Emulsion Lab Report

Group members:

Tan Mei Jie A144393
Cheng Chen Wei A144388
Loh Chen Mun A144409
Nur Syahirah bt Abd Manap A144395
Syahira binti Sabar A144381

TITLE : ASSSESMENT EFFECT OF DIFFERENT INGREDIENTS ON EMULSION FORMULATION

OBJECTIVES:

 1) To determine HLB surfactant on emulsion stability

2) To investigate physical effect and stability on emulsion formulation result from use of different  emulsified agents.

INTRODUCTION:

Emulsion is two phase system that unstable thermodynamically. it has at least two liquid that insoluble each other where one of its (internal phase)homogenously spread in the form of liquid in others (external phase). Emulsion is classified into two types : oil in water emulsion(o/w) or water in oil emulsion (w/o). emulsion is stabilized with addition of emulsifying agents. It can be divided into four types : 1) hydrophilic coloid 2) finely divided solids 3) surfactant.

One method, HLB method (hydrophjilic-lipophilic balance) is used to determine quantity and surfactant types that is needed to prepare one stable emulsion. each surfactant is labelled one number in scale from 1 (lypophililic) to 20 (hydrophilic). Normally, combination of 2 agents isused to prepare more stable emulsion. HLB value for combination emulsifying agents can be determine using following formula.

HLB value =

 (surfactant quantity 1)(surfactant HLB 1)+(surfactantquantiy 2)(surfactant HLB 2)

                                                 Surfactant quantity  1 + surfactant quantity 2

APPARATUS:

8 test tube , 1 measuring cylinder 50 ml ,2 pasture pipet and droppers, vortex mixture,weighing boat , 1 mortar pestle, light microscope, microscope slide, 1 set pipet 5 ml, 1 beaker  50 ml, 1 centrifuge tube 15 ml, coulter counter, centrifuge, viscometer, water bath (45 c), refrigator (4 c)

INGREDIENTS:

Coconut palm oil, arachis oil, olive oil,mineral oil, distilled water, span 20, tween 80, sudan 111 (0.5%), ISOTON 111

PROCEDURES

1. Each test tube was labelled and a straight line was drawn 1cm from the bottom of the test tube.

2. 4mL of the given oil (Schedule 1) for each group and 4mL of distilled water were mixed in the test tube

Group	Test Oil
1, 2	Palm Oil
3,4	Arachis Oil
5,6	Olive Oil
7,8	Mineral Oil

3. Span 20 and Tween 80 were dropped into the mixture. The test tube was sealed and left to the Vortex mixture for 45 seconds. The time taken needed to reach the interface of 1cm was recorded. The HLB value of each samples were determined.

No. of tubes	1	2	3	4	5	6	7	8
Span 20 (drops)	15	12	12	6	6	3	0	0
Tween 80 (drops)	3	6	9	9	15	18	15	0
HLB value	9.67	10.73	11.34	12.44	13.17	14.09	15	0

4. A few drops of Sudan III solution were added to 1g of emulsion that was formed on a weighing boat and flattened. The colour dispersion in the sample was determined and compared under the light microscope. The shape and size of the globules formed were drawn, explained and compared.

5. 50g (of the given oil) Emulsion was prepared by using the wet gum method and the following formula.

Arachis Oil	(Refer Schedule 1)
Acacia	6.25g
Syrup	5mL
Vanillin	2g
Alcohol	3mL
Distilled water (qs)	50mL

6. 40g of the formed emulsion was added into a 50mL beaker and homogenization process was formed for 2 minutes by using a homogenizer.

7. 2g of the formed emulsion (before and after the homogenization) was put on the weighing boat and labelled. A few drops of Sudan III solution was added and flattened. The texture, consistency, degree of the oily shape and colour dispersion of the sample was determined and compared under the light microscope.

 8. The viscosity of the emulsion (15g in 50ml beaker) which is formed after homogenization was measured using viscometer which is calibrated with “Spindle” Type LV-4. The sample was later being exposed to temperature of 45°C (water bath) for 30 minutes and later with temperature 4°C (refrigerator) for 30 minutes. The viscosity of emulsion after the completion of exposure to temperature cycle and the emulsion achieve room temperature (10-15 minutes) was measured.

For 20ml mineral oil:

Reading	Viscosity (cP)
	1	2	3
Before temperature cycle	369.9	379.9	359.9
After temperature cycle	389.9	359.9	369.9

For 25ml mineral oil:

Reading	Viscosity (cP)
	1	2	3
Before temperature cycle	670	700	690
After temperature cycle	1050	1150	1050

For 30ml mineral oil:

Reading	Viscosity (cP)
	1	2	3
Before temperature cycle	144.0	174.0	150.0
After temperature cycle	228.0	330.0	312.0

9. 5g of emulsion which had been homogenized was placed into a centrifuge tube and was centrifuged (4500rpm, 10 minutes, 25°C). The height of separation formed is measured and the ratio of separation height was measured.

	Height (mm)
Emulsion	I			II			III
Group	1	2	3	4	5	6	7	8
Separation phase	15	24	15	13	15	12	5	16
Original emulsion	50	44	50	50	45	50	50	43
Ratio of height	0.30	0.55	0.30	0.26	0.33	0.24	0.10	0.37

DISCUSSION:

1.      What are the HLB values to form a stable emulsion? Discuss.

In order to form a stable oil-in-water emulsions, HLB value should be in low number values which in the range of 8-18, for a stable water-in-oil emulsions, HLB value should be in high number values, in the range of 3-6. For the HLB value between 7-9, emulsifying agents normally act as wetting agent, while between 13-15, they act as detergents, and value of 15-16, they are become solubilizing agent.

Emulsion is a combination of water phase and oil phase, which is totally immiscible among each other. So, selection of an appropriate emulsification system depends on the active ingredients to be incorporated into the product and on the use of the final product. The balance between the hydrophilic and lipophilic properties of emulgent will affect the type of emulsion produced and this tends to be indicated by the relative solubility of the emulgent in polar and non-polar solvents. So, we use ‘hydrophile-lipophile balance’ (HLB) to select appropriate emulsifying agents for a particular system. An ideal emulsion which is stable contains smallest size sphere globules, they are in the closest packing between globules, and they have the largest separation distance or the most consistent color dispersion. Emulsifying agents in this case give maximum effect in solubilising oil phase and aqueous phase. Emulsifying agents are said to form a bridge between the two totally different phases, where the immiscible liquids are now able to ‘mix up’. These surfactants can reduce the surface tension of the interface between the oily and aqueous phase. As a result, the phase of separation will occur slowly. From our experiments, inconsistency of results happened because some of the errors we did during the experiment.

We can determine the stability of an emulsion more easily from the separation phase time. Emulsion which has the longest separation phase time is the most stable emulsion. A stable emulsion contains emulsifying agents added that able to mix and stabilize the two phases well for a very long time.

2.      Compare the physical structures for the turpentine oil emulsions formed and explain. What is the Sudan III Solution? Compare the colour dispersion in the emulsions formed and explain.

Sudan Red 3 test is used to test the presence of lipid in the sample. When the Sudan Red is added to a sample that contain the lipids, the dye will colour the lipid into red. Before homogenisation process, the size of oil globule are not consistent, there are small n large globule and some raw material are not well broken. The colour or the emulsion are not well distributed and some part are darker than the other part. The emulsion is more viscous and greasy. After homogenisation process, the size of oil globule are well consistent, the size of globule are equally small. The colour of the emulsion are well distributed and bright yellowish in colour. The emulsion is less viscous and good flow properties. This changes occur only after homogenisation process. Homogenisation process is the process to reduce the particles and disperse throughout the a fluid. It also used to make the uniformity of consistency.

3. Plot and explain the graph:

a)        Graph of viscosity of sample before and after temperature cycle against amount of mineral oil used.

For 20ml mineral oil:

Reading	Viscosity (cP)			Mean ± SD
	1	2	3
Before temperature cycle	369.9	379.9	359.9	369.9 ± 10.000
After temperature cycle	389.9	359.9	369.9	373.23 ± 15.275
Difference (%)	0.90

For 25ml mineral oil:

Reading	Viscosity (cP)			Mean ± SD
	1	2	3
Before temperature cycle	670	700	690	686.67 ± 15.275
After temperature cycle	1050	1150	1050	1083.33 ± 57.735
Difference (%)	57.76

For 30ml mineral oil:

Reading	Viscosity (cP)			Mean ± SD
	1	2	3
Before temperature cycle	144.0	174.0	150.0	156.0 ± 15.875
After temperature cycle	228.0	330.0	312.0	290.0 ± 54.443
Difference (%)	85.89

Therefore:

Amount of mineral oil (ml)	Average viscosity (cP) ( x ± SD)		Viscosity difference (%)
	Before temperature cycle	After temperature cycle
20	369.9 ± 10.000	373.23 ± 15.275	0.90
25	686.67 ± 15.275	1083.33 ± 57.735	57.76
30	156.0 ± 15.875	290.0 ± 54.443	85.89

In this experiment, three formulation of Mineral Oil Emulsion 50g are prepared by using different amount of mineral oil which are 20ml, 25ml and 30ml. After undergo homogenization, the viscosity of each emulsion is determined by using viscometer. Homogenization is done in order to decrease the globule size. After that, the sample will exposed to 45^oC temperature (water bath) for 30 minutes and then exposed to 4^oC temperature (refrigerator) for another 30 minutes. The viscosity of each emulsion after temperature cycle is determined after it achieve back to room temperature (around 10-15 minutes after temperature cycle) again by using viscometer.

Emulsions are thermodynamically unstable and all emulsions will eventually degrade and separate. The known mechanisms for emulsion degradation are separation (creaming) and oil particle coalescence. This experiment was intended to evaluate the use of temperature cycling as a method of stability testing for mineral oil emulsions.

Based on the graph, it shows that the viscosity of each emulsion will increase after undergo temperature cycle. Theoretically, viscosity of emulsion decreases with an increases temperature. This is because increase in temperature will increase the rate if creaming, owing to a fall in apparent viscosity of the continuous phase. The temperature increase will also cause an increase kinetics motion, both of dispersed droplets and of the emulsifying agent at the oil/water interface. This effect on the dispersed phase will enable the energy barrier to be easily surmounted and thus the number of collision between globules will increase. Increase in motion result in a more expanded monolayer and so coalescence is more likely.

However, when the temperature is decreases back, the viscosity of the emulsion will increase. This is because at low temperature the viscosity of continuos phase will increase and the kinetic energy of system will reduced. This will decrease the rate of migration of globules at disperse phase. Thus, the emulsion become more viscous.

However, based on the graph viscosity of 30ml mineral oil emulsion is lower compared with 20ml and 25ml mineral oil emulsion. This maybe because of wrong technique during handling the procedure. The viscosity of 30ml mineral oil emulsion should be higher than the other because of high oil content will make it more viscous.

b)       Graph of difference viscosity (%) against against amount of mineral oil used. 

 Based on the graph above, the viscosity difference will increase as the amount of mineral oil increase. This is because the higher amount of oil globules in the continuous phase will increases the viscosity of the emulsion. A higher disperse phase concentration would result in hindrance of movement of the droplets and hence reduction in rate of creaming. This also will slows down the phase inversion from occur. That’s why for 30ml mineral oil emulsion will have higher viscosity difference. The higher oil content will prevent the movement of globules thus make it more viscous. However, for 20ml mineral oil emulsion, it has lower viscosity difference because low oil content make the globules easily move in the continuous phase and hence the viscosity can be reduced.

4.      Plot a graph of ratio of separation phase against different amount of mineral oil. Discuss.

	Height (mm)
Emulsion	I			II			III
Group	1	2	3	4	5	6	7	8
Separation phase	15	24	15	13	15	12	5	16
Original emulsion	50	44	50	50	45	50	50	43
Ratio of height	0.30	0.55	0.30	0.26	0.33	0.24	0.10	0.37

Mineral oil (mL)	Ratio of separation phase (x ± SD)
20 (Groups 1, 2 and 3)	0.38 ± 0.14
25 (Groups 4, 5 and 6)	0.28 ± 0.04
30 (Groups 7 and 8)	0.24 ± 0.19

Based on the result obtained, the ratio of separation phase decreases as the amount of mineral oil used increases. However, theoretically, the ratio of separation phase will increase when the amount of oil used increases. This is because as the amount of oil increases, it exceeds the amount of oily phase which will then affect the formation of stable emulsion. So, more separation will be resulted.

Centrifuge is used to separate an emulsion into its aqueous phase and oily phase. After the centrifugation, the oily phase is above the aqueous phase. Ratio of separation phase indicates the stability of an emulsion. The higher the ratio of separation phase, the lower the stability of the emulsion. Therefore, a stable emulsion will have a low ratio of separation phase. Compared to a non-homogenous emulsion, a homogenous emulsion will not separate easily.

From the graph plotted, the result is not accurate. The emulsion produced by using 20 mL of mineral oil supposed to have a lowest ratio of separation phase. However, it showed a highest ratio of separation phase instead of a lowest ratio of separation phase. The situation is same in emulsions formed by using 25 mL and 30 mL of mineral oils. These errors may be due to the method of preparation of emulsion, that is, the wet gum method. May be some of the groups failed to produce good emulsions. This will definitely affect the stability of emulsion which will then affect the result of the experiment.

5.      What are the functions of each ingredient used? How these different ingredients affect the physical characteristics and stability of an emulsion formulation?

Acacia is an emulsifying agent which used to increase the viscosity among the interphase of the oily and aqueous phase. Since acacia is a natural product, this may cause it become a good medium for the microbial growth. Thus, agent antimicrobial such as benzoic acid 0.1% is added to stabilize the emulsion from microbial growth. It is different from the surfactant which reduce the surface tension.

Other than that, vanillin acts as flavoring agent which increases the taste of emulsion. While syrup act as sweetening agent and can be used to increase the viscosity of the emulsion. It also used to mask the unpleasant taste of the mineral oil so that the compliance of the patient is increase. However, we should take into account the quantity of syrup being used. The quantity of syrup is limited to avoid rheological problem and physical properties of the emulsion. This is to avoid the rheological problem in which the emulsion is maybe hard to pour out from the container and some of it may remain and form a layer surrounding the container wall.

Besides, alcohol is preservative which used to prevent microbial growth. Distilled water function as aqueous phase (continuous phase) in oil-in-water emulsion. Mineral oil form the dispersed phase in the oil in water emulsion (o/w emulsion). Some of the oil been used are different in color and this may give rise to different coloration of the emulsion we obtained. Furthermore, the oil such as palm oil has anti-oxidant properties which increase the chemical stability of the emulsion. If increase the quantity of oil exceed the quantity of water in aqueous phase will cause phase inversion. It is also the same for the quantity of water if exceed the quantity of oil will cause the same problem to occur.

CONCLUSIONS:

1. The emulsion stability increases as the HLB value increase. However, after reaching the optimum range of HLB value which is (9.67-11.34), the stability decreases. The droplet size decreases as the increasing HLB value. Then, it decreases after reaching the optimum range HLB values. The same goes for the interfacial force.
2. Homogenization functions to reduce the size of droplets in liquid-liquid dispersions as in this experiment is Mineral Oil Emulsion.
3. Increase in viscosity will increase time for the separation of emulsion into two layers.
4. For the phase separation after centrifugation of emulsion, the more the volume of mineral oil the lower the phase separation ratio.

REFERENCES:

1. Florence, A.T. & Atwood, D. 2011. Physicochemical Principles of Pharmacy. 4^th edition. London: Pharmaceutical Press

2.      Emulsion stability and testing http://www.particlesciences.com/docs/technical_briefs/TB_2011_2.pdf [24 May 2015]

3.      Aulton M.E Pharmaceutics The Design and Manufacture of Medicines, 3^rd ed; Churchhill Livingstone Elsevier: London, 2007

4. http://www.particlesciences.com/news/technical-briefs/2011/emulsion-stability-and-testing.html
5. http://www.lcpe.uni-sofia.bg/publications/2010/2010-10-SC-IL-KG-ND-SJ.pdf
6. http://journal.scconline.org/pdf/cc1968/cc019n10/p00683-p00697.pdf

APPENDICES:

The emulsion is prepared using wet gum method and is poured into a small beaker before homogenization.

Homogenization was carried out by a homogenizer.

The image of emulsion before homogenization under light microscope. 

The image of emulsion after homogenization under light microsope.

The emulsion is centrifuged and the height of separation is measured.