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Phase behavior and stability of nano-emulsions prepared by D phase emulsification method
Accepted Manuscript Phase behavior and stability of nano-emulsions prepared by D phase emulsification method
Wanping Zhang, Yubo Qin, Zihao Gao, Wenhua Ou, Haiyang Zhu, Qianjie Zhang PII: DOI: Reference:
S0167-7322(18)36234-2 https://doi.org/10.1016/j.molliq.2019.04.112 MOLLIQ 10835
To appear in:
Journal of Molecular Liquids
Received date: Revised date: Accepted date:
2 December 2018 9 April 2019 22 April 2019
Please cite this article as: W. Zhang, Y. Qin, Z. Gao, et al., Phase behavior and stability of nano-emulsions prepared by D phase emulsification method, Journal of Molecular Liquids, https://doi.org/10.1016/j.molliq.2019.04.112
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ACCEPTED MANUSCRIPT Phase Behavior and Stability of Nano-Emulsions Prepared by D Phase Emulsification Method Wanping Zhanga, Yubo Qina, Zihao Gaoa, Wenhua Oua, Haiyang Zhub, Qianjie Zhanga,* a
School of Perfume and Aroma Technology, Shanghai Institute of Technology, Shanghai,
People’s Republic of China b
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Shanghai Ruxi Bio-Tech Co., Ltd, Shanghai, People’s Republic of China
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Corresponding author: Name: Qianjie-Zhang
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Email: [email protected]
Address: School of Perfume and Aroma Technology, Shanghai Institute of Technology, No.
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100, Haiquan Road, Fengxian District, Shanghai 201418, People’s Republic of China
ACCEPTED MANUSCRIPT Abstract: D phase emulsification method was applied to prepare nano-emulsions in the Span 80-Tween 80/glycerin/water/mineral oil system. The phase behavior of this system during the entire emulsification process and the stability of the prepared emulsions were investigated by means of a pseudo-ternary phase experimental design. A possible emulsification mechanism was preliminarily proposed on the basis of our experimental results. It was found that the special self-assembled structure of the D phase and its capability for oil phase were the crucial factors in the D phase
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emulsification. Firstly, the surfactant alignment of the D phase was beneficial to the formation of small oil droplets in the O/D phase. Then, a quick movement of the
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surfactants toward the oil-water interface was critical for preparing nano-emulsions. Furthermore, the stability of the nano-emulsions during storage (30 days) was
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predominantly related to the addition amount of surfactants. The higher the content of surfactant in the system, the more stable the nano-particle interface film was formed.
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The stable interface film avoids the aggregation of the particles due to the thermal motion of the molecules, and the nano-particles could remain stable during storage.
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Keywords: D phase emulsification; nano-emulsions; phase behavior; stability Abbreviations:
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PIT: phase inversion temperature
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PIC: phase inversion concentration EIP: emulsion inversion point
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HLB: lipophilic-hydrophilic balance Acknowledgements:
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The research was supported in part by the Shanghai Gaofeng & Gaoyuan Project for University Academic Program Development （1021ZK183011005）and Shanghai Local Capacity Building Projects (19090503600).
ACCEPTED MANUSCRIPT 1.INTRODUCTION Nano-emulsions, also called submicron emulsions [1-2], mini-emulsions [3-4], ultrafine emulsions [5-6] in the literature, are a type of emulsions with uniform and extremely small
droplet
size
in
the
range
20-500nm.
Comparing
with
macro-emulsions, nano-sized particles have less inter-molecular attraction and exhibit higher stability in kinetics against coalescence and sedimentation, hence offering
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increased stability to some extent [7]. The process of preparing nano-emulsions can be roughly divided into two
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categories: high-energy emulsification and low-energy emulsification. The former method works by doing work on the emulsion system, and the emulsion particles are
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broken to the nano-meter level by high-speed stirring, homogenization, or ultrasonication [8-10].
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On the other hand, the low-energy emulsification depends on the spontaneous movement of the surfactants and the formation of low interfacial tensions during the emulsification process. In some literature, the low energy emulsification method is
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simply divided into two categories. One is based on the phase inversion during the emulsification process. The other low-energy emulsification method is the spontaneous emulsification method [11]. Representative low energy emulsification
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methods emcompass phase inversion temperature (PIT) emulsification [12-13], phase
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inversion concentration (PIC) emulsification [14-15], emulsion inversion point (EIP) emulsification [16-17], D phase emulsification method and so on. For nonionic surfactants, PIT methods can be achieved by changing the temperature of system
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which essentially varied the lipophilic-hydrophilic balance (HLB) of the surfactants. Instead of temperature, PIC and EIP methods can be obtained by changing the water
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volume fraction with continually adding water into oil. The emulsification system passes through a relatively viscous state and crosses a point of zero spontaneous curvature, then the fine nano-size particles can be formed. However, these methods are well known for short-chain surfactants with strict requirement for HLB value and don’t work well for most of low-polarity oils. Except these phase inversion emulsifications, homogenous O/W nano-emulsions can be prepared by going through the D-phase which surfactants have self-assembled structure by the intervention of polyol during the emulsification process. Sagitani et al. proposed the method to prepare fine O/W emulsions which called D phase emulsification since the fine O/W emulsions were obtained by diluting an oil-in-surfactant gel emulsion (O/D gel emulsion) comprise of oil droplets surrounded by surfactant phase (D phase) in their
ACCEPTED MANUSCRIPT experiments [18]. Comparing with other low-energy emulsification methods, D phase emulsification is easy to prepare small and narrow distribution emulsion particles. It can emulsify oils with low polarity, small surface tension and poor water solubility. And it is not necessary to control the emulsification temperature and HLB value. Generally, the specific procedure of D phase emulsification is showed as Fig.1. Nonionic surfactant and polyol solution were completely dissolved as one uniform phase (D phase), and then oil was added dropwise to the surfactant solution with
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stirring. An oil-in-surfactant clear gel was formed by dropwise addition of a certain amount of oil. Then, the fine oil-in water emulsion can be obtained by dilution of the
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gel with water. It has been pointed out in literature that the formation of gel phase is the key point to prepare nano-emulsions by D-phase emulsification methods [19].
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Research speculate that it is related to the special arrangement of surfactant, oil and polyol solution, and the dynamic process of its movement determines the formation of
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nano-emulsions. Even the appearance of gel may not be observed, the components should spontaneously undergo the special
form
uniform
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nano-emulsions [20].
arrangement to
In recent years, a number of explorations and attempts were made in preparing nano-emulsions by D phase emulsification method. Most of the research has focused
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on the effect of polyols, saccharide and other polyol-like humectant materials on the properties of surfactants, and therefore on the phase behavior of D phase
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emulsification. Murakami et.al studied the effects of sucrose, D-fructose and D-maltose on surfactant cloud point and D phase emulsification process. It was found that all three kind of sugars can reduce the cloud point of surfactant, enhance its
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hydrophobicity and then influence the emulsification process [21]. Miyahara et.al have prepared nano-emulsions which particle diameter were less than 100 nm by D
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phase emulsification method using nonionic surfactant, EPDME (a novel humectant), water, and mineral oil [22]. Nevertheless, few literatures focused on the phase behavior of entire D phase emulsification process, and the stability of prepared nano-emulsions during the storage. In this paper, the phase behavior of the Span 80-Tween 80/glycerin/mineral oil, Span 80-Tween 80-glycerin/water/mineral oil system in the entire emulsification process was comprehensively investigated by pseudo-ternary phase diagram. The mechanism of the D phase emulsification was expanded by contrasting the phase change during emulsification. In addition, the stability of prepared nano-emulsions was also discussed.
ACCEPTED MANUSCRIPT 2. Experimental 2.1 Materials Span 80 (sorbitan monooleate, HLB=4.3) and Tween 80 (polyoxyethylene sorbitan monooleate, HLB=15.0) were purchased from Sinopham Chemical Reagent Co.,Ltd. The ratio in weight of Span 80 and Tween 80 used was 3:5 (HLB=11). Glycerin and mineral oil were obtained from Infinitus Co.,Ltd and BASF Corp
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respectively. Deionized water was used to prepare all emulsions.
2.2 Preparation of nano-emulsion by D phase emulsification
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Span 80 and Tween 80 were mixed at 3:5 in weight, and then the surfactants were mixed with glycerin at specific ratio which followed by slowly adding mineral
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oil with stirring at 80℃. Stirring lasted for 30min to produce a fine O/D phase. For O/W emulsion preparation, gradual addition of water to the O/D phase was carried out prepared. The process is shown in Fig.2.
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2.3 Pseudo-ternary phase diagram
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with stirring. At specific proportion of oil and water, fine nano-emulsion can be
Samples differing in composition were placed in sample bottle which were then sealed and stored in a thermostat bath at 25℃. A pseudo ternary phase diagram was
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drawn according to the phase state of the sample which was just after prepared and stored after 30 days. Phase state was confirmed by visual observation, conductivity
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measurement, microscopic observation, particle size measurement, and centrifugal stability investigation at 25℃.
Since the electrical conductivity of oil and water was significantly different,
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conductivity measurement (DDS-12DW LIDA) can effectively identify the type of continuous phase produced by emulsification in the experiments involving emulsion
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phase inversion. Microscopic observation (BX-53 Olympus) and particle size measurement (Nano-ZS Malvern) were used to observe the size of the particles, then the macroemulsions and the nano-emulsions could be distinguished. In addition, centrifugal stability (TGL-16aR) of emulsions was also used to distinguish microemulsions from nano-emulsions in the phase diagrams. 2.4 Determination of particle size and polydispersity index Particle size (z-average diameter) were measured using Malvern Zetasizer Nano ZS (Malvern Instruments, UK) at 25℃ by scattering light. The values of the mean diameter were the averages of results obtained for three replicates. 2.5 Transmission electron microscopy
ACCEPTED MANUSCRIPT Nanoemulsions were observed by negative-staining electron microscopy (FEI Tecnai 12) as a direct measure of the droplet size and shape. To perform the TEM observation, a drop of the nano-emulsion was directly deposited on the 300 mesh copper grid and the natural sedimentation lasted for 10 min. Then the sample was negatively stained by floating the grid face-down on a drop of 2% (w/v) phosphotungstic acid for 2 min and dried for 10 min. The images of the sample were obtained by observing the grids in the transmission electron microscope at an
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acceleration voltage of 120 kv. 2.6 Statistical analysis
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their means and standard deviations were reported.
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All measurements were performed on at least three freshly prepared samples and
3. Results and Discussion
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3.1 Phase Behavior of Span 80-Tween 80/Glycerin/Mineral Oil In order to examine the entire process of D phase emulsification clearly and
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intuitively, the phase behavior of the O/D phase formation process was first investigated.
In the Span 80-Tween 80/glycerin/mineral oil (Span 80:Tween 80=3:5) system,
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lipophilic emulsifier Span 80 and hydrophilic emulsifier Tween 80 formed specific
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mixed, oriented, self-assembled structure in glycerin during the emulsification process. The O/D phase formed by adding mineral oil to the surfactant glycerin solution exhibited by different appearance depending on the ratio of added raw materials.
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Pseudo-ternary phase diagram of Span 80-Tween 80/glycerin/mineral oil system were constructed (Fig. 3). Since the polyol used in this system was glycerin (100%), which means there was no water participated in the preparation process of O/D phase, the
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phase region obtained during this preparation process was relatively simple. The prepared O/D phase was divided into different regions and marked as ‘a’, ‘b’, and ‘c’. The sample photos in Fig.3 shows the different appearance of each region. The amount of oil added in ‘a’ region was low and they could be solubilized. Samples in this region presented the same color as surfactants. Few oils added in the samples were solubilized or loaded in the structure formed by surfactants and glycerin. Samples prepared in ‘b’ zone were uniform and translucent. With the increase of oil volume fraction in the system, the surplus oil was produced (‘c’ area). Samples obtained in ‘c’ region were not stable and sedimentation phenomenon occurred immediately after stirring stopped. However, it was found that most samples of ‘c’
ACCEPTED MANUSCRIPT areas could obtained nano-emulsions by diluting with water in subsequent procedure, but the formation and stabilization of nano-emulsions should be discussed separately. It was worth noting that the typical gel emulsion was not observed under our preparation condition which due to the absence of water. Therefore, it is the oriented arranged structure of D phase required stirring constantly during adding oil to supply energy to prompt the forming of small oil drops, which is also the indispensable for nano-emulsions preparation.
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3.2 Phase behavior of Span 80-Tween 80-Glycerin/Water/Mineral Oil System
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To investigated the effect of different O/D phase on the formation of nano-emulsions, different ratio of surfactants and glycerin in weight (two lines in
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Fig.3 marked as ①and ②) were chose to prepare through same emulsification process. The chosen ratio of Span 80-Tween 80 to glycerin (①7.5:2.5 in weight and ②2.1:7.9
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in weight) can coverage the different region of the O/D phase, and the phase behavior of each system were observed. Pseudo-ternary phase diagram of the initial system
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after preparation (Fig.4,7) and after 30 days (Fig.6,8) were obtained respectively. Following the addition of water to the starting O/D structure (‘a’ and ‘c’ regions in Fig.3), micro-emulsions (M), gel emulsions (P), and nano-emulsions (N) were
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prepared while the mineral oil addition ration is less than 75%, which indicated by the filled circles in Fig.4. As shown by the dashed line in Fig.4, micro-emulsions with
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ultrafine particles and transparent appearance were obtained when the added water was less. Semitransparent appearance of the microemulsions benefits from the fine emulsion particles [23]. For example, the particle size at the star label of M region in
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Fig. 4 was 52.35±0.78nm. Transmission electron microscopy (TEM) investigations of the microparticles (Fig.5a) showed the typical appearance of the microemulsions in
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the M area. Comparing with the M region in Fig.6, it could be found that the micro-emulsions were thermodynamically stable system, and there was no instability phenomenon such as creaming or sedimentation occurred during storage. As the addition weight of water increase to 20%~40%, gel emulsions were prepared. Samples in this region showed high viscosity thus perform favorable stability in the process of storage. Continuing increasing the water fraction to 50%, nano-emulsions were formed. The particle size at the star label of N region in Fig.4 was 143.47±2.03nm. The morphology and structure of the prepared sample were also studied using TEM investigations and the result was shown in Fig.5b. As mentioned in the precious section, the O/D phase is a special self-assembled structure formed by
ACCEPTED MANUSCRIPT surfactants and glycerin and oil is dispersed as fine particles in the arrangement. While adding water to the O/D phase, the surfactants move rapidly to the oil-water interface and forming ultrafine O/W emulsions. In the dynamic process of particle motion, the movement speed of mineral oil should be lower than the movement speed of the surfactant, hence the oil droplets can be wrapped by continuous water phase before the coalescence. By comparing the phase behavior in Fig.3 and Fig.4, it was found that the ‘a’ and
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‘c’ region didn’t have strict correspondence with the ‘M’, ‘P’ and ‘N’ region. It indicated that the surplus oil in O/D structure didn’t have obviously effects on the
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transformation of the O/D phase to the O/W emulsion. As described above, although the O/D structure formed during the emulsification process was unstable, and
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sedimentation phenomenon occurred immediately in the prepared O/D samples after the stirring stopped. However, during the preparation of O/W, water was added under
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agitation, and the oriented O/D phase still existed. The added water can quickly surround the dispersed oil droplets to form fine nano-emulsions.
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At the initial system after preparation, W/O emulsions were formed due to the high oil phase weight ratio (E(W/O) region in Fig.4). But considering the proportion of emulsifiers in the system (the weight ratio of lipophilic emulsifier Span-80 was less
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than hydrophilic emulsifier), formed W/O structure was not stable. During storage, samples finally changed to O/W emulsions and excess oil floated to the upper layer
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after 30 days. Furthermore, since nano-emulsions were thermodynamically unstable systems, ultrafine particles were easily aggregated by the influence of Ostwald ripening to cause instability [24-26]. Part of the nano-emulsions (N area in Fig.4)
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were creaming after storage (E(O/W) region in Fig.6). The upper layer of the samples was comprised of large particles which aggregated and coalesced from nano particles
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and the lower layer was still nano-emulsions. Though some samples were unstable due to the influence of Ostwald ripening, most of the prepared nano-emulsions remained stable during storage for 30 days. In this system, stable nano-emulsions can be prepared while the oil content below 80% and water content above 50 wt%. It was mainly benefit by the high proportion of surfactants in the system since there have enough surfactants in forming stable oil-water interface film [27]. Differing with the former case, the system with lower concentration of surfactants presented the polymorphism. More than five phase regions can be obtained by adding water to the samples through line ②in Fig.3. Micro-emulsions (M) were formed while the oil content below 20% and water content between 25% to 50%.
ACCEPTED MANUSCRIPT Comparing with the ‘M’ region in Fig.4, this region was narrower due to the lower surfactant concentration. Nano-emulsions formed in this system had a variety of particle size. The higher the emulsifier content, the smaller the particle size of the emulsions. While the surfactants and glycerin content were relatively low, macro-emulsions were prepared. With the decrease of oil content, W/O emulsions and O/W emulsions were obtained in sequence. However, since the weight ratio of surfactants to glycerin was low (2.1:7.9), the
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stability during 30 days’ observation of prepared emulsions was decreased. Except the ‘M’ region, all other regions occurred unstable phenomenon during storage (as shown
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in Fig.8), and the appearance of final samples were shown in Fig.9. It was notable that, the nano-emulsions prepared in this system showed different unstable results: oil slick
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(O+N); stratification (E+N); and maintained good stability (N). This phenomenon may be related to the different addition of water and oil in the system. By comparing
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the phase behavior in Fig.3, Fig.7 and Fig.8, it could be found that nano-emulsions could be prepared by diluting the most samples in ‘b’ area with water, but the stable
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nano-emulsions only can be formed while the amount of oil added was low and the amount of emulsifier or water added was high. Then oriented O/D phase was formed and the surfactants can maintain the stability of the nano-emulsions.
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In summary, it can be concluded that when the surfactant content in the system was decreased, the amount of oil that can be carried by the self-assembled structure of
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the D phase was also decreased, which due to the oil loading capacity of D phase was mainly related to the amounts of surfactants. Addition of oil beyond the D phase carrying capacity would easily lead to oil droplets aggregating and coalescing, and
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thus nano-emulsions were difficult to prepare.
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4. Conclusion
The entire process of preparing nano-emulsions by D phase emulsification method was discussed on the basis of Span 80-Tween 80/glycerin/water/mineral oil system. The self-assembled structure of emulsifiers formed in polyols plays a decisive role in the formation of nano-emulsions. Through the description of the ternary phase diagram, it could be found that the D phase emulsification preparation process was easy to produce nano-emulsions in this system, and the addition amount of the emulsifier plays a significant role in the stability of the nano-emulsions. Emulsifiers as a key factor in stabilizing the interface can protect nanoparticles against the coalescence caused by molecular thermal motion during storage for 30 days. At
ACCEPTED MANUSCRIPT higher surfactants/glycerin content, nano-emulsions can be prepared while the oil content below 80% and water content above 50 wt%. At lower surfactants/glycerin content, stable nano-emulsion can be prepared while the oil content below 25% and water content above 50 wt% during the 30-day inspection period. These experimental results contribute to deeper understanding of the process and mechanism of D phase emulsification, and also provide a theoretical basis for the preparation of
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nano-emulsions.
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Figure Name： Fig.1 Specific Procedure of D Phase Emulsification Fig.2 Process of the D Phase Emulsification Fig.3 The Ternary Phase Diagram of Span 80-Tween 80/Glycerin/Mineral Oil System at 25℃ after Preparation (‘a’, ‘b’, ‘c’ represent the O/D phase of different appearances) Fig.4 The Ternary Phase Diagram of Span 80-Tween 80-Glycerin/Water/Mineral Oil System
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at 25℃ after Preparation. (The ratio of Span 80-Tween 80: glycerin was 7.5:2.5, M represents the micro-emulsions, P represents gel emulsions, N represents nano-emulsions, E represents
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macro-emulsions)
Fig.5 TEM micrographs of the micro-emulsion (a) and the nano-emulsion (b).
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Fig.6 The Ternary Phase Diagram of Span 80-Tween 80-Glycerin/Water/Mineral Oil System after 30 Days. (The ratio of Span 80-Tween 80: glycerin was 7.5:2.5, M represents the
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micro-emulsions, P represents gel emulsions, N represents nano-emulsions, E represents macro-emulsions)
Fig.7 The Ternary Phase Diagram of Span 80-Tween 80-Glycerin/Water/Mineral Oil System
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at 25℃ after Preparation. (The ratio of Span 80-Tween 80: glycerin was 2.1:7.9, U represents the unstudy area, M represents micro-emulsions, N represents nano-emulsions, E represents macro-emulsions)
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Fig.8 The Ternary Phase Diagram of Span 80-Tween 80-Glycerin/Water/Mineral Oil System
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after 30 Days. (The ratio of Span 80-Tween 80: glycerin was 2.1:7.9, M represents micro-emulsions, N represents nano-emulsions, E represents macro-emulsions, W represents water-soluble component)
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Fig.9 Sample Appearance Obtained from Span 80-Tween 80-Glycerin/Water/Mineral Oil System after Storage for 30 days
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Graphical abstract
Highlights ·Discussed the entire process of D phase emulsification method.
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· Investigated the phase behavior by means of pseudo-ternary phase
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diagram.
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·A possible emulsification mechanism was preliminarily proposed.
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Wanping Zhang, Yubo Qin, Zihao Gao, Wenhua Ou, Haiyang Zhu, Qianjie Zhang PII: DOI: Reference:
S0167-7322(18)36234-2 https://doi.org/10.1016/j.molliq.2019.04.112 MOLLIQ 10835
To appear in:
Journal of Molecular Liquids
Received date: Revised date: Accepted date:
2 December 2018 9 April 2019 22 April 2019
Please cite this article as: W. Zhang, Y. Qin, Z. Gao, et al., Phase behavior and stability of nano-emulsions prepared by D phase emulsification method, Journal of Molecular Liquids, https://doi.org/10.1016/j.molliq.2019.04.112
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ACCEPTED MANUSCRIPT Phase Behavior and Stability of Nano-Emulsions Prepared by D Phase Emulsification Method Wanping Zhanga, Yubo Qina, Zihao Gaoa, Wenhua Oua, Haiyang Zhub, Qianjie Zhanga,* a
School of Perfume and Aroma Technology, Shanghai Institute of Technology, Shanghai,
People’s Republic of China b
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Shanghai Ruxi Bio-Tech Co., Ltd, Shanghai, People’s Republic of China
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Corresponding author: Name: Qianjie-Zhang
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Email: [email protected]
Address: School of Perfume and Aroma Technology, Shanghai Institute of Technology, No.
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100, Haiquan Road, Fengxian District, Shanghai 201418, People’s Republic of China
ACCEPTED MANUSCRIPT Abstract: D phase emulsification method was applied to prepare nano-emulsions in the Span 80-Tween 80/glycerin/water/mineral oil system. The phase behavior of this system during the entire emulsification process and the stability of the prepared emulsions were investigated by means of a pseudo-ternary phase experimental design. A possible emulsification mechanism was preliminarily proposed on the basis of our experimental results. It was found that the special self-assembled structure of the D phase and its capability for oil phase were the crucial factors in the D phase
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emulsification. Firstly, the surfactant alignment of the D phase was beneficial to the formation of small oil droplets in the O/D phase. Then, a quick movement of the
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surfactants toward the oil-water interface was critical for preparing nano-emulsions. Furthermore, the stability of the nano-emulsions during storage (30 days) was
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predominantly related to the addition amount of surfactants. The higher the content of surfactant in the system, the more stable the nano-particle interface film was formed.
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The stable interface film avoids the aggregation of the particles due to the thermal motion of the molecules, and the nano-particles could remain stable during storage.
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Keywords: D phase emulsification; nano-emulsions; phase behavior; stability Abbreviations:
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PIT: phase inversion temperature
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PIC: phase inversion concentration EIP: emulsion inversion point
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HLB: lipophilic-hydrophilic balance Acknowledgements:
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The research was supported in part by the Shanghai Gaofeng & Gaoyuan Project for University Academic Program Development （1021ZK183011005）and Shanghai Local Capacity Building Projects (19090503600).
ACCEPTED MANUSCRIPT 1.INTRODUCTION Nano-emulsions, also called submicron emulsions [1-2], mini-emulsions [3-4], ultrafine emulsions [5-6] in the literature, are a type of emulsions with uniform and extremely small
droplet
size
in
the
range
20-500nm.
Comparing
with
macro-emulsions, nano-sized particles have less inter-molecular attraction and exhibit higher stability in kinetics against coalescence and sedimentation, hence offering
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increased stability to some extent [7]. The process of preparing nano-emulsions can be roughly divided into two
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categories: high-energy emulsification and low-energy emulsification. The former method works by doing work on the emulsion system, and the emulsion particles are
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broken to the nano-meter level by high-speed stirring, homogenization, or ultrasonication [8-10].
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On the other hand, the low-energy emulsification depends on the spontaneous movement of the surfactants and the formation of low interfacial tensions during the emulsification process. In some literature, the low energy emulsification method is
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simply divided into two categories. One is based on the phase inversion during the emulsification process. The other low-energy emulsification method is the spontaneous emulsification method [11]. Representative low energy emulsification
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methods emcompass phase inversion temperature (PIT) emulsification [12-13], phase
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inversion concentration (PIC) emulsification [14-15], emulsion inversion point (EIP) emulsification [16-17], D phase emulsification method and so on. For nonionic surfactants, PIT methods can be achieved by changing the temperature of system
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which essentially varied the lipophilic-hydrophilic balance (HLB) of the surfactants. Instead of temperature, PIC and EIP methods can be obtained by changing the water
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volume fraction with continually adding water into oil. The emulsification system passes through a relatively viscous state and crosses a point of zero spontaneous curvature, then the fine nano-size particles can be formed. However, these methods are well known for short-chain surfactants with strict requirement for HLB value and don’t work well for most of low-polarity oils. Except these phase inversion emulsifications, homogenous O/W nano-emulsions can be prepared by going through the D-phase which surfactants have self-assembled structure by the intervention of polyol during the emulsification process. Sagitani et al. proposed the method to prepare fine O/W emulsions which called D phase emulsification since the fine O/W emulsions were obtained by diluting an oil-in-surfactant gel emulsion (O/D gel emulsion) comprise of oil droplets surrounded by surfactant phase (D phase) in their
ACCEPTED MANUSCRIPT experiments [18]. Comparing with other low-energy emulsification methods, D phase emulsification is easy to prepare small and narrow distribution emulsion particles. It can emulsify oils with low polarity, small surface tension and poor water solubility. And it is not necessary to control the emulsification temperature and HLB value. Generally, the specific procedure of D phase emulsification is showed as Fig.1. Nonionic surfactant and polyol solution were completely dissolved as one uniform phase (D phase), and then oil was added dropwise to the surfactant solution with
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stirring. An oil-in-surfactant clear gel was formed by dropwise addition of a certain amount of oil. Then, the fine oil-in water emulsion can be obtained by dilution of the
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gel with water. It has been pointed out in literature that the formation of gel phase is the key point to prepare nano-emulsions by D-phase emulsification methods [19].
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Research speculate that it is related to the special arrangement of surfactant, oil and polyol solution, and the dynamic process of its movement determines the formation of
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nano-emulsions. Even the appearance of gel may not be observed, the components should spontaneously undergo the special
form
uniform
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nano-emulsions [20].
arrangement to
In recent years, a number of explorations and attempts were made in preparing nano-emulsions by D phase emulsification method. Most of the research has focused
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on the effect of polyols, saccharide and other polyol-like humectant materials on the properties of surfactants, and therefore on the phase behavior of D phase
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emulsification. Murakami et.al studied the effects of sucrose, D-fructose and D-maltose on surfactant cloud point and D phase emulsification process. It was found that all three kind of sugars can reduce the cloud point of surfactant, enhance its
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hydrophobicity and then influence the emulsification process [21]. Miyahara et.al have prepared nano-emulsions which particle diameter were less than 100 nm by D
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phase emulsification method using nonionic surfactant, EPDME (a novel humectant), water, and mineral oil [22]. Nevertheless, few literatures focused on the phase behavior of entire D phase emulsification process, and the stability of prepared nano-emulsions during the storage. In this paper, the phase behavior of the Span 80-Tween 80/glycerin/mineral oil, Span 80-Tween 80-glycerin/water/mineral oil system in the entire emulsification process was comprehensively investigated by pseudo-ternary phase diagram. The mechanism of the D phase emulsification was expanded by contrasting the phase change during emulsification. In addition, the stability of prepared nano-emulsions was also discussed.
ACCEPTED MANUSCRIPT 2. Experimental 2.1 Materials Span 80 (sorbitan monooleate, HLB=4.3) and Tween 80 (polyoxyethylene sorbitan monooleate, HLB=15.0) were purchased from Sinopham Chemical Reagent Co.,Ltd. The ratio in weight of Span 80 and Tween 80 used was 3:5 (HLB=11). Glycerin and mineral oil were obtained from Infinitus Co.,Ltd and BASF Corp
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respectively. Deionized water was used to prepare all emulsions.
2.2 Preparation of nano-emulsion by D phase emulsification
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Span 80 and Tween 80 were mixed at 3:5 in weight, and then the surfactants were mixed with glycerin at specific ratio which followed by slowly adding mineral
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oil with stirring at 80℃. Stirring lasted for 30min to produce a fine O/D phase. For O/W emulsion preparation, gradual addition of water to the O/D phase was carried out prepared. The process is shown in Fig.2.
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2.3 Pseudo-ternary phase diagram
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with stirring. At specific proportion of oil and water, fine nano-emulsion can be
Samples differing in composition were placed in sample bottle which were then sealed and stored in a thermostat bath at 25℃. A pseudo ternary phase diagram was
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drawn according to the phase state of the sample which was just after prepared and stored after 30 days. Phase state was confirmed by visual observation, conductivity
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measurement, microscopic observation, particle size measurement, and centrifugal stability investigation at 25℃.
Since the electrical conductivity of oil and water was significantly different,
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conductivity measurement (DDS-12DW LIDA) can effectively identify the type of continuous phase produced by emulsification in the experiments involving emulsion
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phase inversion. Microscopic observation (BX-53 Olympus) and particle size measurement (Nano-ZS Malvern) were used to observe the size of the particles, then the macroemulsions and the nano-emulsions could be distinguished. In addition, centrifugal stability (TGL-16aR) of emulsions was also used to distinguish microemulsions from nano-emulsions in the phase diagrams. 2.4 Determination of particle size and polydispersity index Particle size (z-average diameter) were measured using Malvern Zetasizer Nano ZS (Malvern Instruments, UK) at 25℃ by scattering light. The values of the mean diameter were the averages of results obtained for three replicates. 2.5 Transmission electron microscopy
ACCEPTED MANUSCRIPT Nanoemulsions were observed by negative-staining electron microscopy (FEI Tecnai 12) as a direct measure of the droplet size and shape. To perform the TEM observation, a drop of the nano-emulsion was directly deposited on the 300 mesh copper grid and the natural sedimentation lasted for 10 min. Then the sample was negatively stained by floating the grid face-down on a drop of 2% (w/v) phosphotungstic acid for 2 min and dried for 10 min. The images of the sample were obtained by observing the grids in the transmission electron microscope at an
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acceleration voltage of 120 kv. 2.6 Statistical analysis
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their means and standard deviations were reported.
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All measurements were performed on at least three freshly prepared samples and
3. Results and Discussion
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3.1 Phase Behavior of Span 80-Tween 80/Glycerin/Mineral Oil In order to examine the entire process of D phase emulsification clearly and
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intuitively, the phase behavior of the O/D phase formation process was first investigated.
In the Span 80-Tween 80/glycerin/mineral oil (Span 80:Tween 80=3:5) system,
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lipophilic emulsifier Span 80 and hydrophilic emulsifier Tween 80 formed specific
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mixed, oriented, self-assembled structure in glycerin during the emulsification process. The O/D phase formed by adding mineral oil to the surfactant glycerin solution exhibited by different appearance depending on the ratio of added raw materials.
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Pseudo-ternary phase diagram of Span 80-Tween 80/glycerin/mineral oil system were constructed (Fig. 3). Since the polyol used in this system was glycerin (100%), which means there was no water participated in the preparation process of O/D phase, the
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phase region obtained during this preparation process was relatively simple. The prepared O/D phase was divided into different regions and marked as ‘a’, ‘b’, and ‘c’. The sample photos in Fig.3 shows the different appearance of each region. The amount of oil added in ‘a’ region was low and they could be solubilized. Samples in this region presented the same color as surfactants. Few oils added in the samples were solubilized or loaded in the structure formed by surfactants and glycerin. Samples prepared in ‘b’ zone were uniform and translucent. With the increase of oil volume fraction in the system, the surplus oil was produced (‘c’ area). Samples obtained in ‘c’ region were not stable and sedimentation phenomenon occurred immediately after stirring stopped. However, it was found that most samples of ‘c’
ACCEPTED MANUSCRIPT areas could obtained nano-emulsions by diluting with water in subsequent procedure, but the formation and stabilization of nano-emulsions should be discussed separately. It was worth noting that the typical gel emulsion was not observed under our preparation condition which due to the absence of water. Therefore, it is the oriented arranged structure of D phase required stirring constantly during adding oil to supply energy to prompt the forming of small oil drops, which is also the indispensable for nano-emulsions preparation.
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3.2 Phase behavior of Span 80-Tween 80-Glycerin/Water/Mineral Oil System
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To investigated the effect of different O/D phase on the formation of nano-emulsions, different ratio of surfactants and glycerin in weight (two lines in
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Fig.3 marked as ①and ②) were chose to prepare through same emulsification process. The chosen ratio of Span 80-Tween 80 to glycerin (①7.5:2.5 in weight and ②2.1:7.9
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in weight) can coverage the different region of the O/D phase, and the phase behavior of each system were observed. Pseudo-ternary phase diagram of the initial system
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after preparation (Fig.4,7) and after 30 days (Fig.6,8) were obtained respectively. Following the addition of water to the starting O/D structure (‘a’ and ‘c’ regions in Fig.3), micro-emulsions (M), gel emulsions (P), and nano-emulsions (N) were
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prepared while the mineral oil addition ration is less than 75%, which indicated by the filled circles in Fig.4. As shown by the dashed line in Fig.4, micro-emulsions with
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ultrafine particles and transparent appearance were obtained when the added water was less. Semitransparent appearance of the microemulsions benefits from the fine emulsion particles [23]. For example, the particle size at the star label of M region in
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Fig. 4 was 52.35±0.78nm. Transmission electron microscopy (TEM) investigations of the microparticles (Fig.5a) showed the typical appearance of the microemulsions in
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the M area. Comparing with the M region in Fig.6, it could be found that the micro-emulsions were thermodynamically stable system, and there was no instability phenomenon such as creaming or sedimentation occurred during storage. As the addition weight of water increase to 20%~40%, gel emulsions were prepared. Samples in this region showed high viscosity thus perform favorable stability in the process of storage. Continuing increasing the water fraction to 50%, nano-emulsions were formed. The particle size at the star label of N region in Fig.4 was 143.47±2.03nm. The morphology and structure of the prepared sample were also studied using TEM investigations and the result was shown in Fig.5b. As mentioned in the precious section, the O/D phase is a special self-assembled structure formed by
ACCEPTED MANUSCRIPT surfactants and glycerin and oil is dispersed as fine particles in the arrangement. While adding water to the O/D phase, the surfactants move rapidly to the oil-water interface and forming ultrafine O/W emulsions. In the dynamic process of particle motion, the movement speed of mineral oil should be lower than the movement speed of the surfactant, hence the oil droplets can be wrapped by continuous water phase before the coalescence. By comparing the phase behavior in Fig.3 and Fig.4, it was found that the ‘a’ and
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‘c’ region didn’t have strict correspondence with the ‘M’, ‘P’ and ‘N’ region. It indicated that the surplus oil in O/D structure didn’t have obviously effects on the
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transformation of the O/D phase to the O/W emulsion. As described above, although the O/D structure formed during the emulsification process was unstable, and
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sedimentation phenomenon occurred immediately in the prepared O/D samples after the stirring stopped. However, during the preparation of O/W, water was added under
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agitation, and the oriented O/D phase still existed. The added water can quickly surround the dispersed oil droplets to form fine nano-emulsions.
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At the initial system after preparation, W/O emulsions were formed due to the high oil phase weight ratio (E(W/O) region in Fig.4). But considering the proportion of emulsifiers in the system (the weight ratio of lipophilic emulsifier Span-80 was less
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than hydrophilic emulsifier), formed W/O structure was not stable. During storage, samples finally changed to O/W emulsions and excess oil floated to the upper layer
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after 30 days. Furthermore, since nano-emulsions were thermodynamically unstable systems, ultrafine particles were easily aggregated by the influence of Ostwald ripening to cause instability [24-26]. Part of the nano-emulsions (N area in Fig.4)
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were creaming after storage (E(O/W) region in Fig.6). The upper layer of the samples was comprised of large particles which aggregated and coalesced from nano particles
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and the lower layer was still nano-emulsions. Though some samples were unstable due to the influence of Ostwald ripening, most of the prepared nano-emulsions remained stable during storage for 30 days. In this system, stable nano-emulsions can be prepared while the oil content below 80% and water content above 50 wt%. It was mainly benefit by the high proportion of surfactants in the system since there have enough surfactants in forming stable oil-water interface film [27]. Differing with the former case, the system with lower concentration of surfactants presented the polymorphism. More than five phase regions can be obtained by adding water to the samples through line ②in Fig.3. Micro-emulsions (M) were formed while the oil content below 20% and water content between 25% to 50%.
ACCEPTED MANUSCRIPT Comparing with the ‘M’ region in Fig.4, this region was narrower due to the lower surfactant concentration. Nano-emulsions formed in this system had a variety of particle size. The higher the emulsifier content, the smaller the particle size of the emulsions. While the surfactants and glycerin content were relatively low, macro-emulsions were prepared. With the decrease of oil content, W/O emulsions and O/W emulsions were obtained in sequence. However, since the weight ratio of surfactants to glycerin was low (2.1:7.9), the
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stability during 30 days’ observation of prepared emulsions was decreased. Except the ‘M’ region, all other regions occurred unstable phenomenon during storage (as shown
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in Fig.8), and the appearance of final samples were shown in Fig.9. It was notable that, the nano-emulsions prepared in this system showed different unstable results: oil slick
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(O+N); stratification (E+N); and maintained good stability (N). This phenomenon may be related to the different addition of water and oil in the system. By comparing
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the phase behavior in Fig.3, Fig.7 and Fig.8, it could be found that nano-emulsions could be prepared by diluting the most samples in ‘b’ area with water, but the stable
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nano-emulsions only can be formed while the amount of oil added was low and the amount of emulsifier or water added was high. Then oriented O/D phase was formed and the surfactants can maintain the stability of the nano-emulsions.
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In summary, it can be concluded that when the surfactant content in the system was decreased, the amount of oil that can be carried by the self-assembled structure of
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the D phase was also decreased, which due to the oil loading capacity of D phase was mainly related to the amounts of surfactants. Addition of oil beyond the D phase carrying capacity would easily lead to oil droplets aggregating and coalescing, and
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thus nano-emulsions were difficult to prepare.
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4. Conclusion
The entire process of preparing nano-emulsions by D phase emulsification method was discussed on the basis of Span 80-Tween 80/glycerin/water/mineral oil system. The self-assembled structure of emulsifiers formed in polyols plays a decisive role in the formation of nano-emulsions. Through the description of the ternary phase diagram, it could be found that the D phase emulsification preparation process was easy to produce nano-emulsions in this system, and the addition amount of the emulsifier plays a significant role in the stability of the nano-emulsions. Emulsifiers as a key factor in stabilizing the interface can protect nanoparticles against the coalescence caused by molecular thermal motion during storage for 30 days. At
ACCEPTED MANUSCRIPT higher surfactants/glycerin content, nano-emulsions can be prepared while the oil content below 80% and water content above 50 wt%. At lower surfactants/glycerin content, stable nano-emulsion can be prepared while the oil content below 25% and water content above 50 wt% during the 30-day inspection period. These experimental results contribute to deeper understanding of the process and mechanism of D phase emulsification, and also provide a theoretical basis for the preparation of
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nano-emulsions.
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Figure Name： Fig.1 Specific Procedure of D Phase Emulsification Fig.2 Process of the D Phase Emulsification Fig.3 The Ternary Phase Diagram of Span 80-Tween 80/Glycerin/Mineral Oil System at 25℃ after Preparation (‘a’, ‘b’, ‘c’ represent the O/D phase of different appearances) Fig.4 The Ternary Phase Diagram of Span 80-Tween 80-Glycerin/Water/Mineral Oil System
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at 25℃ after Preparation. (The ratio of Span 80-Tween 80: glycerin was 7.5:2.5, M represents the micro-emulsions, P represents gel emulsions, N represents nano-emulsions, E represents
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macro-emulsions)
Fig.5 TEM micrographs of the micro-emulsion (a) and the nano-emulsion (b).
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Fig.6 The Ternary Phase Diagram of Span 80-Tween 80-Glycerin/Water/Mineral Oil System after 30 Days. (The ratio of Span 80-Tween 80: glycerin was 7.5:2.5, M represents the
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micro-emulsions, P represents gel emulsions, N represents nano-emulsions, E represents macro-emulsions)
Fig.7 The Ternary Phase Diagram of Span 80-Tween 80-Glycerin/Water/Mineral Oil System
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at 25℃ after Preparation. (The ratio of Span 80-Tween 80: glycerin was 2.1:7.9, U represents the unstudy area, M represents micro-emulsions, N represents nano-emulsions, E represents macro-emulsions)
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Fig.8 The Ternary Phase Diagram of Span 80-Tween 80-Glycerin/Water/Mineral Oil System
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after 30 Days. (The ratio of Span 80-Tween 80: glycerin was 2.1:7.9, M represents micro-emulsions, N represents nano-emulsions, E represents macro-emulsions, W represents water-soluble component)
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Fig.9 Sample Appearance Obtained from Span 80-Tween 80-Glycerin/Water/Mineral Oil System after Storage for 30 days
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Graphical abstract
Highlights ·Discussed the entire process of D phase emulsification method.
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· Investigated the phase behavior by means of pseudo-ternary phase
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diagram.
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·A possible emulsification mechanism was preliminarily proposed.
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