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Long-term stability of crystal-stabilized water-in-oil emulsions
Journal of Colloid and Interface Science 460 (2015) 247–257
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Journal of Colloid and Interface Science journal homepage: www.elsevier.com/locate/jcis
Long-term stability of crystal-stabilized water-in-oil emulsions Supratim Ghosh a,⇑, Mamata Pradhan b, Tejas Patel b, Samira Haj-shafiei b, Dérick Rousseau b,⇑ a b
Department of Food and Bioproduct Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, Canada
g r a p h i c a l a b s t r a c t
a r t i c l e
i n f o
Article history: Received 4 June 2015 Revised 28 August 2015 Accepted 31 August 2015 Available online 1 September 2015 Keywords: Emulsion stability Pickering Network Wax crystallization Interfacial crystallization Coalescence Sedimentation Microstructure Contact angle
a b s t r a c t The impact of cooling rate and mixing on the long-term kinetic stability of wax-stabilized water-in-oil emulsions was investigated. Four cooling/mixing protocols were investigated: cooling from 45 °C to either 25 °C or 4 °C with/without stirring and two cooling rates – slow (1 °C/min) and fast (5 °C/min). The sedimentation behaviour of the emulsions was significantly affected by cooling protocol. Stirring was critical to the stability of all emulsions, with statically-cooled (no stirring) emulsions suffering from extensive aqueous phase separation. Emulsions stirred while cooling showed sedimentation of a waxy emulsion layer leaving a clear oil layer at the top, with a smaller separation and droplet size distribution at 4 °C compared to 25 °C, indicating the importance of the amount of crystallized wax on emulsion stability. Light microscopy revealed that crystallized wax appeared both on the droplet surface and in the continuous phase, suggesting that stirring ensured dispersibility of the water droplets during cooling as the wax was crystallizing. Wax crystallization on the droplet surface provided stability against droplet coalescence while continuous phase wax crystals minimized inter-droplet collisions. The key novel aspect of this research is in the simplicity to tailor the spatial distribution of wax crystals, i.e., either at the droplet surface or in the continuous phase via use of a surfactant and judicious stirring and/or cooling. Knowledge gained from this research can be applied to develop strategies for long-term storage stability of crystal-stabilized W/O emulsions. Ó 2015 Elsevier Inc. All rights reserved.
⇑ Corresponding authors at: Department of Food and Bioproduct Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7J 2X3, Canada (S. Ghosh). Department of Chemistry and Biology, Ryerson University, Toronto, Ontario M5B 2K3, Canada (D. Rousseau). E-mail addresses: [email protected] (S. Ghosh), [email protected] (D. Rousseau). http://dx.doi.org/10.1016/j.jcis.2015.08.074 0021-9797/Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Emulsions in products such as cosmetics, pharmaceuticals and foods rely on the presence of crystallized species such as polymers, paraffins or triacylglycerols for long-term stability [1–6]. Water-in-
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oil (W/O) emulsions used in such applications must satisfy different criteria including: (i) stability against physical breakdown (particularly once in the hands of the consumer), (ii) resistance to changes in environmental conditions (temperature, vibration/ shaking) and (iii) lack of change in quality attributes (texture, etc.) over time. Key factors include the composition and volume fraction of the continuous and dispersed phases, the mean droplet size and distribution of the dispersed phase, the presence and concentration of stabilizing agents (surfactants, thickeners or crystals) and how the emulsion is processed and stored [7]. Finally, the interactions between the various components in an emulsion will also affect their stability [8]. Emulsion processing requires that the droplets be welldispersed within the continuous phase, which is normally achieved via valve homogenization or high-shear mixing. In many thickened or solid-like emulsions, subsequent gelation or crystallization of the continuous phase helps to keep the dispersed droplets in place. Yet, little is known regarding how to tailor the spatial distribution of crystals in oil-continuous emulsions to assist in retarding physical breakdown. If crystals are adsorbed to the oil–water interface, they create a solid shell around individual dispersed droplets which prevents droplet–droplet coalescence [9,10]. Such surface-active species are usually surfactants that solidify directly at the interface or adsorb onto the droplet surface [3]. As they are surfaceinactive, many crystalline species such as wax or triacylglycerols will form a continuous phase network that envelops the dispersed aqueous phase thereby limiting droplet–droplet collisions [8]. We have previously shown that the surfactant glycerol monooleate can promote the heterogeneous interfacial nucleation of hydrogenated canola oil (a surface-inactive fat) at the water droplet surface in W/O emulsions [11]. We also showed that the addition of a dispersed aqueous phase significantly altered the rheological properties of W/O emulsions [12], as has also been reported by others [5]. Visintin et al. characterized emulsion rheology, and showed that presence of a dispersed aqueous phase was a key contributor to emulsion elasticity [13]. Finally, Hodge and Rousseau showed that the addition of crystallized fat prior to, or after, homogenization (and subsequently quench-crystallized) enhanced the stability of surfactant-stabilized W/O emulsions, with the latter conferring greater stability [14,15]. This paper discusses the impact of cooling rate and stirring on the long-term properties of wax-stabilized W/O emulsions. The goal was to investigate how the presence of a dispersed aqueous phase impacted emulsion macroscopic phase separation, wax crystal morphology, solid wax content and droplet size distribution. In particular, we were interested in showing how it is possible to use minimal amounts of surfactant to generate W/O emulsions with long-term stability against physical breakdown. This was achieved through the use of a low concentration of wax in the continuous oil phase that was suitably crystallized. Importantly, we showed that the optimal stabilization of model W/O emulsions depends on the concerted role of interfacial and network wax crystallization, with the combination of stirring and rapid cooling from the melt conferring the highest kinetic stability. The results from this study are relevant to processed products that exist as oil-continuous emulsions and may serve as a guide for the judicious formulation of emulsions where long-term kinetic stability is desired.
surfactant to aid in emulsification, yet not keep the emulsion stable for any length of time. The light mineral oil was obtained from Fisher Scientific (Nepean, ON, Canada). The stated maximum kinematic viscosity at 40 °C was 33.5 mPa s and its density (q) was 0.84 g/ml at 25 °C. A 3.5 wt% sodium chloride (Fisher Scientific, Nepean, ON, Canada) in de-ionized water (resistivity P18.0 MOhm cm) solution was used as the dispersed aqueous phase (q = 1.02 g/ml). The surfactant used was glycerol monooleate (GMO) (Dimodan MO 90Ò, Danisco, New Century, KS, USA). Dimodan MO 90 is a food-grade low-HLB (hydrophilic/lipophilic balance) emulsifier composed of >92% glycerol monooleate, with the rest consisting of small amounts of glycerol, glycerol dioleate and glycerol trioleate. A highly-refined paraffin wax (IGI-1242; m.p. 56.7–58.9 °C) was obtained from The International Group, Inc. (Toronto, ON, Canada). Its n-paraffin composition provided by the manufacturer is given in Fig. 1. 2.2. Emulsion preparation A schematic of the emulsion preparation protocol is shown in Fig. 2. The oil phase contained 0.05 wt% GMO as emulsifier and 5 wt% paraffin wax. This level of emulsifier allowed for the formation of dispersed droplets, but did not prevent them from measurably coalescing within a few days in the absence of stabilizing wax. All emulsions were formulated to initially consist of dispersed aqueous droplets with a mean diameter of 30 lm with a maximum size of 300 lm, which permitted direct comparison between the different W/O emulsions. The aqueous phase contained 3.5 wt% NaCl, which was necessary to prevent undue formation of oil droplets in the water phase (hence double emulsion formation), which we ascribed to charge generation by the intense shearing action of the homogenizer [16]. Pre-mixing was performed by slow addition of the aqueous phase (9 ml/min) to the oil phase in a beaker at 45 °C via magnetic stirring at 500 rpm for 15 min. The pre-mix was quickly transferred to a valve homogenizer (APV-1000, APV, Albertslund, Denmark) preheated to 45 °C and homogenized at 500 psi for 4 cycles. Afterwards, 100 ml samples were collected in 250 ml glass beakers (diameter: 6.5 cm and height: 9 cm) and cooled via one of the four methods denoted at ‘quench-static’, ‘quench-stirred’, ‘slow-static’ or ‘slow-stirred’. For quench cooling, samples were transferred to a 0 °C waterbath whereas slow-cooled samples were placed in a temperaturecontrolled waterbath (Model 1157P, VWR International, Mississauga, ON) at 45 °C and cooled at a rate of 1 °C/min. Stirring during cooling was performed using a magnetic stirrer at 300 rpm. All samples were prepared in triplicate. Emulsion temperatures during the cooling regimes were measured along the centreline of the bea-
2. Materials and methods 2.1. Materials All emulsions consisted of a 20 wt% aqueous phase and a 80 wt% oil phase containing 5 wt% paraffin wax, mineral oil and sufficient
Fig. 1. n-Paraffin compositional analysis of the solid wax sample. Analysis is performed by high temperature gas chromatography yielding a n-paraffin distribution of the wax from C20 to C90.
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2.6. Droplet size distribution The dispersed droplet size distribution of the emulsions was determined at 25 °C and 4 °C using a Bruker Minispec Mq pulsed field gradient nuclear magnetic resonance (pfg-NMR) unit (Bruker Canada, Milton, ON, Canada) that allows unimodal characterization of emulsion droplet size distributions via restricted diffusion measurement [17]. The temperature inside the NMR sample chamber was controlled by an external waterbath pre-set at specific temperatures. The Minispec Mq NMR software version was 2.58 revision 12/NT/XP (Bruker Biospin GmbH, Rheinstetten, Germany) and the water droplet size application was v5.2 revision 4a. The pulsed gradient separation and number of pulse widths were 210 ms and 8 ms, respectively. The oil suppression delay was 85 ms and the magnet gradient strength was 2 T/m. The pfgNMR field gradient strength was calibrated with CuSO4-doped water (diffusion coefficient = 2.3 109 m2 s1 at 25 °C). Emulsion samples (height = 1 cm) in glass tubes (ID = 0.8 cm, L = 20 cm) were placed in the NMR unit and their droplet size distribution was determined by d3,3 (volume-weighted geometric mean diameter) values and the breadth of distribution, r (geometric standard deviation). Destabilized water droplets that phase-separated from the emulsion were calculated as free water and used as an indicator of emulsion (in)stability.
Fig. 2. Schematic of the emulsion preparation and stabilization protocol.
ker using an auto logger thermocouple (K type with thermometer HH2002AL, Omega, Montréal, QC, Canada) and downloaded to a computer using an IR-sensor connection (HHP-2000DL, Omega, Montréal, QC, Canada). Cooling was stopped when the centre of the sample in the beaker had reached the desired temperature (25 °C or 4 °C). 2.3. Emulsion storage All samples were stored at either room temperature (25 ± 1 °C) or at refrigeration temperature (4 ± 1 °C) for 1.5 years (78 wks). At set intervals, samples were analyzed via sedimentation behaviour, microscopy, solid wax content (SWC) and water droplet size determination. 2.4. Sedimentation Emulsion sedimentation behaviour was recorded visually and with a digital camera for 78 wks. The height of the sedimented emulsion layer was calculated from the digital images using Microsoft Office Picture Manager (Microsoft Canada, Mississauga, ON, Canada). Emulsion destabilization was measured based on the percent-emulsified layer (Femulsion) retained during storage:
F emulsion ¼
hemulsion htotal
ð1Þ
where hemulsion and htotal are the heights of the sedimented emulsion layer and the initial sample, respectively. 2.5. Microscopy Samples were taken from the storage beakers with a glass rod, placed on a viewing slide, covered with a cover slip and observed with an inverted Zeiss Axiovert 200M light microscope (Zeiss Inc., Toronto, ON, Canada) equipped with a temperaturecontrolled stage (model TSA02i with STC200 controller, Instec, Boulder, CO, USA) pre-set at the desired temperature. The viewing slides and the cover slips were cooled to the sample temperature prior to sampling. A 20 objective lens was used to record sample microstructure under DIC (Differential Interference Contrast) and PLM (Polarized Light Microscopy) modes.
2.7. Solid wax content (SWC) Emulsions were transferred into NMR tubes to a height of 4 cm. Samples were prepared and stored at both 4 and 25 °C. Samples were analyzed using the Bruker Minispec Mq pulsed NMR unit with the application sfc_lfc v2.51. Temperature of the NMR chamber was controlled using a water bath pre set to desired temperature. 2.8. X-ray diffraction Static wide-angle X-ray diffraction of the emulsions was performed on a Hecus S3-MICROcaliX (Hecus X-ray Systems GmbH, Graz, Austria). The unit uses a GeniX 50 W microsource and customized FOX-3D multi-layer point focussing optics (Xenocs SA, Grenoble, France). Samples (20 ll) were loaded in quartz capillaries (d = 1 mm) and characterized at 25 °C. 2.9. Contact angle measurements A portion of the wax used to stabilize the emulsion was melted at 80 °C with stirring, poured into an aluminium weigh boat at room temperature and allowed to crystallize for 24 h. Solidified wax discs were then cut into 1 cm3 cubes and placed in spectrophotometer cuvettes, with the smooth side facing upwards. Cuvettes were then filled with light mineral oil alone or with GMO. A small water droplet (D 1 mm) was injected from a needle (gauge 22, 0.394 mm nominal ID, Fisher Scientific, Nepean, ON, Canada) onto the surface of the solid wax using a 10 ml syringe and a programmable syringe pump (kd Scientific, Markham, ON, Canada) operated at 0.1 ml/min. Images of sessile water droplets were captured for 24 h using a Teli CCD camera with a macro lens assembly and IDS Falcon/Eagle framegrabber (DataPhysics Instrument GmbH, Filderstadt, Germany). Droplet contact angles against the solid surface were measured digitally (SCA 20 software, version 2.1.5 build 16, DataPhysics Instrument GmbH, Filderstadt, Germany). The oil phase/water interfacial tensions (IT) were determined using ASTM method D971 [18], with the DuNouy ring method (Fisher surface tensiometer Model 21, Fisher Scientific, Nepean, ON, Canada).
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2.10. Statistical analyses Triplicate analyses were performed on all sedimentation and droplet size measurements. Analyses of variance and post hoc tests were performed and statistical differences were deemed significant at p = 0.05. 3. Results and discussion 3.1. Emulsion cooling rates Fig. 3 shows a comparison of the different cooling rates used for emulsions cooled to either 25 °C or 4 °C. A maximum cooling rate of 4.6 °C/min was achieved with the quench-cooling and stirring regime whereas the slowest cooling rate was with slow-static cooling (0.7 °C/min). Stirring helped to maintain a more uniform cooling rate throughout the beaker whereas statically-cooled samples yielded slower temperature drops with some temperature inhomogeneities throughout. None of the cooling rates used were by any means rapid. Thus, it would be incorrect to speak of true ‘quenching’ in any scenario. 3.2. Emulsion sedimentation Control emulsions made with either GMO or wax destabilized within 1 h of preparation. Hence, the presence of both the GMO and paraffin wax was necessary to confer kinetic stability to all emulsions. All wax-containing emulsions nevertheless demonstrated phase separation dependent on composition and cooling/stirring regime (Figs. 4 and 5). When stored at 4 °C, the stirred emulsions (quench or slowly-cooled) showed oil separation and no aqueous phase separation. Separation was evident after 6 wks (Fig. 4). Those slowly and statically-cooled broke down with a clear water layer appearing soon after preparation. The quench-static emulsions demonstrated limited phase separation (not readily visible in the beaker) over 78 wks. These results demonstrated that more rapid formation of a wax crystal network prevented oil or water separation. In regards to stirred emulsions, the encased dispersed phase appeared to sediment along with the wax crystal network, leading to phase separation and oiling-off. The enhanced sedimentation stability of the stirred emulsions with respect to water separation ensured that the water droplets remained dispersed throughout the continuous oil phase during cooling and wax crystallization. Stirring also appeared to promote more direct contact between the aqueous droplets and wax crystals given the
entrainment of the wax crystal network by the dispersed phase in the stirred emulsions. Emulsions cooled to 25 °C were less stable against sedimentation than at 4 °C (Fig. 5). All emulsions demonstrated some phase separation after storage for 1.5 yrs, though the storage vessel used (i.e., beaker vs. NMR tube) was critical to this evaluation. Stirred emulsions (quench or slowly-cooled) showed oil separation after storage for 1 wk, but no water phase separation even after 1.5 yrs. Quiescent emulsions (quench or slowly-cooled) demonstrated some breakdown right after preparation, as the wax crystal network formed was too sparse to encase the dispersed droplets. With time, a supernatant oil phase was also visible. Storage in NMR tubes accentuated the observed destabilization, which has been ascribed to differences in wall effect and convective flow [19]. The 25 °C protocol did not lead to measurable droplets with NMR, further demonstrating the critical role that the wax crystal network played on emulsion stabilization. Too weak a network or too low an SWC resulted in an unstable emulsion, particularly with so little emulsifier. Overall, there was a much slower rate of emulsion sedimentation at 4 °C vs. 25 °C, as there was more solid wax present to prevent density-driven settling of the dispersed water droplets. For comparison, the sedimentation stability of the emulsions’ oil phases was also evaluated separately. All samples remained stable, and did not show any indication of phase separation at 4 °C and 25 °C. This lack of macroscopic phase separation clearly indicated that the dispersed phase played a significant role on phase separation. The continuous phase of all emulsions stored at 25 °C was flowable whereas for those stored at 4 °C, only the stirred samples were flowable, possibly due to the influence of stirring on wax crystal size and inter-crystal interactions [20]. 3.3. Sedimentation kinetics Femulsion (Eq. (1)) plotted vs. storage time (Fig. 6) was used to estimate sedimentation rate. Only stir-cooled emulsions were evaluated as the statically-cooled emulsions rapidly destabilized with clear separation of the aqueous phase. Amongst the stirred emulsions, sedimentation was slowest for those stored at 4 °C (no sedimentation for 1 wk) compared to storage at 25 °C (where emulsions sedimented to 70% of their original heights within the first wk). After 6 wks, the latter emulsions sedimented to 65% to 68% and to 63% to 65% after 1.5 years, respectively for quenched and slowly-cooled systems. By contrast, the 4 °C emulsions sedimented to 82% and 78% of their initial heights after 6 wks for the quenched and slowly-cooled emulsions, respectively, followed by a decrease in height to 69% for the quenched-stirred emulsion and to 65% for the slow-stirred emulsion after 1.5 years. 3.4. Emulsion microstructure
Fig. 3. Temperature profiles of emulsions under the four cooling protocols investigated. Cooling rates were calculated from the average slopes of the cooling curves: quench-stirred (4.6 °C/min), quench-static (1.3 °C/min), slow-stirred (0.9 °C/min), and slow-static (0.7 °C/min). Quench cooling was done in a 0 °C waterbath whereas slow-cooled samples were placed in a 45 °C waterbath and cooled at a rate of 1 °C/min. For cooling to 25 °C samples were withdrawn from the waterbath when the centre of the sample reached desired temperature.
Emulsion microstructure at 4 °C and 25 °C was highlydependent on the cooling/stirring regime used. As noted above, emulsions stored at 4 °C were more stable against sedimentation than those stored at 25 °C. Microscopy results concurred, with evidence of a more stable dispersed aqueous phase at the lower storage temperature. Fig. 7 shows the combined DIC and polarized light micrographs of emulsions stored at 4 °C for 78 wks. Though demonstrating oil separation, the stirred emulsions (quench and slowly-cooled) showed the presence of a relatively stable dispersed aqueous phase, with little increase in droplet size over time. By contrast, the statically-cooled emulsions underwent breakdown with little evidence of dispersed droplets soon after emulsion preparation. Irrespective of cooling rate, with stirring, there was evidence of interfacial wax crystal adhesion (Fig. 7 arrows) suggesting the existence of wax crystal surface activity, and the possi-
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Fig. 4. Sedimentation behaviour of emulsions stored at 4 °C as a function of time.
bility of GMO promoting interfacial wax crystallization. Such a phenomenon was recently observed by us [11]. Fig. 8 shows combined DIC and polarized light micrographs of emulsions stored at 25 °C for 78 wks. The quenched-stirred emulsion was the most stable against changes in droplet size, with a still evident distribution of droplets visible after 1.5 years. The slowstirred emulsions were stable until at least 6 wks, but had broken down after 78 wks. Both statically-cooled emulsions were not stable, with the dispersed phase breaking down soon after homogenization and only a few water droplets can be seen. There was some evidence of interfacially-adsorbed crystals in all cases, which was most apparent with the quenched-stirred emulsions (as per the 4 °C results). By comparing Figs. 7 and 8, it can be seen that initially, 4 °C emulsions consisted of smaller droplets compared with those at 25 °C. After 6 wks, droplet sizes in emulsions at both temperatures were comparable whereas after 1.5 years, only the 4 °C emulsions remained stable with only a minor increase in droplet size. Fig. 9((A) and (B)) shows a close-up view of interfacial wax crystallization promoted by stirring under quench-cooling conditions. Clearly visible are interfacial crystals that stabilized the dispersed phase via Pickering stabilization as well as network stabilization, where crystals contributed to hindering droplet movement by enmeshing them within a continuous crystalline matrix. Together, these two mechanisms heightened the stability of the emulsions.
Macierzanka et al. [3] studied the time-dependent structural evolution of W/O emulsions prepared with mixtures of two different crystalline emulsifiers (partial acyglycerols and fatty acid monodiesters of propylene glycol). The emulsion droplets were initially Pickering-stabilized. With time, interfacial crystals grew into the continuous oil phase, leading to network stabilization and crystalline bridges between the droplets. Fig. 9A is the combined DIC and polarized light image whereas Fig. 9B shows the corresponding polarized light view only. Most crystalline species measured well below 40 lm in length and existed as needles with high aspect ratios. For comparison, Fig. 9C shows a putative crystal morphology where the oil phase of the emulsion was quench-cooled to 4 °C under stirring. Other than the missing interfacial crystals (given that no aqueous phase is present), the continuous phase crystal appearance is quite similar to that observed in the emulsions. 3.5. Evolution of droplet size distribution Emulsion droplet sizes were only measurable for the stirred emulsions cooled to 4 °C. All other emulsions (statically-cooled to 4 °C and all 25 °C emulsions) did not yield droplets measurable via pulsed NMR, either due to their size (too large) or population (too small). At 4 °C, the average droplet size (d33, 50th percentile value) of the quench-stirred and slow-stirred emulsions ranged
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Fig. 5. Sedimentation behaviour of emulsions at 25 °C as a function of time. Statically-cooled emulsions were also stored in NMR tubes to show a clear view of aqueous phase separation for both the quenched and slow-cooled samples (shown at right after 78 weeks’ storage).
free water content confirmed that both emulsions very slowly broke down with time. The initial breadth of the quenchedstirred emulsion droplet size distribution was broader than that of its slowly-stirred counterpart. After 1.5 years, however, the latter’s droplet size distribution narrowed more extensively (i.e., 95% of droplets were between 26 and 42 lm) which was accompanied by an increase in % free water. This narrowing was interpreted as greater emulsion destabilization than in the quenched-stirred emulsion, which saw a smaller increase in free water during this time. 3.6. Solid wax content
Fig. 6. Estimation of emulsion destabilization by sedimentation. Evolution of percent emulsified layer (Femulsion from Eq. (1)) is plotted as a function of time for slow or quenched-cooled stirred emulsions stored at 4 and 25 °C.
between 29 and 33 lm and remained unchanged over 78 wks (Fig. 10) (p > 0.05). During this time, the 97.5th percentile values significantly decreased indicating the loss of larger droplets as free water (i.e., larger than the NMR instrumental limit of 500 lm) or visually phase-separated water (p < 0.05). The gradual increase in
Fig. 11 shows the SWC of stirred emulsions as a function of time. As the statically-cooled emulsions were not stable at either 4 °C or 25 °C, their SWC was not measured. The SWC was much higher at 4 °C compared to 25 °C, with no effect of cooling rate (p > 0.05). After 78 wks, there was a significant increase in SWC at both temperatures due to continued wax solidification (p < 0.05). Though crystallization to 4 °C led to initially higher SWCs, over 78 wks, SWC increased to 60% for the emulsions processed at 25 °C and only 20% for those at 4 °C. Wax crystallization history was an important step in final SWC, and it also contributed to the observed differences in wax crystal
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Cooling protocol
Day 0
Week 1
Week 6
Week 78
Quenchedstirred
Quenchedstatic
Slowstirred
Slowstatic
Fig. 7. Combined DIC and polarized light micrographs of wax-stabilized emulsions at 4 °C as a function of storage time. The presence of both interfacial crystals (shown by arrows) and continuous phase crystals can be seen. The scale bar represents 40 lm.
morphology and population. Irrespective of the stirring/cooling regime, crystallization began when supercooling was sufficient for the highest-melting wax species to nucleate and act as crystallization centres. With more nuclei formed, there was more rapid bulk crystallization. Given the compositional variety of the wax (Fig. 1), low supercooling (i.e., cooling to 25 °C) initiated highmelting wax crystallization first. High supercooling (4 °C) likely resulted in more rapid, mass crystallization of the bulk, with formation of more mixed crystals consisting of lower and highermelting species, with perhaps these crystals acting as secondary nucleation sites. 3.7. Wax crystal structure X-ray diffraction revealed that the lamellar spacing of the solid paraffin wax at 25 °C was 41.57 Å with a weak second-order spacing at 20.39 Å and short spacings at 4.18 Å and 3.74 Å. Little change in both small and long-order spacings were observed for the wax present in all emulsions, with first order spacing at 42.1 to 42.5 Å. Short spacings were identical to the solid wax, with spacings at 4.18 Å and 3.74 Å. Given the alkane diversity present in the paraffin wax (straight-chained, branched, etc.), it would be imprudent to assign specific crystalline structures to each of these samples. Based on Craig et al. for pure alkanes, the 100% paraffin wax existed in the orthorhombic unit cell [21]. The orthorhombic unit cell c/2 value, which corresponds to the dimension of the carbon atom chains
and gaps between the planes of methyl endgroups between two successive molecular layers was determined using:
ðc=ÅÞ=2 ¼ 1:2724nc ¼ 1:8752
ð2Þ
where nc is the average chain length. Based on Eq. (2), a (c/Å)/2 spacing of 41.57 Å indicates an average alkane length (nc) of 31 carbons, which closely matched the compositional analysis of wax with an average alkane chain length of 29 carbons (Fig. 1). 3.8. Wax crystal wetting Contact angles (h) between the solid wax, and aqueous and oily phases were used to understand the ability of wax to promote Pickering crystallization. Equilibrium h of water droplets on the wax surface were measured at 147.5 ± 2.9° in light mineral oil and 130.5 ± 4.1° with the added presence of GMO. It is wellknown that 90° < h < 180° favours stabilization of water droplets in an oil-continuous medium [2]. At h = 90°, particles are equally wetted by the water and oil phases giving rise to the most stable emulsion. At h = 180°, crystals are completely wetted by the oil phase so no association with the water droplets occurs. In the present case, the smallest h measured through the aqueous phase was obtained with GMO-doped mineral oil against wax. The IT of mineral oil against water was 44.9 ± 3.4 mN/m and 4.1 ± 0.3 with added presence of GMO.
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Cooling protocol
Day 0
Week 1
Week 6
Week 78
Quenchedstirred
Quenchedstatic
Slowstirred
Slowstatic
Fig. 8. Combined DIC and polarized light micrographs of wax-stabilized emulsions at 25 °C as a function of storage time. The presence of both interfacial crystals (shown by arrows) and continuous phase crystals can be seen. The scale bar represents 40 lm.
The displacement energy (Edisp), which is directly linked to h, was used as a measure of the energy required to remove wax crystals from the oil/water interface [22] (Eq. (3)):
Edisp ¼ pr 2 cow ð1 cos hÞ2
ð3Þ
where cow is the IT at the oil–water interface and r is the radius of a wax ‘particle’. The sign inside the bracket is negative for displacement into the water phase, and positive for displacement into oil (e.g., movement of wax crystals into the oil phase) [23]. From Eq. (3), Edisp increases with an increase in oil–water IT, but decreases with increase in h. Assuming r 1 lm, Edisp to remove a wax crystal from the water/GMO/mineral oil interface was 3.8 108 kT, which indicates that presence of wax crystals at the water droplet surface is favourable unless a high amount of energy is provided for removal. Of note, Edisp to remove a wax crystal from the water/mineral oil interface (without GMO) was 8.5 108 kT, comparable to the value for GMO. However, as molten wax has no emulsifying activity, it could not be used to form and stabilize water droplets, hence, their movement towards the interface is not favourable. 3.9. Wax crystallization and network structure Effective emulsion stabilization was dependent on the concerted roles of interfacial and network wax crystallization, with both contributing to delaying water droplet sedimentation, flocculation and coalescence. Such ‘dual-mode’ wax crystallization was
itself dependent on temperature and mixing regime during emulsion processing. Crystallization to 4 °C yielded SWCs 3–4 higher than at 25 °C, which markedly impacted emulsion stability. At lower SWCs, there was insufficient solid mass to effectively surround the droplets and physically entrap the aqueous phase whereas the higher SWC at 4 °C retarded phase separation and coalescence. The presence of both interfacial and surrounding crystals kept droplets separate from one another due to the presence of interstitial crystals thus reducing the rate of flocculation. However, wax that crystallized directly at the interface visibly retarded coalescence and sedimentation vis-à-vis network-type crystals, as these crystals enveloped individual droplets thus preventing film drainage and rupture. As wax is not surface-active, the use of the surfactant GMO promoted direct interfacial crystallization. By contrast, bulk network crystallization, though also beneficial in stabilizing the emulsions, did not offer the same level of stabilization against emulsion breakdown, presumably due to gravity-induced droplet movement throughout the wax crystal network. Pickering particle removal from an oil–water interface is a necessary precursor to coalescence. The energy required for crystal displacement will depend on the composition and rheology of the interface as well as crystal wettability [24]. There was no particle desorption from the interface observed even after 78 wks given the high Edisp. Rather, alkane self-organization and crystallization promoted the formation of a solid shell that englobed the dispersed droplets. This suggested that incomplete coverage
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Fig. 9. Combined DIC and polarized light micrograph (A) and corresponding polarized light only micrograph (B) of wax-stabilized emulsions at 4 °C. The bottom picture presents a polarized light micrograph of a wax–oil mixture (C) at 4 °C cooled using a similar protocol to the emulsions. Scale bar represents 40 lm.
Fig. 10. Droplet size distribution and free water content of quenched-stirred and slow-stirred emulsions at 4 °C as a function of time. 2.5th, 50th, and 97.5th percentile droplet size are shown from left to right on day 0 and week 78.
and/or absence of interfacial crystallization in some emulsions was responsible for film drainage and coalescence [25], particularly if little affinity between the droplet surface and wax crystals existed, as observed in the quiescently-cooled samples. Thus, mixing regime was critical to effective emulsion stabilization. In the absence of mixing during cooling, little or no interfacial crystalliza-
tion was observed, given the lack of wax mass transfer towards the GMO-covered droplet surface. As wax is not surface-active, the GMO, other than its role in reducing interfacial tension, promoted wax interfacial adhesion. This behaviour is similar to recent reports showing the concerted role of surfactants and particles in Pickering-type stabilization,
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Fig. 11. Solid wax content of quench-stirred and slow-stirred emulsions at 25 °C and 4 °C as a function of time.
where interaction between the former and latter leads to enhanced emulsion stabilization [26]. However, this primarily occurred with concurrent cooling and mixing, where interaction between the oleic acid in the GMO and aliphatic chains in the wax was promoted. Such chain–chain ordering encouraged heterogeneous nucleation of the wax at the interface, as noted by Davies et al. in their work on GMO-tristearin systems [27]. Similarly, Arima et al. found that high-melting sucrose palmitic acid oligoesters acted as a template for the interfacial nucleation and growth of palm oil mid-fraction at the surface of oil droplets in oil-in-water emulsions [28]. These now-anchored wax crystals provided a physical barrier against flocculation and coalescence. The GMO promoted interfacial crystallization either via modification of the wax’s wetting behaviour or by promoting heterogeneous nucleation. Other efforts in our laboratory have demonstrated that surfactant structural complementarity is an important parameter in whether species such as wax or triacylglycerols will interfacially crystallize or not [17]. 4. Conclusion Though the scientific literature is replete with research efforts on Pickering emulsions stabilized with wax (e.g., Li et al. [29]), little is known about the concerted role of wax crystals simultaneously present at the oil–water interface and within the continuous oil phase. The present research clearly demonstrates that control of the interactions between dispersed crystals and neighbouring aqueous droplets in W/O emulsions may be used as a tool to modulate the long-term stability of W/O emulsions. Such findings are significant given that oil-continuous emulsions are commonplace in cosmetics, personal care products, crude oil and processed foods [30]. General requirements dictating successful emulsion stabilization by Pickering particles include optimization of their spatial arrangement, size and surface activity as well as the extent of droplet coverage [31]. The wax crystals in this study englobed the dispersed aqueous droplets with a thin mechanical barrier. They also performed ‘double-duty’ by embedding themselves within the interface of two or more neighbouring droplets thereby arresting coalescence via either monolayer or multilayer bridging. Wax crystal adhesion to the droplet surface required the presence of a surfactant, in this present case GMO, to confer surface activity to the wax. Beyond the ability of the interfacially-adsorbed crystals to
hinder interdroplet coalescence, wax crystals with little to no affinity for the oil–water interface aggregated to form a network in the continuous oil phase, which reduced water droplet settling due to gravity differences. Finally, to permit effective coverage, the diameter of interfacially-adsorbed particles should be much smaller than the droplet size to permit effective enrobing which, generally speaking, was the case with the wax crystals. The present results showed that the long-term stability of the present wax-stabilized W/O emulsions was highly-dependent on the concerted role of interfacial and network wax crystallization, with the combination of stirring and quench-cooling providing the highest kinetic stability. Further elucidation of the mechanisms at play is necessary to clarify the behaviour of wax species at the oil–water interface, in particular the possible role of surfactantwax molecular complementarity as well as the physical state of the interfacially-bound surfactant. Though solid-state templating is clearly established as a mechanism for heterogeneous nucleation, these results suggest that liquid-state surfactants may also promote such templating. This is an on-going challenge currently being addressed.
Acknowledgment Financial assistance from the Natural Sciences and Engineering Research Council of Canada (NSERC) is gratefully acknowledged.
References [1] A. Beri, J.E. Norton, I.T. Norton, Effect of emulsifier type and concentration, aqueous phase volume and wax ratio on physical, material and mechanical properties of water in oil lipsticks, Int. J. Cosmet. Sci. 35 (6) (2013) 613–621. [2] B. Binks, Particles as surfactants—similarities and differences, Curr. Opin. Colloid Interface Sci. 7 (2002) 21–41. [3] A. Macierzanka, H. Szela˛g, P. Szumała, R. Pawłowicz, A.R. Mackie, M.J. Ridout, Effect of crystalline emulsifier composition on structural transformations of water-in-oil emulsions: emulsification and quiescent conditions, Colloids Surf., A 334 (1–3) (2009) 40–52. [4] D. Rousseau, S. Ghosh, H. Park, Comparison of the dispersed phase coalescence mechanisms in different tablespreads, J. Food Sci. 74 (1) (2009) E1–E7. [5] G. Sun, J. Zhang, H. Li, Structural behaviors of waxy crude oil emulsion gels, Energy Fuels 28 (6) (2014) 3718–3729. [6] D. Tambe, M. Sharma, The effect of colloidal particles on fluid–fluid interfacial properties and emulsion stability, Adv. Colloid Interface Sci. 52 (1994). [7] M.T. Ghannam, Water-in-crude oil emulsion stability investigation, Pet. Sci. Technol. 23 (January) (2005) 649–667. [8] D. Johansson, B. Bergenståhl, E. Lundgren, Wetting of fat crystals by triglyceride oil and water. 1. The effect of additives, J. Am. Oil Chem. Soc. 72 (8) (1995) 921–931. [9] S.M. Hodge, D. Rousseau, Continuous-phase fat crystals strongly influence water-in-oil emulsion stability, J. Am. Oil Chem. Soc. 82 (3) (2005) 159–164. [10] E.H. Lucassen-Reynders, M. van den Tempel, Stabilization of water-in-oil emulsions by solid particles, J. Phys. Chem. 67 (4) (1963) 731–734. [11] S. Ghosh, D. Rousseau, Triacylglycerol interfacial crystallization and shear structuring in water-in-oil emulsions, Cryst. Growth Des. 12 (10) (2012) 4944– 4954. [12] S. Haj-shafiei, S. Ghosh, D. Rousseau, Kinetic stability and rheology of waxstabilized water-in-oil emulsions at different water cuts, J. Colloid Interface Sci. 410 (2013) 11–20. [13] R.F.G. Visintin, T.P. Lockhart, R. Lapasin, P. D’Antona, Structure of waxy crude oil emulsion gels, J. Nonnewton. Fluid Mech. 149 (1–3) (2008) 34–39. [14] S.M. Hodge, D. Rousseau, Flocculation and coalescence in water-in-oil emulsions stabilized by paraffin wax crystals, Food Res. Int. 36 (7) (2003) 695–702. [15] D. Rousseau, S.M. Hodge, Stabilization of water-in-oil emulsions with continuous phase crystals, Colloids Surf., A 260 (1–3) (2005) 229–237. [16] M. Pradhan, D. Rousseau, A one-step process for oil-in-water-in-oil double emulsion formation using a single surfactant, J. Colloid Interface Sci. 386 (1) (2012) 398–404. [17] S. Ghosh, T. Tran, D. Rousseau, Comparison of Pickering and network stabilization in water-in-oil emulsions, Langmuir 27 (11) (2011) 6589–6597. [18] ASTM D971-99a Standard Test Method for Interfacial Tension of Oil Against Water by the Ring Method; see (accessed 01.04.15). [19] P. Mazur, W. van Saarloos, Many-sphere hydrodynamic interactions and mobilities in a suspension, Physica A 82 (1982). 90127-3.
S. Ghosh et al. / Journal of Colloid and Interface Science 460 (2015) 247–257 [20] Y. Zhang, J. Gong, Y. Ren, P. Wang, Effect of emulsion characteristics on wax deposition from water-in-waxy crude oil emulsions under static cooling conditions, Energy Fuels 24 (2) (2010) 1146–1155. [21] S.R. Craig, G.P. Hastie, K.J. Roberts, A.R. Gerson, J.N. Sherwood, R.D. Tack, Investigation into the structures of binary-, tertiary- and quinternary-mixtures of n-alkanes and real diesel waxes using high-resolution synchrotron X-ray powder diffraction, J. Mater. Chem. 8 (1998) 859–869. [22] B.P. Binks, S.O. Lumsdon, Influence of particle wettability on the type and stability of surfactant-free emulsions, Langmuir 16 (23) (2000) 8622–8631. [23] T. Tran, S. Ghosh, D. Rousseau, Spheroidal fat crystal microstructures formed with confined gap shearing, Cryst. Growth Des. 14 (12) (2014) 6383–6390. [24] D. Rousseau, Fat crystals and emulsion stability—a review, Food Res. Int. 33 (2000) 3–14. [25] J. Frelichowska, M.-A. Bolzinger, Y. Chevalier, Effects of solid particle content on properties of o/w Pickering emulsions, J. Colloid Interface Sci. 351 (2) (2010) 348–356.
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[26] A. Nesterenko, A. Drelich, H. Lu, D. Clausse, I. Pezron, Influence of a mixed particle/surfactant emulsifier system on water-in-oil emulsion stability, Colloids Surf., A 457 (2014) 49–57. [27] E. Davies, E. Dickinson, R. Bee, Shear stability of sodium caseinate emulsions containing monoglyceride and triglyceride crystals, Food Hydrocolloids 14 (2000) 145–153. [28] S. Arima, S. Ueno, a. Ogawa, K. Sato, Scanning microbeam small-angle X-ray diffraction study of interfacial heterogeneous crystallization of fat crystals in oil-in-water emulsion droplets, Langmuir: ACS J. Surf. Colloids 25 (17) (2009) 9777–9784. [29] C. Li, Z. Mei, Q. Liu, J. Wang, J. Xu, D. Sun, Formation and properties of paraffin wax submicron emulsions prepared by the emulsion inversion point method, Colloids Surf., A 356 (1–3) (2010) 71–77. [30] B.P. Binks, R. Murakami, Phase inversion of particle-stabilized materials from foams to dry water, Nat. Mater. 5 (11) (2006) 865–869. [31] E. Dickinson, Use of nanoparticles and microparticles in the formation and stabilization of food emulsions, Trends Food Sci. Technol. 24 (1) (2012) 4–12.
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science journal homepage: www.elsevier.com/locate/jcis
Long-term stability of crystal-stabilized water-in-oil emulsions Supratim Ghosh a,⇑, Mamata Pradhan b, Tejas Patel b, Samira Haj-shafiei b, Dérick Rousseau b,⇑ a b
Department of Food and Bioproduct Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, Canada
g r a p h i c a l a b s t r a c t
a r t i c l e
i n f o
Article history: Received 4 June 2015 Revised 28 August 2015 Accepted 31 August 2015 Available online 1 September 2015 Keywords: Emulsion stability Pickering Network Wax crystallization Interfacial crystallization Coalescence Sedimentation Microstructure Contact angle
a b s t r a c t The impact of cooling rate and mixing on the long-term kinetic stability of wax-stabilized water-in-oil emulsions was investigated. Four cooling/mixing protocols were investigated: cooling from 45 °C to either 25 °C or 4 °C with/without stirring and two cooling rates – slow (1 °C/min) and fast (5 °C/min). The sedimentation behaviour of the emulsions was significantly affected by cooling protocol. Stirring was critical to the stability of all emulsions, with statically-cooled (no stirring) emulsions suffering from extensive aqueous phase separation. Emulsions stirred while cooling showed sedimentation of a waxy emulsion layer leaving a clear oil layer at the top, with a smaller separation and droplet size distribution at 4 °C compared to 25 °C, indicating the importance of the amount of crystallized wax on emulsion stability. Light microscopy revealed that crystallized wax appeared both on the droplet surface and in the continuous phase, suggesting that stirring ensured dispersibility of the water droplets during cooling as the wax was crystallizing. Wax crystallization on the droplet surface provided stability against droplet coalescence while continuous phase wax crystals minimized inter-droplet collisions. The key novel aspect of this research is in the simplicity to tailor the spatial distribution of wax crystals, i.e., either at the droplet surface or in the continuous phase via use of a surfactant and judicious stirring and/or cooling. Knowledge gained from this research can be applied to develop strategies for long-term storage stability of crystal-stabilized W/O emulsions. Ó 2015 Elsevier Inc. All rights reserved.
⇑ Corresponding authors at: Department of Food and Bioproduct Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7J 2X3, Canada (S. Ghosh). Department of Chemistry and Biology, Ryerson University, Toronto, Ontario M5B 2K3, Canada (D. Rousseau). E-mail addresses: [email protected] (S. Ghosh), [email protected] (D. Rousseau). http://dx.doi.org/10.1016/j.jcis.2015.08.074 0021-9797/Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Emulsions in products such as cosmetics, pharmaceuticals and foods rely on the presence of crystallized species such as polymers, paraffins or triacylglycerols for long-term stability [1–6]. Water-in-
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oil (W/O) emulsions used in such applications must satisfy different criteria including: (i) stability against physical breakdown (particularly once in the hands of the consumer), (ii) resistance to changes in environmental conditions (temperature, vibration/ shaking) and (iii) lack of change in quality attributes (texture, etc.) over time. Key factors include the composition and volume fraction of the continuous and dispersed phases, the mean droplet size and distribution of the dispersed phase, the presence and concentration of stabilizing agents (surfactants, thickeners or crystals) and how the emulsion is processed and stored [7]. Finally, the interactions between the various components in an emulsion will also affect their stability [8]. Emulsion processing requires that the droplets be welldispersed within the continuous phase, which is normally achieved via valve homogenization or high-shear mixing. In many thickened or solid-like emulsions, subsequent gelation or crystallization of the continuous phase helps to keep the dispersed droplets in place. Yet, little is known regarding how to tailor the spatial distribution of crystals in oil-continuous emulsions to assist in retarding physical breakdown. If crystals are adsorbed to the oil–water interface, they create a solid shell around individual dispersed droplets which prevents droplet–droplet coalescence [9,10]. Such surface-active species are usually surfactants that solidify directly at the interface or adsorb onto the droplet surface [3]. As they are surfaceinactive, many crystalline species such as wax or triacylglycerols will form a continuous phase network that envelops the dispersed aqueous phase thereby limiting droplet–droplet collisions [8]. We have previously shown that the surfactant glycerol monooleate can promote the heterogeneous interfacial nucleation of hydrogenated canola oil (a surface-inactive fat) at the water droplet surface in W/O emulsions [11]. We also showed that the addition of a dispersed aqueous phase significantly altered the rheological properties of W/O emulsions [12], as has also been reported by others [5]. Visintin et al. characterized emulsion rheology, and showed that presence of a dispersed aqueous phase was a key contributor to emulsion elasticity [13]. Finally, Hodge and Rousseau showed that the addition of crystallized fat prior to, or after, homogenization (and subsequently quench-crystallized) enhanced the stability of surfactant-stabilized W/O emulsions, with the latter conferring greater stability [14,15]. This paper discusses the impact of cooling rate and stirring on the long-term properties of wax-stabilized W/O emulsions. The goal was to investigate how the presence of a dispersed aqueous phase impacted emulsion macroscopic phase separation, wax crystal morphology, solid wax content and droplet size distribution. In particular, we were interested in showing how it is possible to use minimal amounts of surfactant to generate W/O emulsions with long-term stability against physical breakdown. This was achieved through the use of a low concentration of wax in the continuous oil phase that was suitably crystallized. Importantly, we showed that the optimal stabilization of model W/O emulsions depends on the concerted role of interfacial and network wax crystallization, with the combination of stirring and rapid cooling from the melt conferring the highest kinetic stability. The results from this study are relevant to processed products that exist as oil-continuous emulsions and may serve as a guide for the judicious formulation of emulsions where long-term kinetic stability is desired.
surfactant to aid in emulsification, yet not keep the emulsion stable for any length of time. The light mineral oil was obtained from Fisher Scientific (Nepean, ON, Canada). The stated maximum kinematic viscosity at 40 °C was 33.5 mPa s and its density (q) was 0.84 g/ml at 25 °C. A 3.5 wt% sodium chloride (Fisher Scientific, Nepean, ON, Canada) in de-ionized water (resistivity P18.0 MOhm cm) solution was used as the dispersed aqueous phase (q = 1.02 g/ml). The surfactant used was glycerol monooleate (GMO) (Dimodan MO 90Ò, Danisco, New Century, KS, USA). Dimodan MO 90 is a food-grade low-HLB (hydrophilic/lipophilic balance) emulsifier composed of >92% glycerol monooleate, with the rest consisting of small amounts of glycerol, glycerol dioleate and glycerol trioleate. A highly-refined paraffin wax (IGI-1242; m.p. 56.7–58.9 °C) was obtained from The International Group, Inc. (Toronto, ON, Canada). Its n-paraffin composition provided by the manufacturer is given in Fig. 1. 2.2. Emulsion preparation A schematic of the emulsion preparation protocol is shown in Fig. 2. The oil phase contained 0.05 wt% GMO as emulsifier and 5 wt% paraffin wax. This level of emulsifier allowed for the formation of dispersed droplets, but did not prevent them from measurably coalescing within a few days in the absence of stabilizing wax. All emulsions were formulated to initially consist of dispersed aqueous droplets with a mean diameter of 30 lm with a maximum size of 300 lm, which permitted direct comparison between the different W/O emulsions. The aqueous phase contained 3.5 wt% NaCl, which was necessary to prevent undue formation of oil droplets in the water phase (hence double emulsion formation), which we ascribed to charge generation by the intense shearing action of the homogenizer [16]. Pre-mixing was performed by slow addition of the aqueous phase (9 ml/min) to the oil phase in a beaker at 45 °C via magnetic stirring at 500 rpm for 15 min. The pre-mix was quickly transferred to a valve homogenizer (APV-1000, APV, Albertslund, Denmark) preheated to 45 °C and homogenized at 500 psi for 4 cycles. Afterwards, 100 ml samples were collected in 250 ml glass beakers (diameter: 6.5 cm and height: 9 cm) and cooled via one of the four methods denoted at ‘quench-static’, ‘quench-stirred’, ‘slow-static’ or ‘slow-stirred’. For quench cooling, samples were transferred to a 0 °C waterbath whereas slow-cooled samples were placed in a temperaturecontrolled waterbath (Model 1157P, VWR International, Mississauga, ON) at 45 °C and cooled at a rate of 1 °C/min. Stirring during cooling was performed using a magnetic stirrer at 300 rpm. All samples were prepared in triplicate. Emulsion temperatures during the cooling regimes were measured along the centreline of the bea-
2. Materials and methods 2.1. Materials All emulsions consisted of a 20 wt% aqueous phase and a 80 wt% oil phase containing 5 wt% paraffin wax, mineral oil and sufficient
Fig. 1. n-Paraffin compositional analysis of the solid wax sample. Analysis is performed by high temperature gas chromatography yielding a n-paraffin distribution of the wax from C20 to C90.
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2.6. Droplet size distribution The dispersed droplet size distribution of the emulsions was determined at 25 °C and 4 °C using a Bruker Minispec Mq pulsed field gradient nuclear magnetic resonance (pfg-NMR) unit (Bruker Canada, Milton, ON, Canada) that allows unimodal characterization of emulsion droplet size distributions via restricted diffusion measurement [17]. The temperature inside the NMR sample chamber was controlled by an external waterbath pre-set at specific temperatures. The Minispec Mq NMR software version was 2.58 revision 12/NT/XP (Bruker Biospin GmbH, Rheinstetten, Germany) and the water droplet size application was v5.2 revision 4a. The pulsed gradient separation and number of pulse widths were 210 ms and 8 ms, respectively. The oil suppression delay was 85 ms and the magnet gradient strength was 2 T/m. The pfgNMR field gradient strength was calibrated with CuSO4-doped water (diffusion coefficient = 2.3 109 m2 s1 at 25 °C). Emulsion samples (height = 1 cm) in glass tubes (ID = 0.8 cm, L = 20 cm) were placed in the NMR unit and their droplet size distribution was determined by d3,3 (volume-weighted geometric mean diameter) values and the breadth of distribution, r (geometric standard deviation). Destabilized water droplets that phase-separated from the emulsion were calculated as free water and used as an indicator of emulsion (in)stability.
Fig. 2. Schematic of the emulsion preparation and stabilization protocol.
ker using an auto logger thermocouple (K type with thermometer HH2002AL, Omega, Montréal, QC, Canada) and downloaded to a computer using an IR-sensor connection (HHP-2000DL, Omega, Montréal, QC, Canada). Cooling was stopped when the centre of the sample in the beaker had reached the desired temperature (25 °C or 4 °C). 2.3. Emulsion storage All samples were stored at either room temperature (25 ± 1 °C) or at refrigeration temperature (4 ± 1 °C) for 1.5 years (78 wks). At set intervals, samples were analyzed via sedimentation behaviour, microscopy, solid wax content (SWC) and water droplet size determination. 2.4. Sedimentation Emulsion sedimentation behaviour was recorded visually and with a digital camera for 78 wks. The height of the sedimented emulsion layer was calculated from the digital images using Microsoft Office Picture Manager (Microsoft Canada, Mississauga, ON, Canada). Emulsion destabilization was measured based on the percent-emulsified layer (Femulsion) retained during storage:
F emulsion ¼
hemulsion htotal
ð1Þ
where hemulsion and htotal are the heights of the sedimented emulsion layer and the initial sample, respectively. 2.5. Microscopy Samples were taken from the storage beakers with a glass rod, placed on a viewing slide, covered with a cover slip and observed with an inverted Zeiss Axiovert 200M light microscope (Zeiss Inc., Toronto, ON, Canada) equipped with a temperaturecontrolled stage (model TSA02i with STC200 controller, Instec, Boulder, CO, USA) pre-set at the desired temperature. The viewing slides and the cover slips were cooled to the sample temperature prior to sampling. A 20 objective lens was used to record sample microstructure under DIC (Differential Interference Contrast) and PLM (Polarized Light Microscopy) modes.
2.7. Solid wax content (SWC) Emulsions were transferred into NMR tubes to a height of 4 cm. Samples were prepared and stored at both 4 and 25 °C. Samples were analyzed using the Bruker Minispec Mq pulsed NMR unit with the application sfc_lfc v2.51. Temperature of the NMR chamber was controlled using a water bath pre set to desired temperature. 2.8. X-ray diffraction Static wide-angle X-ray diffraction of the emulsions was performed on a Hecus S3-MICROcaliX (Hecus X-ray Systems GmbH, Graz, Austria). The unit uses a GeniX 50 W microsource and customized FOX-3D multi-layer point focussing optics (Xenocs SA, Grenoble, France). Samples (20 ll) were loaded in quartz capillaries (d = 1 mm) and characterized at 25 °C. 2.9. Contact angle measurements A portion of the wax used to stabilize the emulsion was melted at 80 °C with stirring, poured into an aluminium weigh boat at room temperature and allowed to crystallize for 24 h. Solidified wax discs were then cut into 1 cm3 cubes and placed in spectrophotometer cuvettes, with the smooth side facing upwards. Cuvettes were then filled with light mineral oil alone or with GMO. A small water droplet (D 1 mm) was injected from a needle (gauge 22, 0.394 mm nominal ID, Fisher Scientific, Nepean, ON, Canada) onto the surface of the solid wax using a 10 ml syringe and a programmable syringe pump (kd Scientific, Markham, ON, Canada) operated at 0.1 ml/min. Images of sessile water droplets were captured for 24 h using a Teli CCD camera with a macro lens assembly and IDS Falcon/Eagle framegrabber (DataPhysics Instrument GmbH, Filderstadt, Germany). Droplet contact angles against the solid surface were measured digitally (SCA 20 software, version 2.1.5 build 16, DataPhysics Instrument GmbH, Filderstadt, Germany). The oil phase/water interfacial tensions (IT) were determined using ASTM method D971 [18], with the DuNouy ring method (Fisher surface tensiometer Model 21, Fisher Scientific, Nepean, ON, Canada).
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2.10. Statistical analyses Triplicate analyses were performed on all sedimentation and droplet size measurements. Analyses of variance and post hoc tests were performed and statistical differences were deemed significant at p = 0.05. 3. Results and discussion 3.1. Emulsion cooling rates Fig. 3 shows a comparison of the different cooling rates used for emulsions cooled to either 25 °C or 4 °C. A maximum cooling rate of 4.6 °C/min was achieved with the quench-cooling and stirring regime whereas the slowest cooling rate was with slow-static cooling (0.7 °C/min). Stirring helped to maintain a more uniform cooling rate throughout the beaker whereas statically-cooled samples yielded slower temperature drops with some temperature inhomogeneities throughout. None of the cooling rates used were by any means rapid. Thus, it would be incorrect to speak of true ‘quenching’ in any scenario. 3.2. Emulsion sedimentation Control emulsions made with either GMO or wax destabilized within 1 h of preparation. Hence, the presence of both the GMO and paraffin wax was necessary to confer kinetic stability to all emulsions. All wax-containing emulsions nevertheless demonstrated phase separation dependent on composition and cooling/stirring regime (Figs. 4 and 5). When stored at 4 °C, the stirred emulsions (quench or slowly-cooled) showed oil separation and no aqueous phase separation. Separation was evident after 6 wks (Fig. 4). Those slowly and statically-cooled broke down with a clear water layer appearing soon after preparation. The quench-static emulsions demonstrated limited phase separation (not readily visible in the beaker) over 78 wks. These results demonstrated that more rapid formation of a wax crystal network prevented oil or water separation. In regards to stirred emulsions, the encased dispersed phase appeared to sediment along with the wax crystal network, leading to phase separation and oiling-off. The enhanced sedimentation stability of the stirred emulsions with respect to water separation ensured that the water droplets remained dispersed throughout the continuous oil phase during cooling and wax crystallization. Stirring also appeared to promote more direct contact between the aqueous droplets and wax crystals given the
entrainment of the wax crystal network by the dispersed phase in the stirred emulsions. Emulsions cooled to 25 °C were less stable against sedimentation than at 4 °C (Fig. 5). All emulsions demonstrated some phase separation after storage for 1.5 yrs, though the storage vessel used (i.e., beaker vs. NMR tube) was critical to this evaluation. Stirred emulsions (quench or slowly-cooled) showed oil separation after storage for 1 wk, but no water phase separation even after 1.5 yrs. Quiescent emulsions (quench or slowly-cooled) demonstrated some breakdown right after preparation, as the wax crystal network formed was too sparse to encase the dispersed droplets. With time, a supernatant oil phase was also visible. Storage in NMR tubes accentuated the observed destabilization, which has been ascribed to differences in wall effect and convective flow [19]. The 25 °C protocol did not lead to measurable droplets with NMR, further demonstrating the critical role that the wax crystal network played on emulsion stabilization. Too weak a network or too low an SWC resulted in an unstable emulsion, particularly with so little emulsifier. Overall, there was a much slower rate of emulsion sedimentation at 4 °C vs. 25 °C, as there was more solid wax present to prevent density-driven settling of the dispersed water droplets. For comparison, the sedimentation stability of the emulsions’ oil phases was also evaluated separately. All samples remained stable, and did not show any indication of phase separation at 4 °C and 25 °C. This lack of macroscopic phase separation clearly indicated that the dispersed phase played a significant role on phase separation. The continuous phase of all emulsions stored at 25 °C was flowable whereas for those stored at 4 °C, only the stirred samples were flowable, possibly due to the influence of stirring on wax crystal size and inter-crystal interactions [20]. 3.3. Sedimentation kinetics Femulsion (Eq. (1)) plotted vs. storage time (Fig. 6) was used to estimate sedimentation rate. Only stir-cooled emulsions were evaluated as the statically-cooled emulsions rapidly destabilized with clear separation of the aqueous phase. Amongst the stirred emulsions, sedimentation was slowest for those stored at 4 °C (no sedimentation for 1 wk) compared to storage at 25 °C (where emulsions sedimented to 70% of their original heights within the first wk). After 6 wks, the latter emulsions sedimented to 65% to 68% and to 63% to 65% after 1.5 years, respectively for quenched and slowly-cooled systems. By contrast, the 4 °C emulsions sedimented to 82% and 78% of their initial heights after 6 wks for the quenched and slowly-cooled emulsions, respectively, followed by a decrease in height to 69% for the quenched-stirred emulsion and to 65% for the slow-stirred emulsion after 1.5 years. 3.4. Emulsion microstructure
Fig. 3. Temperature profiles of emulsions under the four cooling protocols investigated. Cooling rates were calculated from the average slopes of the cooling curves: quench-stirred (4.6 °C/min), quench-static (1.3 °C/min), slow-stirred (0.9 °C/min), and slow-static (0.7 °C/min). Quench cooling was done in a 0 °C waterbath whereas slow-cooled samples were placed in a 45 °C waterbath and cooled at a rate of 1 °C/min. For cooling to 25 °C samples were withdrawn from the waterbath when the centre of the sample reached desired temperature.
Emulsion microstructure at 4 °C and 25 °C was highlydependent on the cooling/stirring regime used. As noted above, emulsions stored at 4 °C were more stable against sedimentation than those stored at 25 °C. Microscopy results concurred, with evidence of a more stable dispersed aqueous phase at the lower storage temperature. Fig. 7 shows the combined DIC and polarized light micrographs of emulsions stored at 4 °C for 78 wks. Though demonstrating oil separation, the stirred emulsions (quench and slowly-cooled) showed the presence of a relatively stable dispersed aqueous phase, with little increase in droplet size over time. By contrast, the statically-cooled emulsions underwent breakdown with little evidence of dispersed droplets soon after emulsion preparation. Irrespective of cooling rate, with stirring, there was evidence of interfacial wax crystal adhesion (Fig. 7 arrows) suggesting the existence of wax crystal surface activity, and the possi-
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Fig. 4. Sedimentation behaviour of emulsions stored at 4 °C as a function of time.
bility of GMO promoting interfacial wax crystallization. Such a phenomenon was recently observed by us [11]. Fig. 8 shows combined DIC and polarized light micrographs of emulsions stored at 25 °C for 78 wks. The quenched-stirred emulsion was the most stable against changes in droplet size, with a still evident distribution of droplets visible after 1.5 years. The slowstirred emulsions were stable until at least 6 wks, but had broken down after 78 wks. Both statically-cooled emulsions were not stable, with the dispersed phase breaking down soon after homogenization and only a few water droplets can be seen. There was some evidence of interfacially-adsorbed crystals in all cases, which was most apparent with the quenched-stirred emulsions (as per the 4 °C results). By comparing Figs. 7 and 8, it can be seen that initially, 4 °C emulsions consisted of smaller droplets compared with those at 25 °C. After 6 wks, droplet sizes in emulsions at both temperatures were comparable whereas after 1.5 years, only the 4 °C emulsions remained stable with only a minor increase in droplet size. Fig. 9((A) and (B)) shows a close-up view of interfacial wax crystallization promoted by stirring under quench-cooling conditions. Clearly visible are interfacial crystals that stabilized the dispersed phase via Pickering stabilization as well as network stabilization, where crystals contributed to hindering droplet movement by enmeshing them within a continuous crystalline matrix. Together, these two mechanisms heightened the stability of the emulsions.
Macierzanka et al. [3] studied the time-dependent structural evolution of W/O emulsions prepared with mixtures of two different crystalline emulsifiers (partial acyglycerols and fatty acid monodiesters of propylene glycol). The emulsion droplets were initially Pickering-stabilized. With time, interfacial crystals grew into the continuous oil phase, leading to network stabilization and crystalline bridges between the droplets. Fig. 9A is the combined DIC and polarized light image whereas Fig. 9B shows the corresponding polarized light view only. Most crystalline species measured well below 40 lm in length and existed as needles with high aspect ratios. For comparison, Fig. 9C shows a putative crystal morphology where the oil phase of the emulsion was quench-cooled to 4 °C under stirring. Other than the missing interfacial crystals (given that no aqueous phase is present), the continuous phase crystal appearance is quite similar to that observed in the emulsions. 3.5. Evolution of droplet size distribution Emulsion droplet sizes were only measurable for the stirred emulsions cooled to 4 °C. All other emulsions (statically-cooled to 4 °C and all 25 °C emulsions) did not yield droplets measurable via pulsed NMR, either due to their size (too large) or population (too small). At 4 °C, the average droplet size (d33, 50th percentile value) of the quench-stirred and slow-stirred emulsions ranged
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Fig. 5. Sedimentation behaviour of emulsions at 25 °C as a function of time. Statically-cooled emulsions were also stored in NMR tubes to show a clear view of aqueous phase separation for both the quenched and slow-cooled samples (shown at right after 78 weeks’ storage).
free water content confirmed that both emulsions very slowly broke down with time. The initial breadth of the quenchedstirred emulsion droplet size distribution was broader than that of its slowly-stirred counterpart. After 1.5 years, however, the latter’s droplet size distribution narrowed more extensively (i.e., 95% of droplets were between 26 and 42 lm) which was accompanied by an increase in % free water. This narrowing was interpreted as greater emulsion destabilization than in the quenched-stirred emulsion, which saw a smaller increase in free water during this time. 3.6. Solid wax content
Fig. 6. Estimation of emulsion destabilization by sedimentation. Evolution of percent emulsified layer (Femulsion from Eq. (1)) is plotted as a function of time for slow or quenched-cooled stirred emulsions stored at 4 and 25 °C.
between 29 and 33 lm and remained unchanged over 78 wks (Fig. 10) (p > 0.05). During this time, the 97.5th percentile values significantly decreased indicating the loss of larger droplets as free water (i.e., larger than the NMR instrumental limit of 500 lm) or visually phase-separated water (p < 0.05). The gradual increase in
Fig. 11 shows the SWC of stirred emulsions as a function of time. As the statically-cooled emulsions were not stable at either 4 °C or 25 °C, their SWC was not measured. The SWC was much higher at 4 °C compared to 25 °C, with no effect of cooling rate (p > 0.05). After 78 wks, there was a significant increase in SWC at both temperatures due to continued wax solidification (p < 0.05). Though crystallization to 4 °C led to initially higher SWCs, over 78 wks, SWC increased to 60% for the emulsions processed at 25 °C and only 20% for those at 4 °C. Wax crystallization history was an important step in final SWC, and it also contributed to the observed differences in wax crystal
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Cooling protocol
Day 0
Week 1
Week 6
Week 78
Quenchedstirred
Quenchedstatic
Slowstirred
Slowstatic
Fig. 7. Combined DIC and polarized light micrographs of wax-stabilized emulsions at 4 °C as a function of storage time. The presence of both interfacial crystals (shown by arrows) and continuous phase crystals can be seen. The scale bar represents 40 lm.
morphology and population. Irrespective of the stirring/cooling regime, crystallization began when supercooling was sufficient for the highest-melting wax species to nucleate and act as crystallization centres. With more nuclei formed, there was more rapid bulk crystallization. Given the compositional variety of the wax (Fig. 1), low supercooling (i.e., cooling to 25 °C) initiated highmelting wax crystallization first. High supercooling (4 °C) likely resulted in more rapid, mass crystallization of the bulk, with formation of more mixed crystals consisting of lower and highermelting species, with perhaps these crystals acting as secondary nucleation sites. 3.7. Wax crystal structure X-ray diffraction revealed that the lamellar spacing of the solid paraffin wax at 25 °C was 41.57 Å with a weak second-order spacing at 20.39 Å and short spacings at 4.18 Å and 3.74 Å. Little change in both small and long-order spacings were observed for the wax present in all emulsions, with first order spacing at 42.1 to 42.5 Å. Short spacings were identical to the solid wax, with spacings at 4.18 Å and 3.74 Å. Given the alkane diversity present in the paraffin wax (straight-chained, branched, etc.), it would be imprudent to assign specific crystalline structures to each of these samples. Based on Craig et al. for pure alkanes, the 100% paraffin wax existed in the orthorhombic unit cell [21]. The orthorhombic unit cell c/2 value, which corresponds to the dimension of the carbon atom chains
and gaps between the planes of methyl endgroups between two successive molecular layers was determined using:
ðc=ÅÞ=2 ¼ 1:2724nc ¼ 1:8752
ð2Þ
where nc is the average chain length. Based on Eq. (2), a (c/Å)/2 spacing of 41.57 Å indicates an average alkane length (nc) of 31 carbons, which closely matched the compositional analysis of wax with an average alkane chain length of 29 carbons (Fig. 1). 3.8. Wax crystal wetting Contact angles (h) between the solid wax, and aqueous and oily phases were used to understand the ability of wax to promote Pickering crystallization. Equilibrium h of water droplets on the wax surface were measured at 147.5 ± 2.9° in light mineral oil and 130.5 ± 4.1° with the added presence of GMO. It is wellknown that 90° < h < 180° favours stabilization of water droplets in an oil-continuous medium [2]. At h = 90°, particles are equally wetted by the water and oil phases giving rise to the most stable emulsion. At h = 180°, crystals are completely wetted by the oil phase so no association with the water droplets occurs. In the present case, the smallest h measured through the aqueous phase was obtained with GMO-doped mineral oil against wax. The IT of mineral oil against water was 44.9 ± 3.4 mN/m and 4.1 ± 0.3 with added presence of GMO.
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Cooling protocol
Day 0
Week 1
Week 6
Week 78
Quenchedstirred
Quenchedstatic
Slowstirred
Slowstatic
Fig. 8. Combined DIC and polarized light micrographs of wax-stabilized emulsions at 25 °C as a function of storage time. The presence of both interfacial crystals (shown by arrows) and continuous phase crystals can be seen. The scale bar represents 40 lm.
The displacement energy (Edisp), which is directly linked to h, was used as a measure of the energy required to remove wax crystals from the oil/water interface [22] (Eq. (3)):
Edisp ¼ pr 2 cow ð1 cos hÞ2
ð3Þ
where cow is the IT at the oil–water interface and r is the radius of a wax ‘particle’. The sign inside the bracket is negative for displacement into the water phase, and positive for displacement into oil (e.g., movement of wax crystals into the oil phase) [23]. From Eq. (3), Edisp increases with an increase in oil–water IT, but decreases with increase in h. Assuming r 1 lm, Edisp to remove a wax crystal from the water/GMO/mineral oil interface was 3.8 108 kT, which indicates that presence of wax crystals at the water droplet surface is favourable unless a high amount of energy is provided for removal. Of note, Edisp to remove a wax crystal from the water/mineral oil interface (without GMO) was 8.5 108 kT, comparable to the value for GMO. However, as molten wax has no emulsifying activity, it could not be used to form and stabilize water droplets, hence, their movement towards the interface is not favourable. 3.9. Wax crystallization and network structure Effective emulsion stabilization was dependent on the concerted roles of interfacial and network wax crystallization, with both contributing to delaying water droplet sedimentation, flocculation and coalescence. Such ‘dual-mode’ wax crystallization was
itself dependent on temperature and mixing regime during emulsion processing. Crystallization to 4 °C yielded SWCs 3–4 higher than at 25 °C, which markedly impacted emulsion stability. At lower SWCs, there was insufficient solid mass to effectively surround the droplets and physically entrap the aqueous phase whereas the higher SWC at 4 °C retarded phase separation and coalescence. The presence of both interfacial and surrounding crystals kept droplets separate from one another due to the presence of interstitial crystals thus reducing the rate of flocculation. However, wax that crystallized directly at the interface visibly retarded coalescence and sedimentation vis-à-vis network-type crystals, as these crystals enveloped individual droplets thus preventing film drainage and rupture. As wax is not surface-active, the use of the surfactant GMO promoted direct interfacial crystallization. By contrast, bulk network crystallization, though also beneficial in stabilizing the emulsions, did not offer the same level of stabilization against emulsion breakdown, presumably due to gravity-induced droplet movement throughout the wax crystal network. Pickering particle removal from an oil–water interface is a necessary precursor to coalescence. The energy required for crystal displacement will depend on the composition and rheology of the interface as well as crystal wettability [24]. There was no particle desorption from the interface observed even after 78 wks given the high Edisp. Rather, alkane self-organization and crystallization promoted the formation of a solid shell that englobed the dispersed droplets. This suggested that incomplete coverage
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Fig. 9. Combined DIC and polarized light micrograph (A) and corresponding polarized light only micrograph (B) of wax-stabilized emulsions at 4 °C. The bottom picture presents a polarized light micrograph of a wax–oil mixture (C) at 4 °C cooled using a similar protocol to the emulsions. Scale bar represents 40 lm.
Fig. 10. Droplet size distribution and free water content of quenched-stirred and slow-stirred emulsions at 4 °C as a function of time. 2.5th, 50th, and 97.5th percentile droplet size are shown from left to right on day 0 and week 78.
and/or absence of interfacial crystallization in some emulsions was responsible for film drainage and coalescence [25], particularly if little affinity between the droplet surface and wax crystals existed, as observed in the quiescently-cooled samples. Thus, mixing regime was critical to effective emulsion stabilization. In the absence of mixing during cooling, little or no interfacial crystalliza-
tion was observed, given the lack of wax mass transfer towards the GMO-covered droplet surface. As wax is not surface-active, the GMO, other than its role in reducing interfacial tension, promoted wax interfacial adhesion. This behaviour is similar to recent reports showing the concerted role of surfactants and particles in Pickering-type stabilization,
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Fig. 11. Solid wax content of quench-stirred and slow-stirred emulsions at 25 °C and 4 °C as a function of time.
where interaction between the former and latter leads to enhanced emulsion stabilization [26]. However, this primarily occurred with concurrent cooling and mixing, where interaction between the oleic acid in the GMO and aliphatic chains in the wax was promoted. Such chain–chain ordering encouraged heterogeneous nucleation of the wax at the interface, as noted by Davies et al. in their work on GMO-tristearin systems [27]. Similarly, Arima et al. found that high-melting sucrose palmitic acid oligoesters acted as a template for the interfacial nucleation and growth of palm oil mid-fraction at the surface of oil droplets in oil-in-water emulsions [28]. These now-anchored wax crystals provided a physical barrier against flocculation and coalescence. The GMO promoted interfacial crystallization either via modification of the wax’s wetting behaviour or by promoting heterogeneous nucleation. Other efforts in our laboratory have demonstrated that surfactant structural complementarity is an important parameter in whether species such as wax or triacylglycerols will interfacially crystallize or not [17]. 4. Conclusion Though the scientific literature is replete with research efforts on Pickering emulsions stabilized with wax (e.g., Li et al. [29]), little is known about the concerted role of wax crystals simultaneously present at the oil–water interface and within the continuous oil phase. The present research clearly demonstrates that control of the interactions between dispersed crystals and neighbouring aqueous droplets in W/O emulsions may be used as a tool to modulate the long-term stability of W/O emulsions. Such findings are significant given that oil-continuous emulsions are commonplace in cosmetics, personal care products, crude oil and processed foods [30]. General requirements dictating successful emulsion stabilization by Pickering particles include optimization of their spatial arrangement, size and surface activity as well as the extent of droplet coverage [31]. The wax crystals in this study englobed the dispersed aqueous droplets with a thin mechanical barrier. They also performed ‘double-duty’ by embedding themselves within the interface of two or more neighbouring droplets thereby arresting coalescence via either monolayer or multilayer bridging. Wax crystal adhesion to the droplet surface required the presence of a surfactant, in this present case GMO, to confer surface activity to the wax. Beyond the ability of the interfacially-adsorbed crystals to
hinder interdroplet coalescence, wax crystals with little to no affinity for the oil–water interface aggregated to form a network in the continuous oil phase, which reduced water droplet settling due to gravity differences. Finally, to permit effective coverage, the diameter of interfacially-adsorbed particles should be much smaller than the droplet size to permit effective enrobing which, generally speaking, was the case with the wax crystals. The present results showed that the long-term stability of the present wax-stabilized W/O emulsions was highly-dependent on the concerted role of interfacial and network wax crystallization, with the combination of stirring and quench-cooling providing the highest kinetic stability. Further elucidation of the mechanisms at play is necessary to clarify the behaviour of wax species at the oil–water interface, in particular the possible role of surfactantwax molecular complementarity as well as the physical state of the interfacially-bound surfactant. Though solid-state templating is clearly established as a mechanism for heterogeneous nucleation, these results suggest that liquid-state surfactants may also promote such templating. This is an on-going challenge currently being addressed.
Acknowledgment Financial assistance from the Natural Sciences and Engineering Research Council of Canada (NSERC) is gratefully acknowledged.
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