Date of experiment
12 March 2014

Title
The effects of differences characteristic of active ingredient in formulation of emulsion

Introduction
        An emulsion is mixture of two liquids that would not normally mix. That is to say, a mixture of two immiscible liquids. By definition, an emulsion contains tiny particles of one liquid suspended in another. Chemically, they are colloids where both phases are liquids. They are typically milky in appearance and the suspended material may be colloidal in nature.
       A classic example of an emulsion is oil and water when mixed slowly under vigorous stirring. However, when the agitation is stopped, the two liquids separate and the emulsion breaks down.
          Stable emulsions can be formed from two immiscible liquids when an emulsifier is used. Such emulsions do not separate out after a change in conditions like temperature or over time.

Objectives
     1) Determine the effect of HLB surfactants on emulsion stability.
     2) Examine the effects of physical and stability of the emulsion formulation with the use of different agent.

Apparatus
8 test tubes, 1 measuring cylinder 50 ml, 2 set pasture pipette and droppers, weighing boat, 1 set mortar and pestle, light microscope, microscope slides, 1 set pipette 5 ml and bulb, 1 beaker 50 ml, viscometer apparatus, water bath, refrigerator, vortex mixture tool

Materials
Mineral oil, distilled water, span 20, tween 80, larutan sudan III, acacia, syrup, vanillin and alcohol

Procedure
     1) 8 test tubes were labeled and straight line were drawn 1 cm from the bottom of each of the test tubes.
     2) Then, 4 ml of oil (Table 1) and 4 ml of distilled water were added into each of the test tubes.

     3) Span 20 and Tween 80 (Table 2) were drops into the mixture of oil and water in each of the test tubes. The test tubes were closed and the mixture was mix using Vortex mixture tool for about 45 seconds. The times taken for the interface to achieve the 1 cm line were recorded. The HLB values for each sample were determined.

     4) 1g of the emulsion that is formed in each test tube was weight in the weighing boat and Sudan III (0.5%) solution were drops to the emulsion. Dispersion of color in the samples were described and compared. A little sample for each of the test tubes was spread on a microscope slide and observed under a light microscope. The shape and size of globular that are formed were drawn and compared.

    5)  By using the wet gum method, a formulation of Mineral Oil Emulsion were prepared using the formula below:

          Wet gum method
           a)      Acacia is added to the mortar and triturated with water until smooth gum is formed.

           b)      The oil is added to the mixture while triturating continuously until a smooth emulsion is obtained.

       6)  40 g of emulsion that are formed was added into a 50ml beaker and homogenizing process was done for 2 minutes using a homogenizing device.

       7)  2 g of emulsion that are formed was taken before and after the homogenizing process and put into the weighing boat and labeled. A few drops of Sudan III solution was added into the emulsion and smooths it. The texture, consistency, shape, degree of greasiness and dispersion color of samples was described and compared under light microscope.
                                                                           
     8) Viscosity of the emulsion (15 g in 50 ml beaker) that are formed after the homogenizing was determined by using the viscometer equipment that has been calibrated at all times by using the "Spindle" type LV-4. The samples are then exposed to a temperature of 45 ⁰C (water bath) for 30 minutes and then at a temperature of 4 ⁰C (refrigerator) for 30 minutes. Viscosity of the emulsion after exposure to temperature cycles completed was determined and emulsion reaches room temperature (10-15 minute).

     9) 5 g of emulsion that have been homogenized was added into a centrifuge tube and it is centrifuged (4500 rpm, 10 minute, 25 ⁰C).  The resulting high separation was measured and the ratio of the separation was determined.

Results

result for mineral oil

       to calculate the HLB value, we use this formula:

         Emulsions contains materials of different densities.During the centrifugation process of emulsion at 4500rpm for 10 minutes, creaming, flocculation and coalescence will occur . Mineral oil has lower density (0.8/cm3)  than water (1.0g/cm3). The mineral oil droplets rise and concentrate, which appears at the top. Finally, the drops will coalesce to form a separate layer of oil on top from water causing phase separation.

Discussion

       The HLB of an emulsifier is related to its solubility. Thus, an emulsifier having a low HLB will tend to be oil-soluble, and one having a high HLB will tend to be water-soluble, although two emulsifiers may have the same HLB and yet exhibit quite different solubility characteristics.

       Generally, the emulsion of  will separate into two phases within shortest time if the surfactants (Span 20 or Tween 80) are not used in the formulation. This is due to the absence of the surfactant which play a role in dispersing the oily phase into the aqueus phase or otherwise. If a single susfactant is used in the emulsion formulation, the product produced is not so stable compared to the emulsion with combination of 2 surfactants.

           In fact, surfactant is used in formulating the emulsion to stabilise both the oil and aqueus phases which are not immiscible. Micelle consists of the hdrophillic head and hydrophobic tail. It will try to keep the hydrophobic drug particles in the core of the micelle with the tail points inward to the center of the micelle and the head will remain in the aqueus phase. The adsorbtion of the surfactant at the interface between the two immiscible phases will lower the interfacial tension, the process of emulsification will be made easier and the stability may be enhanced.

           However, there may be some experimental errors that can make the results inaccurate. The surfactants added are measured by drops which is not so accurate. There maybe also parallax errors when observing the phase separation in the test tubes. Different observers for different test tube may also resulted in inacurate result.

diagrams of mineral oil emulsion under light microscope 

       Sudan solution is used to show the shape and physical characteristic of oily emulsion. It can show the emulsion whether is oily-in-water emulsion or water-in-oil emulsion by comparing the amount of the globules in red color and the colorless globules. Sudan solution, which is red in color, is dissolved in the oily phase in the emulsion. This makes the oily globules red in color.

non-homogenized and homogenized emulsion

                                              Non-Homogenized Emulsion (magnification x10)

                                           Homogenized emulsion (magnification x10)

          Homogenization is a process of increasing the stability of existing emulsion by decreasing the size of droplets either by impact or shearing the emulsion further. During the homogenization process, droplet deformation occurs where the droplets of emulsion are broke up into smaller droplets and the surfactant that previously attach to the droplet is also separated from the droplet. Stabilization  takes place after homogenization where the surfactant will adsorp onto the surfact and prevent the small droplets from fusing back together.

        For non homogenous emulsion, the size of the droplets appears bigger than the homogenized emulsion due to the impact or shearing of the emulsion to smaller size.The average size of the droplets of on to the surfactant homogenized emulsion is less even than the droplet of homogenized emulsion. distribution of the droplets. The texture of a non homogenized emulsion is less creamy than the homogenized emulsion as the dispersed phase is less distributed compared to homogenized emulsion. The non homogenized emulsion is also less viscous than the non homogenized emulsion. The more even distribution of the dispersed phase in homogenized emulsion also made the colour of the homogenized emulsion to appear more even than the non homogenized emulsion. 

        

Plot and discuss

     i) Graph of sample viscosity before and after the temperature cycling vs the different content of Mineral oil.

        The exaggeration of the temperature fluctuations subjected to the emulsion in the normal storage conditions is used to compare the physical instabilities of the emulsion. The emulsion is subject to freeze-thaw cycling which involved heating the sample at 45°C for 30 minutes, freezing it at 4°C for 30 minutes and lastly let the sample to achieve room temperature before measuring its viscosity. From the results, it shows that the viscosity of emulsion increase after the temperature cycling. Viscosity of emulsion increase as the result of extensive droplet aggregation which may cause by a number of physicochemical mechanisms. First, when the emulsion was placed in the freezer, some of its water crystallized. This caused the dispersed oil droplets to come into closer proximity because the oil droplets were confined to the non frozen regions remaining in the aqueous phase. When there was no presence of sufficient free water to fully hydrate the oil droplet surfaces, the droplet–droplet interactions was forced closer together to effect coalescence to occur. Second, ice crystallization led to an increase in the ionic strength of the freeze-concentrated non-frozen aqueous phase surrounding the emulsion droplets. Third, it is possible that ice crystals formed during freezing may have penetrated into the oil droplets and disrupted their interfacial membranes. This allowed the oil droplets more prone to coalescence between them. Fourth, cooling may have caused some of the fat in the emulsion droplets to crystallize promoting partial coalescence due to penetration of a fat crystal from one droplet through the membrane of another droplet.

   ii) Graph of the viscosity difference (%) versus various amount of mineral oil

       In this experiment, emulsion containing 30ml of mineral oil shows the greatest difference in the viscosity, that is 409.62%. The smallest difference in viscosity, which is 1.95%, is shown by the emulsion containing 20ml of mineral oil. The higher the difference in viscosity, the less stable is the emulsion. From the results obtained in this experiment, emulsion III  is the most unstable emulsion while emulsion I is the most stable one. This is because when there is a higher oily phase present in an emulsion (emulsion with 30ml of mineral oil), the emulsion is more unstable as the oil droplets tend to aggregate especially after the freeze-thawing cycle. Theoretically, emulsion IV with 35ml of mineral oil should show highest viscosity difference. However, it only has 19.13% of viscosity difference, this show that error has occurred. 

Plot a ratio separation phase as a result of centrifugation process on different amount of mineral oil. Describe.

      Based on the graph above, the separation phase ratio decreases as the volume of mineral oil increase. When the separation phase ratio of the emulsion decreases, this indicate the stability of emulsion is higher as the emulsion is more stable from undergoing the process of creaming, flocculation, and coalescence through the centrifugation process. Less amount of phase separation will occur. This indicate the higher the amount of mineral oil, the lower the separation phase ratio of the emulsion, and the more stable of the emulsion, until it achieves the optimum amount of mineral oil or ratio of mineral oil with water in the emulsion.


What is the function of each ingredient used in the preparation of the emulsion? How is the usage of different ingredients would affect the physical properties and stability of an emulsion formulation?
        Mineral oil act as the active ingredient of the dosage form. Acacia act as emulsifying agent that can provide electrostatic between the oil droplets to prevent them from coalescence in the vehicle, making the emulsion to be more stable.Syrup functions to provide a sweet flavour as well as masking the unpleasant taste of mineral oil. Vanillin act as flavouring agent and make the emulsion taste better.Alcohol also act as emulsifierand stabilize the emulsion. Distilled water act as solvent and act as vehicle for all the ingredients of the emulsion.

Conclusion
The HLB values for the mineral oil are different from each other due to the differences of quantity tween 80 and span 20 that was added into the sample.  From the result of viscosity test, it shows that the viscosity of emulsion increase after the temperature cycling as the result of extensive droplet aggregation.
For the experiment of viscosity that involving different content of mineral oil, emulsion containing 30ml of mineral oil shows the greatest difference in the viscosity, that is 409.62%. The smallest difference in viscosity, which is 1.95%, is shown by the emulsion containing 20ml of mineral oil. For the experiment of separation phase ratio, the result that we obtained show that the separation phase ratio decreases as the volume of mineral oil increase.


References
1) Lab (7) Preparation of a pharmaceutical emulsion, Jordan University of Science and Technology, Faculty of Pharmacy,Department of Pharmaceutical Technology, Retrieved from http://www.just.edu.jo/~sfg/Lab72.htm

2. R. L.Earle, Emulsification,Unit Operation in Food Processing , Retrieved from http://www.nzifst.org.nz/unitoperations/sizereduction2.htm

3. Klein K., Stability Testing…So What’s Adequate?, Cosmetech Laboratories Inc., Retrieved from
http://www.zenitech.com/documents/Stability%20Testing.pdf

4. Emulsion Stability and Testing, Particle Sciences, Retrieved from
 http://www.particlesciences.com/news/technical-briefs/2011/emulsion-stability-and-testing.html