Effect of salt concentration on n-Dodecane-in-brine nano-emulsions produced by phase inversion temperature method

____________________________________ *Corresponding author: [email protected]

Effect of Salt Concentration on n-Dodecane-in-brine Nano-emulsions Produced by Phase Inversion Temperature Method

Firoozeh Pourjavaheri-Jad1, Rajarathinam Parthasarathy1*, Nhol Kao1 and Yung Ngothai2

1 School of Civil, Environmental and Chemical Engineering

RMIT University, VIC 3001 Australia

2 School of Chemical Engineering The University of Adelaide, SA 5005 Australia

ABSTRACT

Nano-emulsions are a class of very small and uniform nanometric-sized emulsions that can be defined as transparent or translucent liquid dispersions composed of water, oil and surfactants. They are kinetically stable with mean droplet diameters ranging from 20 to 1000 nm. Nano-emulsions can be prepared by a number of high and low energy processes. This study focuses on the preparation of nano-emulsions using one of the low energy methods known as phase inversion temperature (PIT) method which depends on the changes in the solubility of polyoxyethylene-type non-ionic surfactant with temperature. The surfactant is more hydrophilic at low temperatures and lipophilic at high temperatures. PIT point is a temperature in between at which condition the interfacial tension between the oil and water phases becomes extremely low leading to the production of emulsions with very fine droplets. The present work concentrates on the effect of salt concentration on oil-in-water nano-emulsions produced by PIT method using salt concentrations in the range from 0 to 0.1 M. NaCl and KCl were used as the salts. PIT points for the salt solution were found using conductivity measurements. The size distribution and polydispersity of droplets in nano-emulsions formed at PIT point were determined using dynamic light scattering technique. The results specify that both NaCl and KCl salts had no major effect on PIT points possibly due to the narrow salt concentration ranges used in this study. Also the changes in the size and size distribution of droplets with change in salt concentration were not significant within the range of salt concentration used in this study.

INTRODUCTION

Nano-emulsions which are intermediate between normal emulsions and microemulsions are a class of very small and uniform nanometric-sized emulsions. They can be defined as transparent or translucent, similar to microemulsions, liquid and isotropic dispersions composed of water, oil and surfactants that are kinetically stable with mean droplet diameters ranging from 20 to 1000 nm (Anton et al., 2007; Ee et al., 2007; Gutiérrez et al., 2008; Izquierdo et al., 2005; Liew et al., 2008; Solè et al., 2006; Tadros et al., 2004 and Usón et al., 2004). Usually, the droplets in nano-emulsions have an average size between 20 and 200 nm and show narrow size distributions. As mentioned by Gutiérrez et al. (2008), the size range may vary depending on the systems. The terms submicron emulsions (SME), ultra fine emulsions, miniemulsions, nanoparticles, emulsoids, unstable microemulsions and submicrometer-sized emulsions are often referred to and used in the literature as synonyms for this type of liquid/liquid dispersions (Anton et al.,

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2007; Bouchemal et al., 2004; Fernandez et al., 2004; Forgiarini et al., 2001; Izquierdo et al., 2002; Liew et al., 2008 and Sadurní et al., 2005). The term nano-emulsion is increasingly used since it gives a clear idea of the nanoscale size range of the droplets, avoiding misinterpretations with other kinds of dispersions such as microemulsions (Sadurní et al., 2005). The potential applications of nano-emulsions are many. The ultra small droplet sizes, high kinetic stability, low viscosity and visual transparency of nano-emulsions make them very attractive for many industrial applications. Nano-emulsions can be prepared by a number of high- and low-energy processes. High-energy emulsification methods such as high-pressure homogeniser are energy demanding but they allow greater control of droplet sizes and large choice of compositions. On the other hand, low-energy emulsification methods take advantage of the energy stored in the system to promote the formation of small droplets. One of the low-energy methods is phase inversion temperature (PIT) method, which was introduced by Shinoda and Saito (1969). Between all the nano-emulsions preparation techniques, PIT method has been extensively used due to the low energy and surfactant usage (Fernandez et al., 2004; Izquierdo et al., 2002). This method makes use of the chemical energy stored in the components and leads to oil-in-water (O/W) nano-emulsions in the presence of a surfactant and was chosen to produce n-dodecane/brine nano-emulsions in this study. The PIT method depends on the changes in the solubility of polyoxyethylene-type non-ionic surfactant with temperature. The surfactant is more hydrophilic at low temperatures and lipophilic at high temperatures. PIT point is a temperature in between at which condition the interfacial tension between the oil and water phases becomes extremely low leading to the production of emulsions with very fine droplets. However the emulsions formed at the PIT point are extremely unstable and can collapse due to the rapid coalescence of droplets so a quenching process is required to make the emulsion stable (Shinoda and Saito, 1969). Nano-emulsions are stable against creaming and sedimentation because of their small droplet sizes but they are not stable against Ostwald ripening (Taylor & Ottewill, 1994; Solans et al., 2005). Salt is one of the additives used in emulsions prepared in industries. The addition of salt can have different effects on the stability of nano-emulsions depending on the concentration of salt used. A previous study by Srinivasan et al. (2000) focused on the effect of salt concentration on nano-emulsions prepared by high-pressure homogeniser and used particle size measurement to determine nano-emulsion’s stability. Recently Liew et al. (2008) have reported that the effect of added salt in the production of nano-emulsions has not been completely understood. They found that salt had no significant effect on PIT points and the mean size and size distribution of droplets formed in systems with 0 - 0.1 M NaCl are nearly the same. However, Wasan et al. (1988) have reported that phase inversion in emulsions can be achieved by tailoring the salt concentration. It can be seen from the above review, the role of salt in the production and stability of nano-emulsions is still not clear and therefore worthy of further investigation. This study focuses on the effect of salt concentration on oil-in-water nano-emulsions produced by PIT method and determining the optimum salt concentration. PIT points

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for NaCl and KCl solutions with different salt concentration were determined using conductivity measurement. In other words, the hydrophilic-lipophilic balance (HLB) temperature was determined using the electrical conductivity method. Dynamic light scattering technique was used to determine the drop size distribution and polydispersity index of droplets in the prepared nano-emulsions. Results from this study are useful to industries such as cosmetics, pharmaceuticals and food where many modern products are in the form of nano-emulsions.

MATERIALS

The formulation for the nano-emulsions prepared in this work is based on a study by Liew et al. (2008). Nano-emulsions were prepared using n-dodecane (99 % purity from Merck, Australia), a non-ionic surfactant, poly(oxyethylene)(4) lauryl ether (C12E4, Brij30) of technical grade (Sigma-Aldrich), and sodium chloride and potassium chloride (GR for analysis, 99% purity from Merck, Australia). Ultra pure water from Pall Cascada LS system was used to prepare the brine used as the aqueous phase of nano-emulsions. All reagents were used without further modifications.

METHODS

Phase inversion temperature (PIT point) determination

The hydrophilic-lipophilic balance temperature was determined using the electrical conductivity method which is based on the changes in the conductivity of the system as it changes from aqueous continuous system to oil-continuous system (Kunieda et al., 1996). In all experiments, the emulsions were prepared using 19.2 wt% n-dodecane, 4 wt% of C12E4 and the balance is water/brine. The brine used in this work was prepared by dissolving salt (NaCl/KCl) in water. The salt concentration used varied from 0.025 to 0.1 M. The maximum salt concentration was chosen based on the work done by Liew et al. (2008). For each run, 100 g of emulsion was prepared at ambient temperature (~20ºC). First, n-dodecane and C12E4 were mixed in a beaker using a magnetic stirrer. Following that, water/brine was added to the system and agitated for a minimum of 10 minutes to ensure thorough mixing. The mixture was then heated gradually on a hot plate while being stirred continuously. A thermocouple was used to measure the emulsion temperature which was used to control the heating rate of the hot plate. The conductivity of the system was measured continuously as a function of temperature. The HLB temperature was determined as the temperature at which the conductivity decreases sharply which corresponds to a phase inversion from oil-in-water to water-in-oil emulsion. This temperature is also defined as the phase inversion temperature (PIT), which is an average of the temperatures at the maximum and minimum conductivity values (Izquierdo et al., 2002). Producing nano-emulsions using PIT method

Oil-in-water/brine nano-emulsions were produced using a two-step procedure as done by Liew et al. (2008). The system containing oil, water/brine and surfactant was first heated to a temperature of up to 4 - 5oC higher than its PIT point using a hot plate while

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being continuously stirred by a magnetic stirrer. After reaching the aimed temperature, the heated mixture was rapidly cooled to 15oC (a rapid quench) by placing it in an ice bath while still being continuously stirred. All samples were then stored at ~20oC. Droplet size, polydispersity index and stability determination

The mean droplet size and polydispersity index (PDI) of the nano-emulsions were determined by dynamic light scattering (DLS) technique using an ALV-5022F FAST correlator and compact goniometer with a 633 nm red laser and a scattering angle of 90°. Reproducibility of particle size measurement was checked by measuring the mean particle size of different samples of the same emulsion and the percentage error was determined to be ranging from 1.4 to 1.7%. The stability of the nano-emulsions stored at 20oC was assessed by measuring the variations in droplet sizes and PDI with time over a period of 3 days. A sample volume of 5 µL was diluted with the salt solution having the same concentration as that in the nano-emulsion by a factor of 1:1000 and used in these measurements. PDI is an indication of the quality of the dispersion and is basically the number of species per unit of the emulsion. PDI of nano-emulsions can range from 0 to 1.0 but only emulsion with PDl < 0.2 is considered as monodispersed system and suitable for measurements (Liew et al., 2008). Low PDI also suggests a narrow particle size distribution. All measurements were done at 20°C. Nano-emulsions cryo-SEM analysis

Cryo-scanning electron microscopy (Cryo-SEM) technique was used to confirm the droplet size results obtained from DLS. In the sample preparation step, a tiny drop of a freshly made nano-emulsion containing 19.2 wt% of oil, 4 wt% of surfactant and pure water was placed on a sample holder using a pipette. It was then frozen immediately by immersing it into nitrogen slush (a mixture of solid and liquid nitrogen made in Gatan Alto 2100 slushing station). After 1 minute, the stub containing the sample was transferred under vacuum into the cryo-SEM sample preparation chamber (Gatan Alto 2100 cryo-SEM system attached to an FEI Quanta 200 ESEM scanning electron microscope with a Tungsten electron source, FEI UK Ltd., Cambridge, UK). The specimen was fixed at −140°C. Frozen samples were fractured using precision rotary knife inside the Gatan Alto preparation chamber. The sample was then transferred to the SEM where it was imaged while frost was sublimated. The sample was etched in the SEM sample chamber (10 min at –90°C) and transferred back to the Gatan Alto preparation chamber where it was sputter coated with gold (2 min, 11 mA) for about 10 - 15 nm thickness. The sample was then transferred back to the SEM chamber (kept at −140°C) and SEM images were recorded and examined with an accelerating voltage of 30 kV.

RESULTS AND DISCUSSION

Phase inversion temperatures

As mentioned above, the phase inversion temperature (PIT) was determined by taking the average of the temperatures at the maximum and minimum conductivity values. The PIT point also corresponds to the hydrophilic - lipophilic balance temperature of the

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emulsion system as suggested by Liew et al. (2008) and Izquierdo et al. (2002). PIT values for NaCl and KCl systems are shown in Table 1 and Figure 1.

05

10152025303540

0 0.02 0.04 0.06 0.08 0.1 0.12Salt Concentrations (M)

PIT

Poi

nts

(°C

)

NaCl KCl NaCl-Liew et al. (2008)

Figure 1: PIT points for nano-emulsion systems involving NaCl and KCl salt solutions

It can be seen that the PIT points for NaCl system compare well with the data obtained by Liew et al. (2008) for NaCl system. It is clear from the results that salt concentration has no significant effect on PIT values for both NaCl and KCl systems. In the case of systems with NaCl solution, the PIT values remain more or less constant with increase in salt concentration. On the other hand, for KCl systems, the PIT value decreases for the system with 0.025 M of KCl solution and then increases to reach a steady value. Theoretically, the addition of salt dehydrates the surfactant and making it more lipophilic. Therefore the solubility of the aqueous phase should decrease with increase in salt concentration thus depressing the PITs (Shinoda & Takeda, 1970; Kuneida et al., 1989). Although the depression of PIT point was observed for the system with 0.025 M KCl solution, it was not observed for NaCl system or systems with other KCl solutions. It is not clear at this stage why the PIT values for systems with NaCl and KCl (0.075 and 0.1M solutions) do not decrease with increase in salt concentration. Possibly a wider salt concentration range would indicate its effect better on PIT point and further studies are being carried out to confirm the effect of salt concentration.

Table 1: PIT points for nano-emulsion systems with 19.2 wt% n-Dodecane oil and

4 wt% C12E4 surfactant and NaCl/KCl solutions

Salt (M) PIT point (°°°°C)

0 31.5 0.025 NaCl 31.0 0.050 NaCl 32.0 0.075 NaCl 31.9 0.100 NaCl 32.8 0.025 KCl 27.5 0.050 KCl 30.5 0.075 KCl 32.5 0.100 KCl 32.5

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Storage and appearance of nano-emulsions

Freshly prepared nano-emulsions were white opaque in appearance with an opal shadow with extremely low translucency (Figure 2). However they were found to become dull with ageing. Also the storage temperature was found to play an important role on the stability of the emulsions. If the ambient temperature was greater than 20ºC, the samples were found to lose their opal shine a lot faster as compared to samples that were stored at 20ºC or below. These changes in shine and colour were clearly observed by human eyes. The optimum storage temperature for the emulsions prepared in this study was assumed to be about 20°C below the PIT point as mentioned by Ee et al. (2007). This temperature ensured stable emulsions with ultra-small droplet size and low polydispersity indices. Any departure in temperature from this optimum would result in an increase in droplet sizes, polydispersity and instability by Ostwald ripening.

Figure 2: Appearance of nano-emulsions (a) freshly made sample (b) aged sample

Cryo-SEM analysis of nano-emulsions

In order to prove that the dilution of emulsion for the purpose of DLS measurement does not affect the properties of nano-emulsions, cryo-SEM technique was used to determine the particle size in a sample without dilution (Figure 3). The particle size obtained from the cryo-SEM (average diameter of 125 nm) is found to vary ± 4 - 30 nm from the particle size data from DLS (average diameter of 129 nm) indicating that dilution does not affect the particle size measurement significantly. Formation and stability of nano-emulsions

The average droplet size (radius) and polydispersity index values for the various emulsions prepared in this study are shown in Table 2. The size distribution of droplet radius is shown in Figures 4a and 4b for NaCl and KCl systems, respectively. The particle size distribution curves for both systems show that the distributions are monomodal. For the freshly prepared emulsions (Day 1 samples), the mean radius of the droplets varies from 57-66 nm for NaCl systems and 53-66 nm for KCl systems indicating KCl systems leads to relatively smaller drop sizes. It is clear that the mean size of the droplets in Day 1 samples decreases with an increase in salt concentration for both salt systems. It is also clear that the droplet size for all systems increases with

a b

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ageing. The fastest growth rate is for NaCl systems containing 0.075 and 0.1 M solutions and KCl systems containing 0.025 M solution. Among the salt solution systems, 0.1 M NaCl solution system has the smallest droplet size (r = 57.35 nm) in day 1 but is the most unstable one as indicated by the largest increase in droplet size in day 2 (r = 115.13 nm) and day 3 (r = 155.05 nm). The system with pure water, however, shows the most stability with a drop size of 70.87 nm and PDI of 0.032 in day 3. Overall KCl systems show lower particle growth rate than NaCl systems. Also, this study shows that the system containing pure water has the smallest droplet growth rate after 3 days in comparison with NaCl and KCl systems.

Figure 3: Cryo-SEM of a freshly prepared nano-emulsion containing 19.2 wt% n-dodecane oil, 4 wt% C12E4 surfactant and the balance of pure water

Table 2: Average droplet sizes and PDl of nano-emulsions during 3 days of study at 20°C

Average droplet sizes (nm) PDI Salt (M) Day 1 Day 2 Day 3 Day 1 Day 2 Day 3

0 65.60 69.72 70.87 0.046 0.037 0.032 0.025 NaCl 65.08 100.75 113.17 0.073 0.071 0.197 0.050 NaCl 62.10 112.06 128.70 0.026 0.173 0.286 0.075 NaCl 61.53 113.80 133.77 0.060 0.198 0.386 0.100 NaCl 57.35 115.13 155.05 0.054 0.148 0.329 0.025 KCl 65.13 81.23 88.04 0.126 0.078 0.053 0.050 KCl 57.48 64.45 64.52 0.061 0.110 0.017 0.075 KCl 56.15 63.64 65.90 0.033 0.066 0.016 0.100 KCl 53.76 63.55 64.87 0.058 0.065 0.077

PDI values for all systems with 0 and 0.025 M solutions remained below 0.2 indicating the samples of these systems are relatively stable with ageing. The lower PDI values for the systems containing KCl solutions indicate that they lead to relatively stable emulsions after 3 days as compared to NaCl systems. Overall, the 3 days of ageing leads

Air Bubble

Oil Particle

Frozen water

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to decrease in the PDI results for both pure water and KCl systems, but not for NaCl system. These results were confirmed by the quick phase separation for NaCl systems after 3 days which was not noticeable for KCl and pure water systems. This phenomenon indicates that NaCl probably leads to greater dehydration of the surfactant which makes mixture lipophilic especially at higher concentrations. These results indicate that, therefore, pure water and KCl systems are preferred in the preparation of nano-emulsions.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

10 100 1000

Radius (nm) (Log Scale)

Vol

ume

(%)

0 M NaCl0.025 M NaCl0.050 M NaCl0.075 M NaCl0.1 M NaCl

Figure 4a: Particle size distribution for freshly prepared emulsions with 19.2 wt% n-dodecane and 4 wt% C12E4 containing 0 to 0.1 M NaCl

0.0

0.2

0.4

0.6

0.8

1.0

1.2

10 100 1000Radius (nm) (Log Scale)

Vo

lum

e (%

)

0 M KCl0.025 M KCl0.050 M KCl0.075 M KCl0.1 M KCl

Figure 4b: Particle size distribution for freshly prepared emulsions with 19.2 wt% n-dodecane and 4 wt% C12E4 containing 0 to 0.1 M KCl

CONCLUSION

Nano-emulsions were prepared by mixing 19.2 wt% n-dodecane and 4 wt% C12E4 with aqueous solutions containing NaCl or KCl solutions using phase inversion temperature method. The stability of the emulsions was studied using the particle size and PDI data obtained from DLS. The conductivity results suggest that both NaCl and KCl salts had no major effect on PIT points possibly due to the narrow salt concentration range (0 - 0.1 M) used in this study. Nano-emulsions with number average radius ranging from 57-66 and 53-66 nm have been produced for NaCl for KCl systems, respectively. The system without salt produces the most stable nano-emulsions. The system containing KCl solution shows lower drop growth rate as compared to that for NaCl system within 3 days of ageing. PDI values for both pure water and KCl systems decrease with ageing whereas those for NaCl system increase indicating pure water and KCl systems are relatively more stable than the NaCl system. Also the changes in the sizes and size

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distribution of droplets with change in salt concentration were not significant within the range of salt concentration used in this study.

ACKNOWLEDGEMENTS

We gratefully acknowledge Associate Professor Gary Bryant and Mr. Phil Francis from School of Applied Sciences (Physics Dept.) at RMIT University for their great assistance with DLS and cryo-SEM and SLS management at Pall Corporation for their support.

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BIOGRAPHY

Firoozeh Pourjavaheri-Jad has a B.Sc in Food Science and Technology from RMIT University, Melbourne. She has work experience in different food industries such as beverages, dairy, confectionary, spices and packaging. Currently she is working as a validation scientist in the pharmaceutical division of Pall Corporation and doing her Master of Engineering by research on the stability of nano-emulsions produced by phase inversion temperature (PIT) method at RMIT University, Melbourne Australia.