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Stability of monodisperse clove oil droplets prepared by microchannel emulsification
Colloids and Surfaces A: Physicochem. Eng. Aspects 466 (2015) 66–74
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Stability of monodisperse clove oil droplets prepared by microchannel emulsification Nanik Purwanti a,b , Marcos A. Neves c,a , Kunihiko Uemura a , Mitsutoshi Nakajima c,a , Isao Kobayashi a,∗ a
Food Engineering Division, National Food Research Institute, NARO, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan Department of Mechanical and Biosystem Engineering, Bogor Agricultural University, IPB Darmaga Campus, PO. BOX 220, Bogor 16002, Indonesia c Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8572, Japan b
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• O/W emulsion of clove essential • • • •
oil was produced by microchannel emulsification. Initial O/W emulsion droplets are monodisperse. Instability of oil droplets occurs after droplet generation. Instability was explained by diffusion, spontaneous emulsions, and reversed micelles. Diffusion of clove oil is independent of surfactant concentration.
a r t i c l e
i n f o
Article history: Received 1 July 2014 Received in revised form 29 October 2014 Accepted 30 October 2014 Available online 6 November 2014 Keywords: Clove oil Stability Oil-in-water emulsion Microchannel emulsification
a b s t r a c t Monodisperse clove oil droplets in the form of oil-in-water (O/W) emulsions stabilized by 0.1% and 0.5% w/w sodium dodecyl sulphate (SDS) were successfully produced by microchannel (MC) emulsification using a 15 × 15 mm2 MC array plate with 100 parallel MCs fabricated on each side of the plate. The channels have 71 m length, 8.2 m width, and 4 m depth with hydraulic diameter of 4.2 m. The terraces have the same depth size as the channels with 29.1 m length. The average droplet diameter was 17 m with a coefficient of variation around 3%. The droplet size deviated from monodispersity over time and it depended on the droplet configuration on MC array plate. The droplet size became polydisperse within 2 h when the droplets were surrounded by plenty of continuous phase. A higher SDS concentration resulted in more polydisperse droplet size over time. This instability is proposed as the result of diffusion-driven mechanism, spontaneous emulsification, and reversed micelle formation. Instability could be suppressed by saturating both continuous and dispersed phases prior to emulsification. This step may prevent the diffusion that takes place once the oil–water interface is formed. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Essential oil of clove has been widely applied in the pharmaceutical, fragrance, and flavour industries. Its various functionalities,
∗ Corresponding author. Tel.: +81 29 838 8025; fax: +81 838 8122. E-mail address: [email protected] (I. Kobayashi). http://dx.doi.org/10.1016/j.colsurfa.2014.10.058 0927-7757/© 2014 Elsevier B.V. All rights reserved.
some of which have been discovered (e.g., antioxidant [1–3], free-radical scavenger [4,5], anti-stress [6], anti-microbial activities [7], anti-inflammatory, antigiardial, analgesic, and anaesthetic properties [8,9]), may result in wider applications of this oil. To facilitate its application, clove oil can be formulated so that its functionalities are preserved and their release can be controlled. An example is encapsulation of the oil using vehicles such as solid lipid or a polymer matrix. Clove oil can be encapsulated by first
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producing oil-in-water (O/W) emulsions and subsequently solidifying them. Various emulsification techniques are used to produce O/W emulsions. Mechanical emulsification, the traditional technique, produces emulsions by utilizing instruments such as high-speed mixers, colloid mills, high-pressure valves or ultrasonic homogenizers, and micro-fluidizers [10]. Variable shear and pressure fields in such instruments lead to difficulty in controlling the size of emulsions; therefore, the resulting droplets are usually highly polydisperse [11,12]. In addition, high energy input is required to produce emulsions and most of the energy input is dissipated as heat [10]. Other techniques used to produce monodisperse emulsions with narrow size distribution are membrane emulsification [11,13], microchannel (MC) emulsification [14,15], and use of other microfluidic devices such as T-, Y-, or cross junctions [16–18], and an edge-based droplet generation (EDGE) system [19]. Vladisavljevic´ et al. [12] recently systematically reviewed characteristics of emulsions produced using those different techniques. MC emulsification was introduced as a novel emulsification method more than a decade ago [14]. This method produces monodisperse droplets with a coefficient of variation (CV) below 5% using MC arrays. Major aspects that should be considered when producing droplets using this method include design of microchannels (MCs) [12,20,21], selection of surfactants [22–24], operating conditions [15,25], and viscosity ratio between dispersed and continuous phases [26]. MC emulsification is a stable process for producing monodisperse emulsion droplets [23,27,28] and the resulting O/W droplets (triglycerol oil) remain stable for a long time [27,29]. Clove oil can be emulsified using this method. Droplet stability of clove essential oil prepared by MC emulsification was previously reported by Liu et al. [30]. The stability of the oil droplets deposited at the bottom of a sample bottle was reported to depend on surfactant concentration. The oil droplets broke up into smaller droplets after standing for 15 h when the surfactant (sodium dodecyl sulphate (SDS)) concentration in the buffer solution was above its critical micelle concentration (CMC), i.e. 0.2% w/w. To collect oil droplets generated by MC emulsification in a sample bottle, droplets in the MC well (Fig. 1b) should be washed away by the continuous phase. The size of oil droplets should not be influenced by the shearing effect of the continuous phase during droplet collection. However, we recently observed that clove oil droplets stabilized by SDS as the surfactant are unstable after they are generated by an MC array. Consequently, stability of the droplets is further affected during collection and application. This study demonstrates the instability of clove oil droplets generated by MC emulsification and clarifies the mechanism of this instability.
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consists of grooved MC arrays with 100 parallel MCs, fabricated on each side of the plate. The channel and terrace depth (H) is 4 m, the channel length (LC ) is 71 m, the channel width (WC ) is 8.2 m, and the terrace length (LT ) is 29.1 m. The hydraulic diameter (dh ), defined as four times the area over the perimeter, is 4.2 m. The top view of the MCs is depicted in Fig. 1a. Typical views of the crosssection and three-dimensional drawing of the MCs as well as all peripherals for MC emulsification have been described in previous studies [21,26]. 2.3. MC emulsification The following procedure was used to produce clove oil droplets by MC emulsification. The MC plate was initially filled with the continuous phase (i.e., SDS solution). The dispersed phase (i.e., clove oil) was pushed through the reservoir hole of the plate by slowly lifting the oil chamber until the minimal breakthrough pressure, the minimum pressure required to produce oil droplets, was achieved. Typical generation of clove oil droplets in SDS solution by MC emulsification is depicted in Fig. 1b. The generated droplets are located on the well of the MC array plate surrounded by the continuous phase (Fig. 1b). The continuous phase was SDS solution with a concentration of 0.1% w/w (below the CMC) and 0.5% w/w (above the CMC), which was filtered using a 0.2 m hydrophilic syringe filter (Sartorius Minisart® RC25, Sartorius AG, Göttingen, Germany) prior to use. Three conditions of clove oil droplets were produced and analyzed: (1) clove oil droplets were generated until the MC well was completely filled with the droplets (close-packed droplets, Fig. 1c); (2) clove oil droplets were generated forming non-closepacked droplets (Fig. 1d); and (3) the condition was the same as condition (2), but the dispersed phase and the continuous phase were saturated with each other prior to MC emulsification (Fig. 1e). The production of oil droplets by MC emulsification was repeated twice with two independent batches of continuous and dispersed phases. 2.4. Pre-treatment of saturated phases
Extra pure reagent clove oil (Nacalai Tesque, Inc., Kyoto, Japan) was used as the dispersed phase. SDS (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was used as the surfactant. Milli-Q water (resistivity 18.2 M cm at 25 ◦ C, total oxidizable carbon < 4 ppb, Merck-Millipore, Tokyo, Japan) was used to prepare the continuous phase.
The dispersed phase and the continuous phase were saturated with each other using the following steps. A mixture of clove oil and 0.1% w/w SDS solution was stirred at a weight ratio of 1:3 for 1 h. Next, the mixture was centrifuged at 760 × g for 15 min and then at 1070 × g for 10 min (KN-70, Kubota, Tokyo, Japan). The supernatant (SDS solution) was then harvested carefully and the remaining mixture was further centrifuged at 1880 × g for 10 min. The oil phase was harvested carefully and the remaining emulsion was discarded. The harvested SDS solution was filtered using a 0.2 m hydrophilic syringe filter and the oil was filtered using a PFTE hydrophobic syringe filter (Dismic 25 JP, Advantec Toyo Kaisha, Ltd., Tokyo, Japan) prior to MC emulsification. When 0.5% SDS solution was used, the ratio of clove oil and SDS solution was 1:1 and the stirring time was only 5 min to avoid complete conversion of the oil into emulsion. About 80% of the initial weight of the SDS solution and 20–25% of the oil could be recovered from the saturation step. The emulsification was performed at room temperature (25 ◦ C). The size changes of oil droplets inside the MC plate were monitored every 30 min for 2 h.
2.2. MC array plate and peripherals
2.5. Droplet size determination
Droplets of clove oil were produced by a silicon dead-end MC array plate (MS2A4). This MC array plate enables collecting of generated droplets on the well for inline observation; therefore, circulation of the continuous phase to collect the droplets outside the MC array plate is not needed. The 15 mm × 15 mm MC array plate
The diameter of oil droplets was analyzed by measuring the captured images of droplets using image processing software (WinROOF version 6.5, Mitani Co., Fukui, Japan). For each condition, 200 droplets were measured, except for clove oil droplets in 0.1% w/w SDS solution condition (2), for which 300 droplets were measured.
2. Materials and methods 2.1. Materials
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Fig. 1. (a) Top view of three MCs and their dimensions in m. (b) Generation of clove oil droplets from MCs. Configuration or conditions of droplets generated on the MC array plate just after formation (0 h) and after 2 h (represented by the droplets stabilised by 0.5% w/w SDS solution): (c) Close-packed droplets, (d) non-close-packed droplets, and (e) non-close-packed droplets in which both phases were saturated with each other prior to MC emulsification with a scale bar of 20 m.
2.6. Viscosity measurement Viscosities of the continuous and the dispersed phases were measured using a glass capillary viscometer (SO, Shibata Scientific Technology Ltd., Tokyo, Japan) at 25 ◦ C. A capillary tube with constant 0.00388 cSt/s was used for the continuous phase and one with constant 0.0144 cSt/s was used for the dispersed phase. A sample volume of 10 mL was inserted in the capillary tube. The time required for the meniscus of the liquid to move from the upper to the lower mark in the capillary was recorded. The dynamic viscosity (, mPa s) was calculated using the formula = C × t × , where C is the constant of the capillary tube, t is time (s), and is density (g/cm3 ). Each phase was measured with two independent batches. Each batch was measured in duplicate and the measurement of each replicate was repeated three times. 2.7. Density measurement Densities of the continuous and the dispersed phases were measured using a portable density/specific gravity metre (DA-130N, KEM, Kyoto Electronics, Japan) at room temperature (25 ◦ C). Each phase was measured with two independent batches and each batch was measured five times. 2.8. Determination of interfacial tension Interfacial tension between a drop of clove oil (pure and saturated conditions) and the continuous phase was measured using the pendant drop method (PD-W, Kyowa Interface Science Co., Ltd., Saitama, Japan). A drop of clove oil was suspended in SDS solution (saturated and unsaturated) and each drop was measured twice. Ten drops were made from two independent batches, except for pure clove oil in 0.5% SDS solution from which 20 drops were produced and measured.
needle. The drop was illuminated from the side in order to clearly visualise inside the drop and its surroundings. The changes of the drop and its surroundings during the first 5 min were captured using the interfacial tensiometer. The volume of the oil drop was 7 L for all conditions, except for the clove oil drop in 0.5% w/w SDS solution where a volume of only 1 L could be formed. At least two drops prepared from two independent batches were observed for each condition.
2.10. Solubility of clove oil in SDS solution The solubility of clove oil in different concentrations of SDS solution was determined by first placing a ratio of 1:1 clove oil and SDS solutions in glass tubes. Next, 5 mL of SDS solution was poured into the tube and then, 5 mL of clove oil was added. Clove oil passed through the SDS solution and remained at the bottom of the tube because of its higher density. The tubes were tightly closed, completely covered by aluminium foil, and then stored in a dark chamber set at 25 ◦ C. After 24 h, the SDS solution containing clove oil was collected from the tubes using a long needle. This supernatant was centrifuged at 1100 × g for 25 min and then, filtered with a 0.2 m membrane filter unit to remove micelles or spontaneously formed emulsion droplets. The supernatant was diluted five times with fresh SDS solution of corresponding concentration. The absorbance of diluted SDS solution containing clove oil was measured using a spectrophotometer at 300 nm. SDS solution of different concentrations was measured as blank samples. The value of solubility was determined using a standard calibration curve that was also set based on absorbance at 300 nm. Solubility was further calculated in terms of the modified partition coefficient that we defined as k=
Cin water phase Cin oil phase
2.9. Visual observation of oil drops in aqueous phase Clove oil was pushed through a needle immersed in the aqueous phase until forming a drop and this drop was held to hang onto the
and then expressed as a logarithm of this coefficient. Here, k is the partition coefficient and C is the concentration of clove oil. Concentration in the oil phase was assumed to be 100%.
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Fig. 2. Size distribution of clove oil droplets stabilised by 0.1% w/w SDS solution: (a) at 0 h and (b) after 2 h. (:) Close-packed droplets. ( Droplets with saturated phases.
3. Results and discussion Clove oil in the form of O/W emulsion droplets stabilized by SDS solution was generated using MC emulsification. Conditions of the generated droplets on the well of the MC array plate initially were set as indicated in Fig. 1c–e (0 h). The droplets generated by MCs were initially monodisperse regardless of the concentrations of surfactant and droplet conditions on the well. Initially, the average size of the droplets was 17 m with maximum CV of 3%.
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) Non-close-packed droplets. (:)
The droplets also had a narrow size distribution (Figs. 2a and 3a) indicating monodispersity. Close-packed clove oil droplets stabilized by 0.1% and 0.5% w/w SDS solutions were homogenous and stable over 2 h on the well, as indicated by the narrow size distribution (Figs. 2b and 3b ()) and stable droplet size over time (Fig. 4a () and b ()). An overview of the droplets after 2 h is presented in Fig. 1c. The close-packed droplets mimicked emulsion droplets collected outside the MC well in which clove oil-in-water emulsion would be deposited at the
Fig. 3. Size distribution of clove oil droplets stabilised by 0.5% w/w SDS solution: (a) at 0 h and (b) after 2 h. (:) Close-packed droplets. ( Droplets with saturated phases.
) Non-close-packed droplets. (:)
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Fig. 4. Size changes of clove oil droplets in (a) 0.1% w/w and (b) 0.5% w/w SDS solution over time. (:) and () indicate oil droplets that were in close-packed configuration, ( ) and ( ) indicate those that were in non-close-packed configuration, () and (♦) indicate those in which the dispersed and continuous phases were saturated each other prior to droplet formation. The dashed-lines are visual guides.
bottom of a sample bottle. In order to collect emulsion droplets, a continuous phase should be flushed continuously throughout the MC well. During the collection period, the droplets flow through the MC well as groups of droplets or individual droplets. These configurations were simulated with non-close-packed droplets on the well. Non-close-packed droplets were polydisperse and unstable over a period of 2 h. Droplet sizes decreased significantly in the first 30 min; then the decrease was subtle until 2 h when 0.1% w/w SDS solution was used (Fig. 4a ( )). This size decrease indicates that the clove oil droplets shrink over time. The decreased droplet size was also confirmed by size distribution peak shifting to the left (Fig. 2b ( )). When a higher surfactant concentration was applied, the droplets shrank faster and the droplet sizes were more polydisperse (Fig. 4b ( )), as demonstrated by the error bars. These facts are confirmed by the size distribution being located more to the left and broader (Fig. 3b ( )). There was a broader size distribution after 2 h when a higher concentration of surfactant was applied (Fig. 3b ( )). An overview of the droplets after 2 h confirmed the data (Fig. 1d). Based on droplet configurations on the well (i.e., close-packed and non-close-packed), instability seemed to occur when the droplets were surrounded by plenty of continuous phase. The droplets were stable when they are packed with each other. Droplet instability when surrounded by excessive continuous phase implies that clove oil droplets collected in a container outside the MC well may differ in size from those inside the MC well. Reasons for instability were investigated by observing changes that occurred in a drop of clove oil in the aqueous phase. A fresh oil drop held in 0.1% w/w or 0.5% w/w SDS solution appears clean and homogenous internally, indicated by the homogenous black colour of the drop (Fig. 5a and b (0 min)). Diffusion of clove oil into the aqueous phase was observed immediately after the drop was fully formed in 0.1% SDS solution or immediately during formation of the drop in 0.5% SDS solution. Oil diffusion was indicated as eddies around the drop (Fig. 5a (1 min) and the inserted image in Fig. 5b). Oil may diffuse to the aqueous phase because the clove oil–aqueous system is chemically unstable. Clove oil is slightly soluble in water because of its high eugenol content. When this oil contacts with the aqueous phase, a difference in chemical potential arises, as the concentration of oil in the two phases is not in equilibrium. Thus, there is a driving force for oil to diffuse to the aqueous phase when the oil–water (O/W) interface is formed, as can be observed in Fig. 5a (1 min) and the inserted image in Fig. 5b. Diffusion is an instantaneous process in the clove oil–aqueous system; thus, this process
might be proposed as the first mechanism that is responsible for instability of clove oil droplets. No diffusion of clove oil into the aqueous phase could be observed during MC emulsification. However, spontaneous formation of small droplets in the continuous phase, in front of the O/W interface, was observed in the MCs. Formation of these droplets was observed in both SDS concentrations, and it was especially clear when 0.5% w/w SDS solution was used. Small droplets were formed together with the generation of regular droplets from MCs. Small droplets could be clearly observed in front of the O/W interface at 0 h, and more droplets formed during 2 h (Fig. 6a and b). These droplets are considered to be oil droplets. Small droplets inside the oil phase, which are regarded as aqueous droplets, were also observed (Fig. 6c). This was confirmed when a drop of oil formed in the aqueous phase and was held for a few minutes. Whitish aggregates were observed to form inside the oil drop after 1 min in 0.1% w/w SDS solution and 30 s in 0.5% w/w SDS solution. The white aggregates inside the oil drop could be small droplets of the aqueous phase (Fig. 5a and b). Liu et al. [30] indicated that there were waterin-oil (W/O) droplets or other droplet structures in the oil phase when the SDS concentration exceeded its CMC. SDS molecules can diffuse from the water phase into the oil phase when the SDS concentration exceeds its CMC. The molecules form reverse micelles in the oil phase, which may cause the oil to break up. The current study confirms that the droplets observed in the oil phase were W/O droplets; however, these droplets existed regardless of the concentration of SDS applied. SDS molecules seem to diffuse to the oil phase even when their concentration is below their CMC. Oil droplet formation in front of the O/W interface and aqueous droplet formation in the dispersed phase observed in MC emulsification resemble the behaviour of spontaneous emulsification (SE). This process might take place without an external energy supply (e.g., stirring) if two immiscible liquids are not initially at equilibrium [31]. Davies [32] explained the “diffusion and stranding” mechanism for SE in the ternary system by gently bringing a solution of ethanol in toluene into contact with water. The ethanol in the oil phase diffuses into the water and the ethanol carries some oil to the water phase. The oil, in the form of emulsion droplets, is stranded in the water while the ethanol can diffuse further into the water phase. Emulsion of water droplets could also form in the oil phase since ethanol in the oil may permit some water to dissolve. This mechanism was also confirmed by Ruschak and Miller [33] who proposed a “diffusion path” theory that enables predicting SE. Inter-diffusion between two non-equilibrium phases
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Fig. 5. Visualisation of internal oil droplet and the surrounding aqueous phase over time. The inserted image in (b) (a clove oil drop in 0.5% w/w SDS solution) confirms the eddies around the clove oil drop.
creates a super-saturation region that facilitates drop growth. SE in surfactant systems is also explained by the diffusion mechanism [31,34,35]. SE may occur above the threshold concentration of surfactant (CSE ) [34] and SE in the form of water droplets at the water/oil (W/O) interface occurs when 0.006% or more Span 80 is applied. SE can be suppressed by adding salt or gelatine to the aqueous phase. Other mechanisms proposed for SE include Marangoni effects [36], chemical reactions at the interface [37], and bursting of swollen bilayers [38]. However, the diffusion mechanism seems to be in line with phenomena observed in the clove oil–water system containing SDS. One supporting fact regarding this mechanism is that instantaneous diffusion takes place once the O/W interface is formed. Diffusion creates a film layer near the interface that is saturated by oil. Free surfactant molecules
may re-arrange in this layer, forming small droplets in front of the interface. The above observations indicate that instability of clove oil droplets in the continuous phase is driven mainly by a chemically unstable system. Driving forces of oil to diffuse into the continuous phase and surfactant molecules to diffuse into the oil phase lead to droplet instability. This instability might be reduced by creating a chemically stable system. To create such a system, the oil phase and the continuous phase are saturated with one another. The size distributions of clove oil droplets (Figs. 2a () and 3a()) indicate that monodisperse clove oil droplets formed initially, regardless of the SDS concentrations applied. When the saturation step was taken prior to droplet formation, the sizes of clove oil droplets were more stable over time than when
Fig. 6. Oil droplet formation by spontaneous emulsification at (a) 0 h and (b) 2 h, as well as (c) the aqueous droplets inside the oil phase observed on the MC array plate. The photos were taken from oil droplet formation in 0.5% w/w SDS solution without the saturation step.
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Table 1 Properties of clove oil and SDS solutions: viscosity (), density (), and interfacial tension (). Sample
(mPa s)
(kg/m3 )
(mN/m)
Clove oil 0.1% w/w SDS solution 0.5% w/w SDS solution Clove oil in 0.1% w/w SDS solution Clove oil in 0.5% w/w SDS solution Clove oil saturated with 0.1% w/w SDS solution Clove oil saturated with 0.5% w/w SDS solution 0.1% w/w SDS solution saturated with clove oil 0.5% w/w SDS solution saturated with clove oil
6.71 (±0.042) 0.89 (±0.005) 0.92 (±0.002) – – 7.40 (±0.018) 7.45 (±0.021) 0.90 (±0.005) 0.96 (±0.002)
1037 (±0.28) 998 (±0.13) 997 (±0.17) – – 1039 (±0.29) 1039 (±0.48) 998 (±0.16) 998 (±0.15)
9.03 (±0.08)a – – 2.82 (±0.13) 0.70 (±0.06) 3.16 (±0.08)b 2.05 (±0.25)b – –
a b
Measured in water. Measured in respected continuous phase used for saturation; the values inside the brackets are standard deviations.
no saturation step was taken (Figs. 4a () and b (♦)). Observation indicated that the oil drop in the aqueous phase contained whitish aggregates from the beginning (Fig. 5c and d). However, there was no indication that more aggregates formed inside the oil droplet over time. Diffusion of oil to the aqueous phase, indicated by eddies around the drop, could not be observed anymore even in 0.5% w/w SDS solution. However, the size of an oil drop in 0.5% w/w SDS solution decreased slightly after 5 min, indicating that diffusion of oil was still occurring even when the saturation step was taken. The saturation step resulted in changes of physical properties of clove oil as the dispersed phase (Table 1). The viscosities and densities of clove oil saturated with 0.1% and 0.5% w/w SDS solutions exceed those of pure clove oil. However, those values are the same for oils saturated with different concentrations of SDS. Interfacial tension between clove oil and SDS solution increased after the saturation step. This increment was especially high between clove oil and 0.5% w/w SDS solution. Without the saturation step, interfacial tension between clove oil and 0.5% w/w SDS solution was 0.70 mN/m. This small value was reflected by the small size of the oil drop that could form (1 L volume) (Fig. 5b). The interfacial tension after the saturation step was 2.05 mN/m; therefore, a larger oil drop could form (Fig. 5d). This study demonstrated that instability occurs instantaneously in clove oil droplets prepared by MC emulsification. Observation
indicated that instability of clove oil droplets might be governed by diffusion of oil phase into the aqueous phase, and vice versa. Diffusion might cause spontaneous emulsification. Fig. 7 schematically depicts the proposed mechanism for diffusion-driven instability of clove oil droplets in SDS solution. This scheme initially illustrates the moment when the O/W interface forms. The surfactant in the continuous phase immediately moves towards and covers the interface. However, at the same time, the oil diffuses to the continuous phase because of a chemically unstable system, and this process creates a stagnant oil layer in the continuous phase (Fig. 7a). This process eventually causes backward movement of the O/W interface, which might explain the shrinking of the oil droplets. In the stagnant oil layer, surfactant molecules might rearrange before reaching the interface, forming O/W emulsions spontaneously (Fig. 6a and b). However, after reaching the interface, some surfactant molecules are also able to diffuse to the oil phase, forming reversed micelles that can be seen as whitish aggregates in Fig. 5a and b (Fig. 7b). Furthermore, within an undetermined time, the reverse micelles might lead to the breakup of oil droplets, contributing to the appearance of more small O/W droplets in front of the O/W interface and further shrinkage of O/W emulsion droplets. When both the continuous and the dispersed phases are saturated with each other, there is a chance of diffusion to and from both phases because there is still the possibility of repetitive
Fig. 7. Schematic depiction of the proposed mechanism for instability of clove oil droplets in SDS solution over time.
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References
Fig. 8. Solubility of clove oil in different concentrations of SDS solution, expressed as a logarithm of modified partition coefficient (k).
formation of spontaneous O/W emulsions, reversed micelles, and droplet breakup. However, the transport of both phases in saturated condition takes a considerably longer time to reach the same size of oil droplet as in the unsaturated system. The mechanism depicted in Fig. 7a might be neglected once the O/W interface formed when the saturation step was taken. The mechanism depicted in Fig. 7b seems to be the dominating factor affecting the stability of emulsion droplets over time, in which the concentration of surfactant is an important parameter in saturated conditions. This is supported by the solubility of clove oil in different concentrations of SDS (Fig. 8). The solubility of clove oil in SDS solution is relatively constant, regardless of the concentrations of SDS. This may indicate that diffusion of clove oil into the continuous phase depends solely on the diffusivity and concentration gradient of clove oil in both phases. The concentration of surfactant in the continuous phase contributes to other side effects, such as spontaneous emulsion, reversed micelle formation, and further droplet breakup. 4. Conclusions Clove oil as monodisperse O/W emulsion droplets could be formed by MC emulsification. The emulsion droplets exhibited stability over time when the droplets were closely packed on the well of the MC array plate. However, clove oil droplets became instable instantaneously when the droplets were non-close-packed. Stability improved when both dispersed and continuous phases were saturated. Instability of clove oil droplets on the well of the MC array plate could be explained by diffusion-driven instability, spontaneous emulsions, and reversed micelle formation. Diffusion is explained solely by the concentration gradient of clove oil in the oil phase and the continuous phase. Concentrations of SDS as surfactant contribute to the subsequent effect of diffusion (i.e., spontaneous emulsion formation in the continuous phase and reversed micelle formation in the oil phase). This study further suggests that pre-caution should be taken in order to develop stable clove oil-based products in the form of emulsion or encapsulated products. Acknowledgment This research was supported by Kirin Holdings Co., Ltd., Tokyo, during the UNU-Kirin Fellowship Programme at the National Food Research Institute, Tsukuba, Japan in 2013–14.
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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Stability of monodisperse clove oil droplets prepared by microchannel emulsification Nanik Purwanti a,b , Marcos A. Neves c,a , Kunihiko Uemura a , Mitsutoshi Nakajima c,a , Isao Kobayashi a,∗ a
Food Engineering Division, National Food Research Institute, NARO, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan Department of Mechanical and Biosystem Engineering, Bogor Agricultural University, IPB Darmaga Campus, PO. BOX 220, Bogor 16002, Indonesia c Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8572, Japan b
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• O/W emulsion of clove essential • • • •
oil was produced by microchannel emulsification. Initial O/W emulsion droplets are monodisperse. Instability of oil droplets occurs after droplet generation. Instability was explained by diffusion, spontaneous emulsions, and reversed micelles. Diffusion of clove oil is independent of surfactant concentration.
a r t i c l e
i n f o
Article history: Received 1 July 2014 Received in revised form 29 October 2014 Accepted 30 October 2014 Available online 6 November 2014 Keywords: Clove oil Stability Oil-in-water emulsion Microchannel emulsification
a b s t r a c t Monodisperse clove oil droplets in the form of oil-in-water (O/W) emulsions stabilized by 0.1% and 0.5% w/w sodium dodecyl sulphate (SDS) were successfully produced by microchannel (MC) emulsification using a 15 × 15 mm2 MC array plate with 100 parallel MCs fabricated on each side of the plate. The channels have 71 m length, 8.2 m width, and 4 m depth with hydraulic diameter of 4.2 m. The terraces have the same depth size as the channels with 29.1 m length. The average droplet diameter was 17 m with a coefficient of variation around 3%. The droplet size deviated from monodispersity over time and it depended on the droplet configuration on MC array plate. The droplet size became polydisperse within 2 h when the droplets were surrounded by plenty of continuous phase. A higher SDS concentration resulted in more polydisperse droplet size over time. This instability is proposed as the result of diffusion-driven mechanism, spontaneous emulsification, and reversed micelle formation. Instability could be suppressed by saturating both continuous and dispersed phases prior to emulsification. This step may prevent the diffusion that takes place once the oil–water interface is formed. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Essential oil of clove has been widely applied in the pharmaceutical, fragrance, and flavour industries. Its various functionalities,
∗ Corresponding author. Tel.: +81 29 838 8025; fax: +81 838 8122. E-mail address: [email protected] (I. Kobayashi). http://dx.doi.org/10.1016/j.colsurfa.2014.10.058 0927-7757/© 2014 Elsevier B.V. All rights reserved.
some of which have been discovered (e.g., antioxidant [1–3], free-radical scavenger [4,5], anti-stress [6], anti-microbial activities [7], anti-inflammatory, antigiardial, analgesic, and anaesthetic properties [8,9]), may result in wider applications of this oil. To facilitate its application, clove oil can be formulated so that its functionalities are preserved and their release can be controlled. An example is encapsulation of the oil using vehicles such as solid lipid or a polymer matrix. Clove oil can be encapsulated by first
N. Purwanti et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 466 (2015) 66–74
producing oil-in-water (O/W) emulsions and subsequently solidifying them. Various emulsification techniques are used to produce O/W emulsions. Mechanical emulsification, the traditional technique, produces emulsions by utilizing instruments such as high-speed mixers, colloid mills, high-pressure valves or ultrasonic homogenizers, and micro-fluidizers [10]. Variable shear and pressure fields in such instruments lead to difficulty in controlling the size of emulsions; therefore, the resulting droplets are usually highly polydisperse [11,12]. In addition, high energy input is required to produce emulsions and most of the energy input is dissipated as heat [10]. Other techniques used to produce monodisperse emulsions with narrow size distribution are membrane emulsification [11,13], microchannel (MC) emulsification [14,15], and use of other microfluidic devices such as T-, Y-, or cross junctions [16–18], and an edge-based droplet generation (EDGE) system [19]. Vladisavljevic´ et al. [12] recently systematically reviewed characteristics of emulsions produced using those different techniques. MC emulsification was introduced as a novel emulsification method more than a decade ago [14]. This method produces monodisperse droplets with a coefficient of variation (CV) below 5% using MC arrays. Major aspects that should be considered when producing droplets using this method include design of microchannels (MCs) [12,20,21], selection of surfactants [22–24], operating conditions [15,25], and viscosity ratio between dispersed and continuous phases [26]. MC emulsification is a stable process for producing monodisperse emulsion droplets [23,27,28] and the resulting O/W droplets (triglycerol oil) remain stable for a long time [27,29]. Clove oil can be emulsified using this method. Droplet stability of clove essential oil prepared by MC emulsification was previously reported by Liu et al. [30]. The stability of the oil droplets deposited at the bottom of a sample bottle was reported to depend on surfactant concentration. The oil droplets broke up into smaller droplets after standing for 15 h when the surfactant (sodium dodecyl sulphate (SDS)) concentration in the buffer solution was above its critical micelle concentration (CMC), i.e. 0.2% w/w. To collect oil droplets generated by MC emulsification in a sample bottle, droplets in the MC well (Fig. 1b) should be washed away by the continuous phase. The size of oil droplets should not be influenced by the shearing effect of the continuous phase during droplet collection. However, we recently observed that clove oil droplets stabilized by SDS as the surfactant are unstable after they are generated by an MC array. Consequently, stability of the droplets is further affected during collection and application. This study demonstrates the instability of clove oil droplets generated by MC emulsification and clarifies the mechanism of this instability.
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consists of grooved MC arrays with 100 parallel MCs, fabricated on each side of the plate. The channel and terrace depth (H) is 4 m, the channel length (LC ) is 71 m, the channel width (WC ) is 8.2 m, and the terrace length (LT ) is 29.1 m. The hydraulic diameter (dh ), defined as four times the area over the perimeter, is 4.2 m. The top view of the MCs is depicted in Fig. 1a. Typical views of the crosssection and three-dimensional drawing of the MCs as well as all peripherals for MC emulsification have been described in previous studies [21,26]. 2.3. MC emulsification The following procedure was used to produce clove oil droplets by MC emulsification. The MC plate was initially filled with the continuous phase (i.e., SDS solution). The dispersed phase (i.e., clove oil) was pushed through the reservoir hole of the plate by slowly lifting the oil chamber until the minimal breakthrough pressure, the minimum pressure required to produce oil droplets, was achieved. Typical generation of clove oil droplets in SDS solution by MC emulsification is depicted in Fig. 1b. The generated droplets are located on the well of the MC array plate surrounded by the continuous phase (Fig. 1b). The continuous phase was SDS solution with a concentration of 0.1% w/w (below the CMC) and 0.5% w/w (above the CMC), which was filtered using a 0.2 m hydrophilic syringe filter (Sartorius Minisart® RC25, Sartorius AG, Göttingen, Germany) prior to use. Three conditions of clove oil droplets were produced and analyzed: (1) clove oil droplets were generated until the MC well was completely filled with the droplets (close-packed droplets, Fig. 1c); (2) clove oil droplets were generated forming non-closepacked droplets (Fig. 1d); and (3) the condition was the same as condition (2), but the dispersed phase and the continuous phase were saturated with each other prior to MC emulsification (Fig. 1e). The production of oil droplets by MC emulsification was repeated twice with two independent batches of continuous and dispersed phases. 2.4. Pre-treatment of saturated phases
Extra pure reagent clove oil (Nacalai Tesque, Inc., Kyoto, Japan) was used as the dispersed phase. SDS (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was used as the surfactant. Milli-Q water (resistivity 18.2 M cm at 25 ◦ C, total oxidizable carbon < 4 ppb, Merck-Millipore, Tokyo, Japan) was used to prepare the continuous phase.
The dispersed phase and the continuous phase were saturated with each other using the following steps. A mixture of clove oil and 0.1% w/w SDS solution was stirred at a weight ratio of 1:3 for 1 h. Next, the mixture was centrifuged at 760 × g for 15 min and then at 1070 × g for 10 min (KN-70, Kubota, Tokyo, Japan). The supernatant (SDS solution) was then harvested carefully and the remaining mixture was further centrifuged at 1880 × g for 10 min. The oil phase was harvested carefully and the remaining emulsion was discarded. The harvested SDS solution was filtered using a 0.2 m hydrophilic syringe filter and the oil was filtered using a PFTE hydrophobic syringe filter (Dismic 25 JP, Advantec Toyo Kaisha, Ltd., Tokyo, Japan) prior to MC emulsification. When 0.5% SDS solution was used, the ratio of clove oil and SDS solution was 1:1 and the stirring time was only 5 min to avoid complete conversion of the oil into emulsion. About 80% of the initial weight of the SDS solution and 20–25% of the oil could be recovered from the saturation step. The emulsification was performed at room temperature (25 ◦ C). The size changes of oil droplets inside the MC plate were monitored every 30 min for 2 h.
2.2. MC array plate and peripherals
2.5. Droplet size determination
Droplets of clove oil were produced by a silicon dead-end MC array plate (MS2A4). This MC array plate enables collecting of generated droplets on the well for inline observation; therefore, circulation of the continuous phase to collect the droplets outside the MC array plate is not needed. The 15 mm × 15 mm MC array plate
The diameter of oil droplets was analyzed by measuring the captured images of droplets using image processing software (WinROOF version 6.5, Mitani Co., Fukui, Japan). For each condition, 200 droplets were measured, except for clove oil droplets in 0.1% w/w SDS solution condition (2), for which 300 droplets were measured.
2. Materials and methods 2.1. Materials
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Fig. 1. (a) Top view of three MCs and their dimensions in m. (b) Generation of clove oil droplets from MCs. Configuration or conditions of droplets generated on the MC array plate just after formation (0 h) and after 2 h (represented by the droplets stabilised by 0.5% w/w SDS solution): (c) Close-packed droplets, (d) non-close-packed droplets, and (e) non-close-packed droplets in which both phases were saturated with each other prior to MC emulsification with a scale bar of 20 m.
2.6. Viscosity measurement Viscosities of the continuous and the dispersed phases were measured using a glass capillary viscometer (SO, Shibata Scientific Technology Ltd., Tokyo, Japan) at 25 ◦ C. A capillary tube with constant 0.00388 cSt/s was used for the continuous phase and one with constant 0.0144 cSt/s was used for the dispersed phase. A sample volume of 10 mL was inserted in the capillary tube. The time required for the meniscus of the liquid to move from the upper to the lower mark in the capillary was recorded. The dynamic viscosity (, mPa s) was calculated using the formula = C × t × , where C is the constant of the capillary tube, t is time (s), and is density (g/cm3 ). Each phase was measured with two independent batches. Each batch was measured in duplicate and the measurement of each replicate was repeated three times. 2.7. Density measurement Densities of the continuous and the dispersed phases were measured using a portable density/specific gravity metre (DA-130N, KEM, Kyoto Electronics, Japan) at room temperature (25 ◦ C). Each phase was measured with two independent batches and each batch was measured five times. 2.8. Determination of interfacial tension Interfacial tension between a drop of clove oil (pure and saturated conditions) and the continuous phase was measured using the pendant drop method (PD-W, Kyowa Interface Science Co., Ltd., Saitama, Japan). A drop of clove oil was suspended in SDS solution (saturated and unsaturated) and each drop was measured twice. Ten drops were made from two independent batches, except for pure clove oil in 0.5% SDS solution from which 20 drops were produced and measured.
needle. The drop was illuminated from the side in order to clearly visualise inside the drop and its surroundings. The changes of the drop and its surroundings during the first 5 min were captured using the interfacial tensiometer. The volume of the oil drop was 7 L for all conditions, except for the clove oil drop in 0.5% w/w SDS solution where a volume of only 1 L could be formed. At least two drops prepared from two independent batches were observed for each condition.
2.10. Solubility of clove oil in SDS solution The solubility of clove oil in different concentrations of SDS solution was determined by first placing a ratio of 1:1 clove oil and SDS solutions in glass tubes. Next, 5 mL of SDS solution was poured into the tube and then, 5 mL of clove oil was added. Clove oil passed through the SDS solution and remained at the bottom of the tube because of its higher density. The tubes were tightly closed, completely covered by aluminium foil, and then stored in a dark chamber set at 25 ◦ C. After 24 h, the SDS solution containing clove oil was collected from the tubes using a long needle. This supernatant was centrifuged at 1100 × g for 25 min and then, filtered with a 0.2 m membrane filter unit to remove micelles or spontaneously formed emulsion droplets. The supernatant was diluted five times with fresh SDS solution of corresponding concentration. The absorbance of diluted SDS solution containing clove oil was measured using a spectrophotometer at 300 nm. SDS solution of different concentrations was measured as blank samples. The value of solubility was determined using a standard calibration curve that was also set based on absorbance at 300 nm. Solubility was further calculated in terms of the modified partition coefficient that we defined as k=
Cin water phase Cin oil phase
2.9. Visual observation of oil drops in aqueous phase Clove oil was pushed through a needle immersed in the aqueous phase until forming a drop and this drop was held to hang onto the
and then expressed as a logarithm of this coefficient. Here, k is the partition coefficient and C is the concentration of clove oil. Concentration in the oil phase was assumed to be 100%.
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Fig. 2. Size distribution of clove oil droplets stabilised by 0.1% w/w SDS solution: (a) at 0 h and (b) after 2 h. (:) Close-packed droplets. ( Droplets with saturated phases.
3. Results and discussion Clove oil in the form of O/W emulsion droplets stabilized by SDS solution was generated using MC emulsification. Conditions of the generated droplets on the well of the MC array plate initially were set as indicated in Fig. 1c–e (0 h). The droplets generated by MCs were initially monodisperse regardless of the concentrations of surfactant and droplet conditions on the well. Initially, the average size of the droplets was 17 m with maximum CV of 3%.
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) Non-close-packed droplets. (:)
The droplets also had a narrow size distribution (Figs. 2a and 3a) indicating monodispersity. Close-packed clove oil droplets stabilized by 0.1% and 0.5% w/w SDS solutions were homogenous and stable over 2 h on the well, as indicated by the narrow size distribution (Figs. 2b and 3b ()) and stable droplet size over time (Fig. 4a () and b ()). An overview of the droplets after 2 h is presented in Fig. 1c. The close-packed droplets mimicked emulsion droplets collected outside the MC well in which clove oil-in-water emulsion would be deposited at the
Fig. 3. Size distribution of clove oil droplets stabilised by 0.5% w/w SDS solution: (a) at 0 h and (b) after 2 h. (:) Close-packed droplets. ( Droplets with saturated phases.
) Non-close-packed droplets. (:)
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Fig. 4. Size changes of clove oil droplets in (a) 0.1% w/w and (b) 0.5% w/w SDS solution over time. (:) and () indicate oil droplets that were in close-packed configuration, ( ) and ( ) indicate those that were in non-close-packed configuration, () and (♦) indicate those in which the dispersed and continuous phases were saturated each other prior to droplet formation. The dashed-lines are visual guides.
bottom of a sample bottle. In order to collect emulsion droplets, a continuous phase should be flushed continuously throughout the MC well. During the collection period, the droplets flow through the MC well as groups of droplets or individual droplets. These configurations were simulated with non-close-packed droplets on the well. Non-close-packed droplets were polydisperse and unstable over a period of 2 h. Droplet sizes decreased significantly in the first 30 min; then the decrease was subtle until 2 h when 0.1% w/w SDS solution was used (Fig. 4a ( )). This size decrease indicates that the clove oil droplets shrink over time. The decreased droplet size was also confirmed by size distribution peak shifting to the left (Fig. 2b ( )). When a higher surfactant concentration was applied, the droplets shrank faster and the droplet sizes were more polydisperse (Fig. 4b ( )), as demonstrated by the error bars. These facts are confirmed by the size distribution being located more to the left and broader (Fig. 3b ( )). There was a broader size distribution after 2 h when a higher concentration of surfactant was applied (Fig. 3b ( )). An overview of the droplets after 2 h confirmed the data (Fig. 1d). Based on droplet configurations on the well (i.e., close-packed and non-close-packed), instability seemed to occur when the droplets were surrounded by plenty of continuous phase. The droplets were stable when they are packed with each other. Droplet instability when surrounded by excessive continuous phase implies that clove oil droplets collected in a container outside the MC well may differ in size from those inside the MC well. Reasons for instability were investigated by observing changes that occurred in a drop of clove oil in the aqueous phase. A fresh oil drop held in 0.1% w/w or 0.5% w/w SDS solution appears clean and homogenous internally, indicated by the homogenous black colour of the drop (Fig. 5a and b (0 min)). Diffusion of clove oil into the aqueous phase was observed immediately after the drop was fully formed in 0.1% SDS solution or immediately during formation of the drop in 0.5% SDS solution. Oil diffusion was indicated as eddies around the drop (Fig. 5a (1 min) and the inserted image in Fig. 5b). Oil may diffuse to the aqueous phase because the clove oil–aqueous system is chemically unstable. Clove oil is slightly soluble in water because of its high eugenol content. When this oil contacts with the aqueous phase, a difference in chemical potential arises, as the concentration of oil in the two phases is not in equilibrium. Thus, there is a driving force for oil to diffuse to the aqueous phase when the oil–water (O/W) interface is formed, as can be observed in Fig. 5a (1 min) and the inserted image in Fig. 5b. Diffusion is an instantaneous process in the clove oil–aqueous system; thus, this process
might be proposed as the first mechanism that is responsible for instability of clove oil droplets. No diffusion of clove oil into the aqueous phase could be observed during MC emulsification. However, spontaneous formation of small droplets in the continuous phase, in front of the O/W interface, was observed in the MCs. Formation of these droplets was observed in both SDS concentrations, and it was especially clear when 0.5% w/w SDS solution was used. Small droplets were formed together with the generation of regular droplets from MCs. Small droplets could be clearly observed in front of the O/W interface at 0 h, and more droplets formed during 2 h (Fig. 6a and b). These droplets are considered to be oil droplets. Small droplets inside the oil phase, which are regarded as aqueous droplets, were also observed (Fig. 6c). This was confirmed when a drop of oil formed in the aqueous phase and was held for a few minutes. Whitish aggregates were observed to form inside the oil drop after 1 min in 0.1% w/w SDS solution and 30 s in 0.5% w/w SDS solution. The white aggregates inside the oil drop could be small droplets of the aqueous phase (Fig. 5a and b). Liu et al. [30] indicated that there were waterin-oil (W/O) droplets or other droplet structures in the oil phase when the SDS concentration exceeded its CMC. SDS molecules can diffuse from the water phase into the oil phase when the SDS concentration exceeds its CMC. The molecules form reverse micelles in the oil phase, which may cause the oil to break up. The current study confirms that the droplets observed in the oil phase were W/O droplets; however, these droplets existed regardless of the concentration of SDS applied. SDS molecules seem to diffuse to the oil phase even when their concentration is below their CMC. Oil droplet formation in front of the O/W interface and aqueous droplet formation in the dispersed phase observed in MC emulsification resemble the behaviour of spontaneous emulsification (SE). This process might take place without an external energy supply (e.g., stirring) if two immiscible liquids are not initially at equilibrium [31]. Davies [32] explained the “diffusion and stranding” mechanism for SE in the ternary system by gently bringing a solution of ethanol in toluene into contact with water. The ethanol in the oil phase diffuses into the water and the ethanol carries some oil to the water phase. The oil, in the form of emulsion droplets, is stranded in the water while the ethanol can diffuse further into the water phase. Emulsion of water droplets could also form in the oil phase since ethanol in the oil may permit some water to dissolve. This mechanism was also confirmed by Ruschak and Miller [33] who proposed a “diffusion path” theory that enables predicting SE. Inter-diffusion between two non-equilibrium phases
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Fig. 5. Visualisation of internal oil droplet and the surrounding aqueous phase over time. The inserted image in (b) (a clove oil drop in 0.5% w/w SDS solution) confirms the eddies around the clove oil drop.
creates a super-saturation region that facilitates drop growth. SE in surfactant systems is also explained by the diffusion mechanism [31,34,35]. SE may occur above the threshold concentration of surfactant (CSE ) [34] and SE in the form of water droplets at the water/oil (W/O) interface occurs when 0.006% or more Span 80 is applied. SE can be suppressed by adding salt or gelatine to the aqueous phase. Other mechanisms proposed for SE include Marangoni effects [36], chemical reactions at the interface [37], and bursting of swollen bilayers [38]. However, the diffusion mechanism seems to be in line with phenomena observed in the clove oil–water system containing SDS. One supporting fact regarding this mechanism is that instantaneous diffusion takes place once the O/W interface is formed. Diffusion creates a film layer near the interface that is saturated by oil. Free surfactant molecules
may re-arrange in this layer, forming small droplets in front of the interface. The above observations indicate that instability of clove oil droplets in the continuous phase is driven mainly by a chemically unstable system. Driving forces of oil to diffuse into the continuous phase and surfactant molecules to diffuse into the oil phase lead to droplet instability. This instability might be reduced by creating a chemically stable system. To create such a system, the oil phase and the continuous phase are saturated with one another. The size distributions of clove oil droplets (Figs. 2a () and 3a()) indicate that monodisperse clove oil droplets formed initially, regardless of the SDS concentrations applied. When the saturation step was taken prior to droplet formation, the sizes of clove oil droplets were more stable over time than when
Fig. 6. Oil droplet formation by spontaneous emulsification at (a) 0 h and (b) 2 h, as well as (c) the aqueous droplets inside the oil phase observed on the MC array plate. The photos were taken from oil droplet formation in 0.5% w/w SDS solution without the saturation step.
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Table 1 Properties of clove oil and SDS solutions: viscosity (), density (), and interfacial tension (). Sample
(mPa s)
(kg/m3 )
(mN/m)
Clove oil 0.1% w/w SDS solution 0.5% w/w SDS solution Clove oil in 0.1% w/w SDS solution Clove oil in 0.5% w/w SDS solution Clove oil saturated with 0.1% w/w SDS solution Clove oil saturated with 0.5% w/w SDS solution 0.1% w/w SDS solution saturated with clove oil 0.5% w/w SDS solution saturated with clove oil
6.71 (±0.042) 0.89 (±0.005) 0.92 (±0.002) – – 7.40 (±0.018) 7.45 (±0.021) 0.90 (±0.005) 0.96 (±0.002)
1037 (±0.28) 998 (±0.13) 997 (±0.17) – – 1039 (±0.29) 1039 (±0.48) 998 (±0.16) 998 (±0.15)
9.03 (±0.08)a – – 2.82 (±0.13) 0.70 (±0.06) 3.16 (±0.08)b 2.05 (±0.25)b – –
a b
Measured in water. Measured in respected continuous phase used for saturation; the values inside the brackets are standard deviations.
no saturation step was taken (Figs. 4a () and b (♦)). Observation indicated that the oil drop in the aqueous phase contained whitish aggregates from the beginning (Fig. 5c and d). However, there was no indication that more aggregates formed inside the oil droplet over time. Diffusion of oil to the aqueous phase, indicated by eddies around the drop, could not be observed anymore even in 0.5% w/w SDS solution. However, the size of an oil drop in 0.5% w/w SDS solution decreased slightly after 5 min, indicating that diffusion of oil was still occurring even when the saturation step was taken. The saturation step resulted in changes of physical properties of clove oil as the dispersed phase (Table 1). The viscosities and densities of clove oil saturated with 0.1% and 0.5% w/w SDS solutions exceed those of pure clove oil. However, those values are the same for oils saturated with different concentrations of SDS. Interfacial tension between clove oil and SDS solution increased after the saturation step. This increment was especially high between clove oil and 0.5% w/w SDS solution. Without the saturation step, interfacial tension between clove oil and 0.5% w/w SDS solution was 0.70 mN/m. This small value was reflected by the small size of the oil drop that could form (1 L volume) (Fig. 5b). The interfacial tension after the saturation step was 2.05 mN/m; therefore, a larger oil drop could form (Fig. 5d). This study demonstrated that instability occurs instantaneously in clove oil droplets prepared by MC emulsification. Observation
indicated that instability of clove oil droplets might be governed by diffusion of oil phase into the aqueous phase, and vice versa. Diffusion might cause spontaneous emulsification. Fig. 7 schematically depicts the proposed mechanism for diffusion-driven instability of clove oil droplets in SDS solution. This scheme initially illustrates the moment when the O/W interface forms. The surfactant in the continuous phase immediately moves towards and covers the interface. However, at the same time, the oil diffuses to the continuous phase because of a chemically unstable system, and this process creates a stagnant oil layer in the continuous phase (Fig. 7a). This process eventually causes backward movement of the O/W interface, which might explain the shrinking of the oil droplets. In the stagnant oil layer, surfactant molecules might rearrange before reaching the interface, forming O/W emulsions spontaneously (Fig. 6a and b). However, after reaching the interface, some surfactant molecules are also able to diffuse to the oil phase, forming reversed micelles that can be seen as whitish aggregates in Fig. 5a and b (Fig. 7b). Furthermore, within an undetermined time, the reverse micelles might lead to the breakup of oil droplets, contributing to the appearance of more small O/W droplets in front of the O/W interface and further shrinkage of O/W emulsion droplets. When both the continuous and the dispersed phases are saturated with each other, there is a chance of diffusion to and from both phases because there is still the possibility of repetitive
Fig. 7. Schematic depiction of the proposed mechanism for instability of clove oil droplets in SDS solution over time.
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References
Fig. 8. Solubility of clove oil in different concentrations of SDS solution, expressed as a logarithm of modified partition coefficient (k).
formation of spontaneous O/W emulsions, reversed micelles, and droplet breakup. However, the transport of both phases in saturated condition takes a considerably longer time to reach the same size of oil droplet as in the unsaturated system. The mechanism depicted in Fig. 7a might be neglected once the O/W interface formed when the saturation step was taken. The mechanism depicted in Fig. 7b seems to be the dominating factor affecting the stability of emulsion droplets over time, in which the concentration of surfactant is an important parameter in saturated conditions. This is supported by the solubility of clove oil in different concentrations of SDS (Fig. 8). The solubility of clove oil in SDS solution is relatively constant, regardless of the concentrations of SDS. This may indicate that diffusion of clove oil into the continuous phase depends solely on the diffusivity and concentration gradient of clove oil in both phases. The concentration of surfactant in the continuous phase contributes to other side effects, such as spontaneous emulsion, reversed micelle formation, and further droplet breakup. 4. Conclusions Clove oil as monodisperse O/W emulsion droplets could be formed by MC emulsification. The emulsion droplets exhibited stability over time when the droplets were closely packed on the well of the MC array plate. However, clove oil droplets became instable instantaneously when the droplets were non-close-packed. Stability improved when both dispersed and continuous phases were saturated. Instability of clove oil droplets on the well of the MC array plate could be explained by diffusion-driven instability, spontaneous emulsions, and reversed micelle formation. Diffusion is explained solely by the concentration gradient of clove oil in the oil phase and the continuous phase. Concentrations of SDS as surfactant contribute to the subsequent effect of diffusion (i.e., spontaneous emulsion formation in the continuous phase and reversed micelle formation in the oil phase). This study further suggests that pre-caution should be taken in order to develop stable clove oil-based products in the form of emulsion or encapsulated products. Acknowledgment This research was supported by Kirin Holdings Co., Ltd., Tokyo, during the UNU-Kirin Fellowship Programme at the National Food Research Institute, Tsukuba, Japan in 2013–14.
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