservoir surface immediately after their formation (and hence give the raspberry-like aspect in Figs. 6c and 6d). This might be related to wetting of the gelatin by SE drops that are only partially coated by Span on the oil side, assisted by a depletion force due to Span micelles. The diameters of the surface-linked SE droplets turned out to be close to those found for the free SE droplets released from pure water reservoirs, as can be seen from a close examination of Figs. 6d and 7a. Other macroemulsion samples were left in the original formulation (with 1% Span80), and inspected as a function of time. A large number of samples showed that, after 2 days,

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(a)

(b1)

(b2)

Fig. 7. Gelled (10% gelatin) droplets in 1% surfactant solution, after 2 days (a) and 7 days (b1, b2) from preparation. In the b1 and b2 images, the same position is imaged and only the focus was varied as to show better both the SE (small) droplets and the big gelled mother-reservoir. For (a) the objective’s focus was adjusted as to see better the small SE droplets attached to the surface of the big (middle) G-drop.

macro-emulsion G-drops with attached SE small droplets are rare (Fig. 7a). They are though observable due to the reduced transparency of the macro-drop. Samples taken from the sediment of the macroemulsion after 7 days show a massive population of free SE droplets (Figs. 7b1 and 7b2). These findings indicate that, while the SE process goes slower at 1% Span80 than at 20%, the process is still ongoing at 1%. Also (much larger) gelatin-containing single droplets (like in Figs. 1–3) were studied for spontaneous emulsification. These samples were made by injecting µl-quantities of hot gelatin solution into a continuous phase at room temperature as explained in the experimental section. Contrary to the macro-emulsion case, for these ‘single droplet’ systems, the Span80 concentration can be made low without the penalty of coalescence with other mother droplets. This allowed to measure the critical Span80 concentration, below which no SE occurs: CSE , by sim-

ply diluting the oil phase with pure dodecane. It turned out that, for mother droplets containing 10% gelatin, no SE occurred below 0.08% Span80, while for droplets containing 30% gelatin, 0.46% Span80 was minimally needed for SE. For gelatin-free droplets we already found earlier a critical Span80 concentration of 0.006%. The ‘threshold values’ are true constants and have been determined (for each composition) from observations done in time, up to 1 week. The cut-off is sharp. All these observations clearly point out that more gelatin inside the water reservoir protects better the interface against SE. Another interesting observation is that, for surfactant concentrations only slightly above the CSE , for both gelatin-rich and pure water reservoirs, the dimensions of the fresh SE droplets are larger (around 1 µm) than the diameters measured at higher Span concentrations (about 600 nm). On the long term, the SE droplets liberated from gelled single mother droplets behave much alike

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complexity of the problem at the small (dimensional) scales involved, we performed AFM experiments. 3.2. AFM measurements on gelatin gel/dodecane interfaces in presence of Span80

Fig. 8. Gelatin gels decrease their initial weight due to SE-stimulated water transport. The weight loss depends on both the Span80 and the gelatin concentrations. Gelatin contents are: 10% (2) and 30% (!). Data were calculated as percentages of the initial gel weight.

the ones prepared from pure water. Again, around 3 months are enough for the total disappearance of the satellite SE droplets. After reaching the equilibrium, the gelatin containing systems are separated in 3 phases: the oil, the partially dried gel and the water phase. Finally, we also studied spontaneous emulsification for macroscopic gelatin gels in contact with dodecane/Span80 continuous phases. While optical access to the gel/oil interface was precluded for these samples, the water expulsion (via SE) by these gels could still be measured. Analysis of the expelled liquid with SDS-PAGE indicated the absence of gelatin. The results are summarized in Fig. 8. After 24 h, the gel surface was covered with a dense layer of ‘trapped’ SE droplets. The thickness of this layer is by far higher than the average diameter of a single SE droplet. Longer (than 24 h) exposure of the gel to the oil phase did not lead to an increased amount of expelled water. For the 10% gelatin gel, a dependence on the surfactant concentration was found. Increasing the gelatin concentration to 30%, the SE phenomenon was strongly suppressed, though not completely eliminated, at least for high surfactant concentrations. For a 10% gel in salted (0.3 M NaCl) water, only 2% weight loss was registered. This value is relatively close to the one measured (around 1%) for water exudation from gels stored under pure dodecane. Thus, through a combined effect, both salt and gelatin act as strong inhibitors of the SE process. Summarizing our observations up to this point, surfactantmediated water transport is clearly influenced by: (a) the concentration of Span80 dissolved in dodecane, (b) composition of the aqueous phase (pure or with additives: salt, gelatin). Besides spontaneous emulsification, for polymer networks there is also another mechanism that can provide a water supply in the proximity of the surfactant-protected interface with oil. This second mechanism involves water exudation via gel syneresis. Its extent and rate should be very small compared with spontaneous emulsification. Both mechanisms are prone to change the properties of a gel at the interface with the oil. In what follows we will elaborate more on the modifications of the interface region of the gelatin gels affected by each of the two water-transport mechanisms. To be able to capture the

In the first part of this paper, the surfactant concentration was seen to affect the transport of water from a gelatin gel phase to the Span80/dodecane oil phase. In this second part we will discuss the temporal evolution of the gel, focusing on two related properties: the local elasticity of the gel, and the local wettability of the gel surface for (relatively) hydrophilic objects. Both were measured using an Atomic Force Microscope (AFM) with a Si3 N4 tip as an indenter. By performing shallow indentations (<1 µm deep) through the interface, information can be obtained from the compression and wetting forces experienced by the tip. The experiments were performed with macroscopic pieces of (10% gelatin) gel, which were set and kept at 20 ◦ C under dodecane with surfactant. Both low and high Span80 concentrations were investigated. Similar experiments, but in the complete absence of Span80 have been described in [38]. 3.2.1. AFM on gels at low Span80 concentration (CSE gel limit) Based on our earlier finding that, for a single large (10% gelatin) droplet, the critical Span80 concentration for SE was 0.08%, we first prepared macroscopic gelatin gels at 0.01 and 0.02% of this surfactant in dodecane. While no visible water separation from the gel was seen, the AFM measurements indicated that the gel/oil interface changes in time. To facilitate the analysis of these changes, let us first consider a typical AFM experiment on a gelatin gel, as compared to the reference case of an (inert) hard substrate. In a force– distance experiment, a piezo tube drives the AFM tip towards the sample from a starting position far above it. No interaction forces from the substrate are felt by the tip, hence the cantilever deflection (D) equals zero. For a hard substrate (like glass), a measurable, non-zero deflection value is only found after the tip makes direct contact. Since for hard surfaces the indentation (i.e., the deformation of the material) is negligible, in the contact regime the cantilever deflection is practically equal to the displacement (Z) of the piezo tube. Only elastic deformations play a role in this case, hence the retraction curve superimposes with the approach curve (see Fig. 9a). For 10% gelatin gels (aged for 24 h, i.e., the minimal time before experiments can be done), the typical ‘mechanical fingerprint’ looks rather different. Again, on approaching the sample, the tip feels no interaction before contact with the gel. In the compression region, the D–Z curve has a smooth increase with a slope smaller than 1, which indicates that a substantial deformation takes place in the gel. On retraction, a partial wetting of the tip by the soft gel generates a capillary bridge that extends far more than the initial position of the sample surface before loading. (A pictorial snapshot of the gel behavior during this retraction stage can be found in Fig. 6d of Ref. [38].) Finally, a rupture of the gel–tip contact returns the deflection value to zero. Despite the obvious adhesion between the gel

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(a) (a)

(b) (b) Fig. 9. AFM approach (1) and retraction (2, ") curves recorded for a regular (blunt-cone) tip in contact with (a) a hard substrate (b) a 10% gelatin gel. Both substrates were immersed in (dodecane +0.01% Span80).

and the AFM tip, it turned out that after the jump-off-contact (at the end of the retraction stage), the indenter was left behind without any detectable contamination by gelatin chains or small pieces of gel [38,48]. These findings indicate that gelatin gels under a compressive load show predominantly elastic behavior. This is confirmed by the absence of a significant hysteresis in the force curve for F > 0, as shown in Fig. 9b, and was already more rigorously proven in [38], through the lack of a dependence of the force curve on the compression speed. To quantify the elasticity, relative Young moduli (E ∗ ) can be determined by fitting the force curves with the Hertz model (after recalculation of the initial D–Z data in terms of force—Z curves and, further, into force (F )—indentation (δ) graphs). The detailed expression linking E ∗ with the indentation depth for loading a soft sample with a blunt-cone hard tip can be found in [38]. In this earlier study, we found that E ∗ showed a dependence on the indentation depth. While for gels compressed up to indentation depths (δ) smaller than 200 nm, scattered E ∗ data were found, for higher δ, a saturation of E ∗ to a lower plateau value was obtained. This was termed: “surface hardening.” In the light of these earlier findings, we chose to take the E ∗ found for an indentation depth ∗ of 500 nm depth (Eδ=500 nm ), as the measure for the elasticity in the present paper, where gels aged for different times are compared.

(c) Fig. 10. Examples of AFM curves displaying additional features (like multiple peaks and snap-in behavior), measured on 10% gelatin/water gels, aged for 2 days (a), 4 days (b) and 7 days (c) under oil with 0.01% surfactant. The arrows indicate the first contact of the tip with the soft sample. The jump-in events in the approach curves are consistent with the formation of free-water pockets on top of the gel.

One complication was that, for gels which were aged for 2 or more days (under dodecane with 0.01% Span80), additional features showed up in the force curves. The total set of AFM recordings taken at different locations (see Fig. 10, for examples), showed a considerable number of curves which did not have a continuous (ascendant) shape in the compression regime (as in Fig. 9b). Multiple measurements performed at the same (X, Y ) location demonstrated that the Z location of the jumpin is not reproducible. These jump-in events could be associated with (i) changes in the tip–sample contact area and/or (ii) attraction due to additional water on the top of the gel [48]. To still

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(a)

(b) Fig. 11. Time-dependence of the apparent elastic modulus E ∗ for 10% gelatin gels (a) without salt and (b) with 0.3 M NaCl, aged in dodecane with 0.01% Span80. The graphs include calculated E ∗ values based on macrorheology experiments (2, ") for the respective samples. The thick line in (b) is copied from (a). The E ∗ -values found with AFM are distributed, as indicated by the dashed regions.

characterize the “elasticity” of these more complex materials with relative Young moduli E ∗ , we chose to use only curves without instabilities during gel loading. Indentation depths as high as 500–700 nm could be reached without jump-ins for a number of curves. While the 1-day-old 10% gelatin gels in the presence of 0.01% Span80 showed a more or less constant E ∗ for various XY locations, the samples aged for 2 or more days showed substantial variations in E ∗ , depending on the XY location. (Note: also the dependence of E ∗ on δ showed an interesting trend for these samples. While the E ∗ values are still scattered for indentations below 200 nm and develop into a plateau at larger indentations, the E ∗ (δ) curves at the XY locations with small ∗ Eδ=500 nm also show less data scattering at small indentations.) In our opinion, the dependence of E ∗ on the XY location indicates that the interfacial region of the gel (with the oil) does not age in a spatially homogeneous manner. The scatter of E ∗ measured at different aging times is illustrated in Fig. 11. A noteworthy detail is that the salt-free (Fig. 11a) and the salted gels (Fig. 11b) behaved slightly different in this respect: for the samples with 0.3 M NaCl, it occasionally occurred that the measured E ∗ values after longer

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Fig. 12. Approach curves recorded for a fixed location on a salted (with 0.3 M NaCl) 10% gel, after gelation for 7 days. The measurements were done on the sample as prepared (with 0.01% Span80, open symbols) and after replacing the liquid phase with fresh dodecane (2). The calculated E ∗ (= 8 kPa) for the undisturbed gel corresponds to the last point (!) in Fig. 11b. The insert is a zoom in the region were the curve switch its behavior to the one measured on the gel.

aging times, were even lower than those of the samples aged for 24 h. This is remarkable, especially considering that the macroscopic elastic modulus, measured for the same samples in a rheometer (see [38] for details about these measurements) shows a steady increase over time. This apparent contradiction (for a few measurements) could suggest that at the surface, not only a hardening occurs, but at some places also a local softening. To further investigate these aged gels, we subjected some of them to a treatment in which the continuous phase was carefully washed away and replaced by pure dodecane. One example of (many times performed) AFM recordings on a fixed XY location for an aged sample, before and after flushing is given in Fig. 12. While the two curves approach the same slope in the end, it is clear that the sample before flushing has a softer response at indentations up to ≈600 nm. The slope of curve for the same sample after the flushing corresponds to E ∗ values near the upper limit of the E ∗ -domain (in Figs. 11a and 11b). This could suggest that during the flushing, structures or entities are removed, that previously caused an apparent softening of gel (interface). Other findings are also worth to mention in this context. Some of the AFM recordings on aged samples that had not been washed, showed large snap-in forces prior to compression into the gel (curves not shown). We think that this points at the presence of free water at the gel–oil interface. Another noteworthy finding is that the small force-hysteresis observed between approach and retract curves under compressed conditions (data not shown), disappears completely after the oil replacement. Finally, it is also interesting to note that the Z-location where (for the non-flushed samples) deviations from a simple elastic behavior occur, was at the most 500 nm above the onset of the usual ‘Hertzian’ behavior. Summarizing all our findings for the (2 days or more) aged gels, i.e., (i) the apparent decrease of E ∗ in time (at some locations), (ii) the existence of AFM curves with snap-in events and (iii) the differences in the AFM curves measured before and af-

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ter oil replacement, we are led to conclude that water syneresis from the gels creates small water pools at the sample/oil interface. If these later ones are small (<600 nm—which appears to be the typical size for the SE droplets), no direct indication (like jump-ins) of their presence will be sensed during compression into the gel. However, the existence of these submicronic water pools will then still be reflected in slopes of the AFM compression curves (like in Fig. 12) and, hence, in the E ∗ data (in Figs. 11a and 11b). This reduction of the E ∗ due to interfacial free water would also explain the different magnitudes for ‘the elastic modulus’ measured for gelatin gels, measured with conventional rheometry and with AFM [38]. To understand why the E ∗ as measured with AFM will be reduced by the presence of a small water pool at the interface, one could imagine a simple qualitative picture, in which both the water pool and the gelatin gel are considered as elastic springs (it was already shown in previous studies (e.g. [48]) that water droplets under compression behave like springs, via their surface tension). Provided that the water pool is large enough, and/or the indentation is small enough, the whole force of the AFM cantilever has to be transmitted to the gelatin gel via the water pool. This situation clearly corresponds to a serial arrangement of the elastic springs of the water-pool and of the gelatin-gel. In that case, the measured apparent ‘spring constant’ will always be smaller than that of the gel alone, since extra deformation takes place in the water pool. 3.2.2. AFM on gels with high Span80 concentration (CSE gel limit) When the surfactant concentration (in the oil covering the gel) exceeded the SE limit, the samples quickly became covered with thick layers of SE-satellite droplets. Attempts to perform AFM measurements on such samples were fruitless and the only option was to try and remove the interfering droplets. Our first attempt to do this was by flushing the gel surface with copious amounts of dodecane and allowing the gel interface to re-equilibrate before measuring AFM force–distance curves. The gel (interface) thus obtained turned out to be soft, with E ∗ moduli of O (1–10 kPa). We also gave the gels a more rigorous treatment, in which the washing with dodecane was followed by a complete removal of the oil phase (by wiping the gel surface with a dry tissue), then covering the gels with fresh dodecane and measuring after a set re-equilibration time. The AFM curves (not shown) did not show significant hysteresis and the obtained E ∗ values were high, i.e., more or less comparable to those of the long-aged samples at very low Span80 concentrations (Fig. 11a). Extending the re-equilibration time from 24 h to 6 days, no further changes in the E ∗ values were found. This indicates that the gel stiffening at the interface, as caused by Spontaneous Emulsification, occurs relatively fast (compared to syneresis). It may in fact already occur during the gelation itself. The results, summarized in Fig. 13, are consistent with a densification of the gel’s top region, caused by the loss of water to the ‘oil phase.’ The more Span80 is present in the continuous phase, the more the extraction of water is facilitated. This is in line with both the data in Fig. 8 on the amount of extracted

Fig. 13. Water expulsion caused by SE generates a hardening of the gel’s surface, which can however be masked by the presence of SE droplets. It is only after wiping with a tissue that the surface hardening can be made visible via the E ∗ values.

water and with the E ∗ data in Fig. 13, which show a stronger stiffening for gels in the presence of higher Span80 concentrations. Water–transport outside the polymer network leaves at the interface a tougher zone. The depth of this partially dried ‘skin’ was found to be at least 600 nm (i.e., the maximum indentation depth in our AFM experiments), a depth which also renders unlikely alternative explanations (for the change in stiffness) involving molecular lengthscales. Compared with the gradual increase in elasticity during gel aging (see Fig. 11a), the surface hardening generated by Spontaneous Emulsification (Fig. 13) is a rather quick way to ‘encapsulate’ the gelatin network. Choosing this last option has important implications for industrial/practical applications. A few of them are mentioned in the following section. 4. Conclusions We studied Spontaneous Emulsification (SE) in W/O systems (i.e. with a water reservoir immersed in oil), to map out the conditions where it occurs and to assess the implications for research on such emulsions. In terms of composition, the simplest W/O system studied by us consists of water, dodecane oil and the Span80 surfactant. Below 0.006% (w/w) surfactant in oil, SE remains absent, but at higher concentrations it invariably occurs. The existence of such a threshold concentration suggests that SE will proceed until the (oil) bulk and (water/oil) surface concentrations of the surfactant reach values which are in equilibrium with respect to each other. Experiments in which other components (like salt or gelatin) were added to the water phase demonstrate that, also with respect to the water, an equilibrium applies: In extracting water from the aqueous reservoir, the surfactant now has to compete with the additives, which tend to keep the water inside the reservoir. This explains why the critical Span80 concentration increases for higher gelatin or salt content. In the presence of gelatin in the reservoir drop, the satellite droplets behave differently. For this last case, the satellite droplets tended to adhere to the reservoir drop. The SE phenomenon could be monitored very well with video-microscopy, due to the formation of satellite droplets ap-

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proximately 600 nm in size. Since this is close to the optical resolution of the microscope, it remains to be seen whether this is a characteristic size, or just ‘a prominent size’ within the range accessible to optical microscopy. The sustained presence of satellite droplet material (evidenced via turbidity) up to several mm above the substrate (in spite of their higher mass density) suggests that also smaller droplets were formed. Overall, AFM in force–distance mode proved to be a suitable method to investigate the subtle chemical and structure changes at the interface which are involved in syneresis and spontaneous emulsification. Indications were found that these processes are accompanied by simultaneous local densifications of the gel network and the formation of small water-pools. While the local density of the gelatin network is reflected in the elastic stiffness, the presence of water pools is manifested most clearly via (capillary) snap-in and snap-out forces. The reduction of the apparent elastic modulus by the water pools may still require a more rigorous proof, but taking all the AFM observations together the picture as just presented, looks consistent. We note in passing that the found here ‘surface sensitivity’ of AFM in force–distance mode, also underlines the need for carefulness in comparing apparent Young moduli from AFM with macroscopic bulk moduli [48] as measured with classical rheology. One potentially important outcome of our investigations is that the SE does no longer affect the water transport in the gelled systems for time scales larger than 24 h, due to the formation of an interfacial gelatin layer that is so dense that the rate of further water transport is reduced practically to zero. 4.1. Implications for related research and practical applications Knowing the conditions where SE can (or cannot) be avoided is of key importance for fundamental studies aimed at materials characterization, like measurement of the surface tension of the W/O interface or the bulk elasticity of gelatin containing reservoirs (with AFM). In experimental situations when SE does occur, the characteristic timescale of the process may be of importance too. Different processes have to take place to keep the SE going: (i) transport of water and surfactant towards the W/O interface and (ii) transport of the satellite droplets away from this interface. Depending on composition and the time after system formulation, different steps may be rate-limiting. For the case of pure water, we imagine that the formation and subsequent transport of the satellite drops will be rate determining. In the case of gelatin-containing drops, the slow release of water due to the contraction of the gelatin network may be rate-limiting. None of the experiments in the present study was done by externally imposing a flow, hence forcing the water reservoir droplets to move with respect to the oil phase. Also in studies different from ours (see Refs. [12–16]), the SE process was seen to operate for ‘immobilized reservoirs.’ In the case that oil would be flowing around the reservoirs, capillary waves at the water/oil interface would become suppressed, thus decreasing the probability for SE occurrence. In the experiments of Link et al. [49], where 2–30 µm-sized water droplets stabilized with

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Span80 in hexadecane were produced in microfluidic flow devices, SE was never observed. As corollary to the present study, we would like to add few comments with relevance for large-scale production and application of emulsions with a gelled internal phase. For such emulsions, the formation of satellite droplets (via SE) and/or the existence of tiny water-pockets (formed via gel syneresis) could have several consequences. First, when water accumulates in the interface region, the surface interactions between droplets change. Supposing that two droplets come in close proximity in a configuration which favors the coalescence of the surface water inclusions, these later may connect trough a capillary neck. If the scale of this bridging extends, it may turn an initially stable dispersion into an aggregated particulate network. Second, for controlled-release applications, in which a precise dosage of water-soluble active compounds must be delivered with constant or at least predictable rate, the SE phenomenon could be highly detrimental. Compared to the gelled reservoirs, the SE-satellites will have a different load of the beneficial reagents. Also, surface hardening (due to SE) will impede on the diffusion of the active substances still trapped inside the gelatin gel. A tougher interface can shield completely the inner substances from the liquid environment outside the polymer-containing drop. Despite the potential drawbacks generated by SE, gelled droplets can be used as carriers if some precautions are taken in their preparation. If the reservoirs containing the gel precursor are kept in (strong enough) flow during gelation, it should be possible to suppress SE. Actually, we have already used a formulation (with water/gelatin/dodecane/Span80) above the SE threshold concentration to prepare stable emulsion droplets under flow conditions [50]. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]

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