Laboratory Experiment on Emulsions: Study of the Effect of Osmotic Pressure on Double Emulsions Preparation

Abstract

Double emulsions are ternary systems commonly used in several disciplines in areas such as food technology, applied chemistry, chemical engineering, materials science, pharmacology and environmental science. In several courses related to these areas, the implementation of laboratory experiment is required to strengthen the knowledge acquired by students during the theoretical lessons. However, it is difficult to find published practical experiments in this field. This work presents a four-hour hands-on laboratory experiment in which students can easily formulate and prepare water-in-oil-in-water double emulsions for vitamin B₁₂ encapsulation. In this experiment, students can analyze the effect of the osmotic pressure produced by the addition of different NaCl concentrations in each aqueous phase, which could lead to the swelling and deswelling phenomena of the inner aqueous droplets and, therefore, affect the encapsulation efficiency of the formulated systems. The double emulsions are analyzed by the students in terms of size and encapsulation efficiency.

1. Introduction

In science education, laboratory experimentation plays a vital role in students’ learning, enriching their knowledge and potential by increasing their interest, attention, and critical capacity, while validating the theoretical knowledge they have acquired during their lessons [1,2]. Laboratory activities have a central role in science curriculum, as they are many benefits from engaging students in a science laboratory. Since the early 1960s, laboratory experiments in science education have been used to engage students in investigation, discovery, inquiry, and problem-solving activities [3]. In this sense, in some chemistry and technical degrees, a final degree project is also carried out in a research laboratory [4]. This type of methodology represents a source of promising new insights on research in science [5]. Furthermore, laboratory lessons can also contribute to the development of students’ manipulation, observation, cooperation, and social communication skills [6]. Moreover, a laboratory environment also offers a more pleasant environment, which is often less monotonous and less tense than that of a theoretical class environment.

Due to the wide applications of emulsions and other typical colloidal systems such as vesicles, hydrogels, colloidosomes, and hollow particles, the preparation and formulation of these systems are studied in several types of degrees and master’s programs related to chemistry, chemical engineering, food technology, and pharmacology.

One of the eligible subjects of the Master in Chemical Engineering at the University of Oviedo (Spain) is Emulsions and Suspensions Technology, in which general knowledge about the synthesis and preparation of emulsions is explained during theoretical lessons. This is intended to strengthen the connection between the formulations learned in class and the manual skills acquired in the lab, by developing one of the three experiments performed in the 10 h laboratory classes as part of the 34 h design of the eligible subject. Moreover, one of the theoretical hours is used for an introduction to the lab experiments, explaining the theoretical background, materials, and methods.

Due to the scarce information regarding the didactic material for colloidal systems, it is difficult to find practical experimental lab designs [7]. This article presents one of the laboratory experiments designed for students to acquire practical knowledge about the formulation of water-oil-water (W₁/O/W₂) double emulsions for the encapsulation of a bioactive compound (vitamin B₁₂) and the stability/instability mechanisms involved.

Different formulations were tested using sodium chloride (NaCl) to study the importance of the osmotic pressure equilibrium between both aqueous phases for the stability and release of the formulated W₁/O/W₂ double emulsions. Each formulation contains exactly the same number of stabilizers and water/oil ratios. However, the NaCl concentration in each aqueous phase differs from one formulation to another, which affects the biocompound encapsulation efficiency of the formulated double emulsions.

2. Theoretical Background

An emulsion is defined as a two-phase system consisting of at least two immiscible liquids with different compositions: one of them dispersed as drops in the other [8,9]. The drop-shaped liquid is called the dispersed phase or inner phase, whereas the liquid in which these drops are suspended is called the continuous phase or external phase. The structure of an emulsion is highly dependent on the volume fraction of the water, the oil, and a third component called the emulsifying agent or emulsifier, as well as the nature of the interfacial film. The most common situation corresponds to the simple emulsions formed by two liquids, one hydrophilic (water) and the other hydrophobic (oil).

Emulsions play an important role in the formulation of foods for the production of simple oil-in-water (O/W) emulsions (e.g., dressings or artificial milks) as well as for the preparation of water-in-oil (W/O) emulsions (e.g., margarine and low-fat spreads). Some emulsions are final products (e.g., coffee creamers), and other emulsions can be used as ingredients, helping to form the structures of more complex products (e.g., yogurts that must interact with other food ingredients but should not be destabilized in the process). However, emulsion droplets can also create new structures in a product (i.e., ice creams) where emulsion destabilization is required for this purpose [9].

Emulsions also have the ability to transport or solubilize hydrophilic or hydrophobic substances in a water phase, in which it is interesting to encapsulate bioactive compounds since these substances (vitamins and antioxidants, among others), presented as natural constituents in food, provide health benefits beyond the basic nutritional value of the product.

One of the most common type of emulsions used for encapsulation are multiple emulsions, which were first reported in 1925 by Seifriz [10]. The simplest multiple emulsions are double emulsions: they are ternary systems where the dispersed droplets contain smaller droplets of a different phase. They have either a water-in-oil-in-water (W₁/O/W₂) structure, where W₁ is the internal aqueous phase, O is the oily phase, and W₂ is the external aqueous phase, or an oil-in-water-in-oil (O₁/W/O₂) structure [8], where O₁ is the internal oily phase, W is the intermediate water phase, and O₂ is the external oily phase. The former (W₁/O/W₂) is the most commonly used type of double emulsions.

Double emulsions are complex systems with higher instabilities than simple emulsions due to their large and different interfacial areas. In addition to the conventional Ostwald ripening and the coalescence or creaming phenomena observed in simple emulsions, other instabilities related to swelling or deswelling can be observed in double emulsions. The swelling phenomena is related to the fact that the inner aqueous drops grow due to the migration of the external aqueous phase through the oil drops [11,12]. On the contrary, the deswelling phenomena is related to the migration of the inner water drops to the external aqueous phase, which decreases the concentration of the internal aqueous droplets and reduces their size (Figure 1).

3. Lab Experiment

This lab experiment was performed in a 4 h session. The work was done in groups of two or three students each. Before the 4 h session, a lesson on emulsion theory was explained. Moreover, a one-hour session was included, where practical information was presented about the lab experiment.

3.1. Emulsions Preparation

The procedures used to prepare double emulsions and the selection of the components are similar to those used in previous research works [13,14,15], where satisfactory results were obtained in terms of stability, particle size, and encapsulation efficiency (EE). First, the three separate phases must be prepared: the internal aqueous phase (W₁), the oily phase (O), and finally the external aqueous phase (W₂). Vitamin B₁₂ was selected as the biocompound to be encapsulated, since it is not an expensive material, it is feasible to be detected by conventional spectrophotometry techniques, and satisfactory results were obtained when it was encapsulated with these types of colloidal systems in previous studies [15]. The experimental protocol is described in detail in the student experimental handout (Appendix A).

3.1.1. Dispersed and Continuous Phases Preparation

W₁ phase (100 mL) was prepared with 0.5 M NaCl and 2500 mg/L vitamin B₁₂ in MilliQ water. The oily phase (250 g) was composed of Miglyol^® 812 (density 945 kg/m³ at 20 °C, Sasol GmbH, Hamburg, Germany) and 5% w/w of polyglycerol polyricinoleate (PGPR, C₂₁H₄₂O₆, Brenntag AG, Essen, Germany), which acts as stabilizer of the W₁/O emulsion. Miglyol^® 812 is a neutral oil formed by esters of caprylic and capric fatty acids and glycerol. PGPR was prepared by the esterification of condensed castor oil fatty acids and is a powerful water-in-oil emulsifying agent commonly used with limited concentration in food formulations; it is highly effective for stabilizing W₁/O emulsions, as demonstrated by several studies [11,12,13,14,15,16]. The critical micelle concentration (CMC) value for PGPR was estimated in previous studies by the Wilhelmy plate method and was found to be between 0.76 and 1.5% w/w in the oily phase [17]. PGPR and Miglyol^® 812 were previously mixed under magnetic stirring for 30 min to ensure total solubilization of the stabilizer in the oil.

The W₂ phase (100 mL) consisted of MilliQ water and 2% w/v of Tween 20 nonionic surfactant (polyoxyethylene sorbitan monolaurate, Sigma-Aldrich, St. Louis, MO, USA) that acts as stabilizer of the O/W₂ interface. Tween 20 is a polyoxyethylene sorbitol ester that belongs to the polysorbate family, and its CMC is 60 mg/L.

Three different salt (NaCl) concentrations were used in the external aqueous phase W₂ to study the effect of osmotic pressure equilibrium between both aqueous phases: 0.1, 0.5, and 1 M. Each laboratory group of two students prepared a different concentration. All W₂ components were previously mixed under magnetic stirring for 10 min.

3.1.2. W₁/O Emulsion Preparation

The preparation of the internal emulsion (W₁/O) was performed in glass vessels by high-shear mixing (Silentcrusher M Homogenizer, Heidolph, Schwabach, Germany) using a 6 mm dispersing tool at 20,000 rpm for 5 min. The final emulsion had a total volume of 30 mL, with a volumetric W₁/O ratio of 20/80.

3.1.3. W₁/O/W₂ Double Emulsions Preparation

The primary W₁/O emulsion was used as the internal phase of the final double emulsion by redispersing it in an external aqueous phase (W₂). The ratio between W₁/O internal emulsion and W₂ was 20/80. For this second emulsification step, the Silentcrusher M homogenizer was also used but at a lower speed: 5000 rpm for 2 min. Emulsification was carried out in 15 mL graduated tubes.

Figure 2 summarizes the procedure described to prepare double emulsions by mechanical agitation in a two-step process.

3.2. Particle Size Characterization

Droplet size distributions of the prepared double emulsions were analyzed by laser light scattering using a Mastersizer S long bench apparatus (Malvern Instruments, Ltd., Malvern, UK). A refractive index of 1.54 was used. Samples were first diluted with deionized water to prevent multiple scattering effects.

Micrographs of the emulsions were obtained with an Olympus BX50 light microscope (Olympus, Tokyo, Japan) at 10–100× magnification using UV–Vis and fluorescence lamps, in order to confirm the particle sizes measured by the laser light scattering technique.

3.3. Measurement of the Clarification Layer

The clarification layer that appeared at the bottom of the graduated tube used for the second emulsification step was measured to determine the amount of W₂ present in the double emulsion and, therefore, whether a portion of W₁ was migrating to W₂. Samples were centrifuged at 10,000 rpm for 10 min to improve phase separation. This observation allowed students to evaluate the effect of the osmotic pressure on the difference in salt concentration between W₁ and W₂.

3.4. Encapsulation Efficiency Determination

The encapsulation efficiency (EE) of double emulsions is defined as the percentage of compound of interest in W₁ that remains in the primary emulsion (W₁/O) after the second emulsification step [18,19]. It can be calculated using Equation (1) [20].

$$EE=100-\frac{C_{recovered}\times 100}{C_0{R}_y}$$

(1)

The encapsulation (or entrapment) efficiency of the bioactive compound should be determined after separation of the external aqueous phase. To do this, the external aqueous phase must be previously recovered by centrifugation at low speed (1000 rpm for 20 min) and filtration with a 0.22 μm polyvinylidene difluoride (PVDF) syringe filter, to eliminate all the cream oily phase still present. Then, the concentration of biocompound of interest in the recovered aqueous phase (C_recovered) is determined by measuring the absorbance in the external phase (W₂), which is correlated with its concentration using a calibration curve. An increase in the absorbance indicates the release of the inner phase to the external phase. It is necessary to have previously prepared a calibration curve of absorbance vs. biocompound concentration (Appendix B).

However, the recovery yield (R_y) must be determined to measure if any amount of the biocompound of interest was retained (loss) during the centrifugation and filtration processes, to avoid an overestimation of the EE values obtained [18]. Therefore, a standard emulsion, where 100% of the W₁ is present in W₂, was required for its determination. For this purpose, an oil-in-water emulsion (O/W₂) was prepared using the same formulation as in the experiments. This O/W₂ emulsion was then diluted at the same ratio with W₁, which contained the appropriate amount of biocompound of interest. For this analysis, a blank reference also needed to be prepared, consisting of an O/W₂ emulsion diluted with W₁, in which the marker (biocompound) was not present. Finally, the R_y was calculated by measuring the C_recovered in the external aqueous phases using Equation (2):

$$R_y(\%)=\frac{C_{recovered}\times 100}{C_0}$$

(2)

where C₀ is the maximum concentration of biocompound expected in the external aqueous phase.

Vitamin B₁₂ concentration in the aqueous phase, C_recovered, was determined at 361 nm wavelength using a T80 UV/VIS spectrophotometer (PG Instruments Ltd., Wibtoft, UK). A calibration curve was previously calculated following the instructions detailed in Appendix B.

Differences in NaCl concentration of W₁ and W₂ phases affect the EE and the corresponding release [20,21]. Therefore, several formulations were prepared by modifying the salt content in both aqueous phases, and then EE for vitamin B₁₂ of the corresponding W₁/O/W₂ double emulsion was determined.

3.5. Hazardous

No hazardous materials are used in this lab experiment, so no additional requirement is needed.

4. Running the Experiment

The laboratory experiment is performed after 20 h of theoretical and practical exercise course so that the students have a background on the theoretical aspects of the formulation, preparation, and characterization of the emulsions. In addition to the theoretical concepts, the experiment is easy to run, and only basic lab skills are required. The student experimental handout used and the corresponding data collection sheet are detailed in Appendix A and Appendix C.

In the prelab hour, the procedure to be followed during the experiment is explained in detail to the students, indicating the instruments used, the concentration of each chemical, and the final amounts of the prepared simple and double emulsions.

Hence, before starting the lab experiment, the students must calculate the amounts of each chemical needed to prepare the W₁/O and W₁/O/W₂ emulsions. The experimental handout (Appendix A) is supplied to the students, as well as a data collection sheet (Appendix C) where they should indicate the amount of each component used for the preparation of each phase and emulsion.

Once each group has prepared the three necessary phases, the simple and double emulsions are formulated following the indicated procedure.

Double emulsions are characterized by laser light scattering in terms of mean droplet size and droplet size distribution. Microscopy is also used to assess the feasibility of the laser technique for droplet size measurement.

The formulated double emulsions are observed to evaluate the clarification layer of each emulsion after centrifugation takes place, to promote the migration of the oil droplets to the top of the graduated tube. This information could give the students an idea of the amount of W₁ that had migrated to W₂.

Once both aqueous phases are ready, each laboratory group should obtain the calibration curve for vitamin B₁₂ concentration by using spectrophotometry. Figure 3 shows an example of a calibration curve obtained at 361 nm.

Each group evaluated the concentration of vitamin B₁₂ in the external continuous phase (W₂) of the emulsion they had prepared. Centrifuged samples were used for this purpose. To ensure that the clarification aqueous layer was collected without any oily phase droplets, plastic tubes that can be easily pierced with a pin from the bottom were used to ensure no contamination.

The calibration curve was used to calculate the amount of free vitamin B₁₂ that was not encapsulated in the emulsion droplets. Figure 4 indicates the schematic protocol.

The Ry obtained after averaging the results achieved by all the groups of students had a value of 99.5 ± 1; being close to 100%, this value was similar to those previously reported [16,18].

5. Interpreting Student Observations

All of the experimental results obtained by each lab group were evaluated in the final report of each student, despite the fact that each group prepared one of the emulsions with a different salt concentration in the external aqueous phase.

The students used particle size measurements to study the effect of the difference in the salt concentration between both aqueous phases to evaluate the swelling and deswelling phenomena that occurred in the emulsions prepared by all the groups. Emulsions with a higher salt concentration in W₂ presented a lower mean droplet size value, caused by the deswelling of the W₁ droplets.

Figure 5 shows an example of the particle size distribution of the three double emulsions that were prepared.

Table 1. Mean particle size of oil droplets in double emulsions and absorbances obtained by analyzing the external aqueous phase in each case.

NaCl Concentration	D[4,3]	d(0.5)	Absorbance in W2
0 M	485.83 µm	83.81 µm	0.080
0.5 M	61.64 µm	54.49 µm	0.197
1 M	44.02 µm	43.97 µm	0.339

shows an example of the volume mean (D_[4,3]) and median (d_(0.5)) diameters of the oil droplets in the formulated double emulsions. D_[4,3] is the volume-weighted mean diameter, which is calculated by Equation (3):

$$D_{\left[4,3\right]}=\frac{{\displaystyle \sum }{n}_i{d}_i^4}{{\displaystyle \sum }{n}_i{d}_i^3}$$

(3)

where d_i is the droplet diameter, and n_i the number of droplets with diameter di; d_(0.5) means that 50% of the total droplets are smaller than this size, i.e., it is the median particle size.

The students could observe that the gently centrifuged samples (Figure 6) showed appreciable differences in their clarification layers. Furthermore, the students were able to easily identify the migration of W₂ to W₁ and vice versa, which was produced by the osmotic pressure induced by the salt concentration difference between both aqueous phases and was responsible for the swelling and deswelling phenomena.

Pierced tubes were used to collect W₂ from each sample and analyze it spectrophotometrically, in order to further calculate the concentration of vitamin B₁₂. Table 1 shows an example of the obtained results, where the increasing absorbance values were obtained as the concentration of NaCl on the continuous phase increased, indicating a lower EE of vitamin B₁₂.

6. Evaluation

After one week of running this experiment, each student presented an individual report, so their comprehension of the data obtained in the lab can be evaluated. The final report must contain the following items: (i) the goal of the experiment, (ii) a materials and methods section, (iii) the results and a discussion, and (iv) the conclusions. In this final report, the results of the particle size, particle size distribution, and calculations regarding the migration of W₁ to W₂ (or of W₂ to W₁) and the vitamin B₁₂ EE should be presented, analyzed, and discussed.

The work carried out by each student was evaluated in three sections: (i) pre-lab work regarding the calculations of the amounts of chemicals required for the preparation of emulsions (10% of the final grade); (ii) laboratory work, followed with care and precision, and demonstrated teamwork (40%); (iii) an individual report by the student (50%).

7. Conclusions

The implementation of lab experiments and interactive science laboratory experiments such as the one presented in this work can enrich the students’ interests in emulsions, suspensions, and colloidal fields. In addition, with this type of practical class, the knowledge of the concepts introduced in the theoretical sessions is enriched.

This experiment serves as an ideal candidate for providing students with laboratory skills training on colloidal systems.

Moreover, the presented experiment can be used as an open laboratory experiment that could also enrich the students’ critical ability, by using the problem-based learning model. In this case, the students must select the appropriate compounds to use in order to create osmotic pressure differences between both aqueous phases. Further, they must determine the optimal conditions to optimize the encapsulation efficiency of the biocompound.