Title:
ASSAY ON THE EFFECTS OF DIFFERENCE IN CONTENT ON THE CHARACTERISTICS OF A EMULSION FORMULATION
Introduction:
Emulsion is a two-phase system which is not stable in thermodynamic. It contains at least 2 liquids which are not miscible to each other (internal/dispersed phase) dispersed homogeneously in another liquid (external/continuous phase). Emulsions can be classified into 2 types: oil in water emulsion (o/w) and water in oil emulsion (w/o). Emulsions become stable with the addition of emulsifying agents. Emulsifying agents can be classified into 3 types; they are (1) hydrophilic colloid, (2) finely divided solid, (3) surface active agents or surfactants.
HLB system (Hydrophilic – Lipophilic Balance) is used to determine the quantity and type of surfactant that is needed to prepare a stable emulsion. Each surfactant is given a number in HLB scale from 1 (lipophilic) to 10 (hydrophilic). Commonly, combination of 2 emulsifying agents is used to produce a more stable emulsion preparation. HLB value for combination of emulsifying agents can be determined using the formula below:
HLB value =
((Quantity of surfactant 1)(HLB of surfactant 1)+(Quantity of surfactant 2)(HLB of surfactant 2))
(Quantity of surfactant 1 +Quantity of surfactant 2)
Objective:
1. To determine the effect of HLB of surfactants on the stability of the emulsions.
2. To study the effects of physical and stability on the emulsion formulation using different content of emulsifying agents.
Apparatus:
8 test tubes
1 50 ml measuring cylinder
2 sets of pasture pipettes and droppers
Vortex mixer
Weighing balance
1 set of mortar & pestle
Light microscope
Microscope slide
1 set of 5 ml pipette and bulb
1 50 ml beaker
Coulter counter machine
Centrifuge machine
Viscometer
Water bath (45°C)
Fridge (4ºC)
Materials:
Palm oil
Arachis oil
Olive oil
Mineral oil
Distilled water
Span 20
Tween 80
Sudan III solution (0.5%)
ISOTON III solution
Procedures: (Palm Oil)
1. Each test tube was labeled and 1 straight line was sketched 1 cm from the bottom of the test tubes.
2. 4 ml oil was mixed with 4 ml distilled water in the test tubes.
Table 1
Group
|
Oil
|
1, 2
|
Palm
oil |
3, 4
|
Arachis oil
|
5, 6
|
Olive oil
|
7, 8
|
Mineral oil
|
3. For mixture of oil and water, a few drops of Span 20 and Tween 80 was added (refer to Table 2).The test tubes were closed and mixed with vortex mixer for 45 seconds. The time needed for the interface to reach the 1 cm line was recorded. The HLB value for each sample was determined.
Group
|
Oil
|
1, 2
|
Palm
oil |
3, 4
|
Arachis oil
|
5, 6
|
Olive oil
|
7, 8
|
Mineral oil
|
Table 2A ( for palm oil)
Tub No.
|
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
|
6.73
|
6.20
|
12.44
|
13.17
|
14.09
|
15
|
0
|
Time of phase separation (min)
|
8
|
8
|
65
|
38
|
62
|
61
|
40
|
1
|
Stability
|
Less stable
|
Less stable
|
Most stable
|
intermediate
|
Most stable
|
Most stable
|
intermediate
|
Least stable
|
Table 2B ( Arachis Oil)
Table 2C (Olive Oil)
Tube No.
|
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
|
15.00
|
0.00
|
Phase separation
time (min)
|
98
|
92
|
64
|
67
|
75
|
89
|
32
|
1.5
|
Stability
|
Most stable
|
stable
|
Intermediate
|
intermediate
|
intermediate
|
stable
|
Least stable
|
Least stable
|
Table 2D (Mineral Oil)
4. A few drops of Sudan III solution were added to 1 g of emulsion formed in a weighing bot and was flattened. The spreading of colour in the sample was described and compared. Some sample was spread on a microscope slide and was observes under light microscope. The shapes and sizes of globules formed were drawn, described and compared.
Test Tube
|
Description
|
1
|
The color is more difficult to spread, the size of droplets is almost similar, near to each other’s and distributed evenly.
|
2
|
The color easier to spread. The droplet's size is almost similar and slightly apart from each other.
|
3
|
The color still difficult to spread. The droplets' size is slightly not similar but still has near to each other.
|
4
|
The color still difficult to spread. The size of droplets is different and slightly near to each other.
|
5
|
The color easier to spread. The droplets are slightly different in size and near to each other.
|
6
|
The color is easy to spread. The size of droplets is slightly different. Some of the droplets are slightly far apart.
|
7
|
The color is easier to spread. The size of droplets is not same and some of them are bigger in size.
|
8
|
The color is very easy to spread. The size of droplets varies, not regular and the droplets are not evenly distributed.
|
5. By using wet gum method, a formulation of Mineral Oil Emulsion (50 g) was prepared using the formula below:
Mineral oil
|
refer Table 3
|
Acacia
|
6.25 g
|
Syrup
|
5 ml
|
Vanillin
|
2 g
|
Alcohol
|
3 ml
|
Distilled water
|
50 ml
|
Table 3
Emulsion
|
Group
|
Mineral oil (ml)
|
I
|
1, 2
|
20
|
II
|
3, 4
|
25
|
III
|
5, 6
|
30
|
IV
|
7, 8
|
35
|
6. 40 g emulsion formed was placed in a 50 ml beaker and homogeneous process was carried out for 2 minutes with homogeneous machine.
7. 2 g emulsion formed was taken (before and after homogeneous process) in a weighing bot and was labeled. A few drops of Sudan solution were added and flattened. The textures, consistency, oily shape degree and spreading of colour of the sample were observed and described under light microscope.
8. Viscosity of emulsion (15 g in 50 ml beaker) form after homogeneous process was determined using viscometer calibrated using “Spindle” type LV-4. The sample was exposed to 45°C (water bath) for 30 minutes and then to 4 ºC (fridge) for 30 minutes. Viscosity of emulsions was determined after heat exposure and the emulsion had achieved room temperature (10 - 15 minutes)
Palm Oil :
Readings
|
Viscosity (cP)
|
Mean + SD
| ||||
1
|
2
|
3
| ||||
Before heat exposure
|
10
|
15
|
13
|
12.67 ± 2.05
| ||
After heat exposure
|
31
|
40
|
37
|
36±3.741
| ||
Differences (%)
|
184.14
| |||||
Arachis Oil :
Amount
of mineral oil (ml)
|
Average
Viscosity (cP)
|
Difference
in viscosity(%)(X±SD)
|
|
Before
cycle
|
After
cycle
|
||
20(G2)
|
409.23±17.32
|
805.83±45.09
|
96.91
|
25(G6)
|
498.67±1.53
|
87.67±1.96
|
82.42
|
Olive oil:
Amount of mineral oil (ml)
|
Average Viscosity (cP)
|
Difference in viscosity(%)(X±SD)
|
|
Before cycle
|
After cycle
|
||
20(G3)
|
33.33±5.77
|
212±23.90
|
536.06
|
30(G7)
|
161.67±49.14
|
413.33±90.18
|
155.66
|
Mineral oil:
Amount of mineral oil (ml)
|
Average Viscosity (cP)
|
Difference in viscosity(%)(X±SD)
|
|
Before cycle
|
After cycle
|
||
25(G4)
|
486.23±40.87
|
295.50±80.84
|
39.23
|
30(G8)
|
173.33±70.27
|
300±69.28
|
73.08
|
9. 5 g emulsion that was homogenized was placed in a centrifuge tube and was centrifuged (4500 rpm). 10 minutes, (25 0 C). The height of the separation formed was measured and the ratio was determined.
Mineral oil (ml)
|
Separation phase (mm)
|
Initial emulsion (mm)
|
Ratio of separation phase
|
20
|
32
|
49
|
0.6531
|
34
|
52
|
0.6538
| |
25
|
36
|
54
|
0.6667
|
35
|
53
|
0.6604
| |
30
|
38
|
55
|
0.6909
|
37
|
54
|
0.6852
|
Discussion:
1. What are the HLB values to form a stable emulsion? Discuss.
HLB stands for Hydrophilic-Lipophilic Balance. The values is a ratio of polar and non-polar group in the surfactant which is shown by the balance of oil soluble substance and water soluble substance in a surface active agent. Surfactants were used to create emulsion. A stable emulsion will not separate. By using the formula,
HLB ( A + B ) = ( Ax + By ) / ( x + y )
The HLB number of a mixture composed of x% of surfactants of HLB A and y% of surfactants of HLB B can be obtained by the following formula. According to our result, the HLB values that produce stable emulsion are 6.20, 13.17 and 14.09 as the time taken for phase separation of these 3 values are the longest which are 65, 62 and 61minutes. 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. The SPAN emulsifiers are usually lipophilic and the TWEEN products are usually hydrophilic. Both are used to reduce the surface tension of water and stabilize the oil and aqueous phase. Surfactant act by adsorbing on the surface between oil and aqueous phase in low concentration. Thus, this adsorption increases the time for phase separation. HLB value of zero has no surfactant added. As a result, the time for phase separation is just 1 min. Apart from that, tube 7 which consists of one type of surfactant, Tween 80 is also not as stable as other tubes which contain two types of surfactants. Therefore, test tube 7 also has a short time for separation which is 40 minutes.
2. Compare the physical appearance of Mineral Oil Emulsion formed and give comments. What is Sudan Test III? Compare the colour spreading in the emulsion formed and give comments.
For the physical appearance of Mineral Oil emulsion, the globules are in varied sizes before homogenization. Homogenization process is to mix the oil and other substances by using external stimuli. The effect of homogenization on the emulsion is the changes in the viscosity of the emulsion. The viscosity of all the emulsion after the homogenization is much greater than before homogenization. Both before and after homogenization, the texture of the mineral oil is smooth and cloudy. Besides, size of the mineral oil droplet was not consistent before the homogenization due to the mixture is not well mix when there is no force and some are clumped together. But, after the homogenization, the size of the droplets becomes more consistent. Moreover, the colour of the mixture before the homogenization was not evenly dispersed while after the homogenization the colour more evenly dispersed. The texture of the globules seems spacious, which is not homogenous. It is greasier before homogenization. After homogenization, globules are relatively uniform in size, homogenous (packed), and appears to be less greasier compared to before homogenization. Besides, crystals are equally distributed, more consistent, and well dispersed.
Sudan III test or Sudan red test is used to examine the presence of lipid or fat in an emulsion. The test uses Sudan III solution which is an oil soluble dye to mix with the lipid phase and colour it to reddish brown. Besides by acting as an indicator, Sudan III can also differentiate types of emulsion formed, whether it is oil in water or water in oil emulsion. Red globules which produce a clear background indicate an oil in water emulsion whereas clear globules in a red background indicates an water in oil emulsion.
In this experiment, the Sudan dye is used to show the position of the lipid in the mixture. Since the mixture is not an ionic mixture, the Sudan dye can work appropriately. Thus, the function of Sudan dye can be explained. The red stain of Sudan dye in Emulsions Ι, ΙΙ, ΙΙΙ and ΙV is uneven before homogenization as the globules are varied in size and less consistently distributed. We can observe that the spreading of reddish brown colour is not consistent. Some parts are stained darker than the other part. Therefore, we can say that these emulsions formed are water-in-oil emulsion. However, after homogenization, the red stain is even. Red globules have been seen in uniform dispersion on a colourless background. The size of the globules is small. Hence, oil in water emulsion is formed after homogenization. Here, phase inversion occurred.
3) Plot the graph and give explanation.
Mineral oil contents
(mL)
|
Viscosity (cP)
(x ± SD)
|
Viscocity difference
(%)
(x±SD)
| |
Before temperature cycle
|
After temperature cycle
| ||
20
|
12.67 ± 2.05
|
36±3.741
|
184.14
|
25
|
486.23±33.37
|
658.67 ± 38.96
|
35.46
|
30
|
173.33 ± 57.35
|
300 ± 43.20
|
73.08
|
a) Graph of viscosity of emulsion before and after temperature cycle against different mineral oil contents
Graph above shows that difference in viscosity versus different amount of different oil. From the graph it is shown that there will be increase in viscosity after temperature cycle. It is because, viscosity of emulsion will decrease when put in water bath of 450c for 30 minutes. By increasing the temperature, it causes kinetic motion of dispersed oil droplets and decrease viscosity of continuous phase. After that, emulsion is put into freezer of 450c for 30 minutes. At the low temperature, kinetic energy of droplets decreases and viscosity of continuous phase increase. So after the temperature cycle, there will be an increase in viscosity of emulsion.
b) Graph of viscosity difference (%) against mineral oil content
Graph above shows that viscosity difference (%) against mineral oil content. Theoretically, as the amount of mineral oil in emulsion increases, the viscosity difference will also increase. It is because increasing mineral oil content causes amount of oil globules (dispersed phase) to increase, then viscosity of emulsion should also be increased. However, there is some error with our experimental result because the emulsion III with 30% oil content has lower viscosity difference than emulsion I with 20% of oil content. Emulsion I should have lowest viscosity difference than II and III.
Experimental error that might be occur such as during the preparation of emulsion, the amount of materials used was not in the exact proportion, eg: excipients and active ingredients. Another error is of the viscometer, continue using the machine without washing properly after using it will lead to the inaccuracy of the data. In order to get a more accurate result, we should use the same type of oil in all emulsion sample and only varies in oil content.
Mineral oil (ml)
|
Separation phase (mm)
|
Initial emulsion (mm)
|
Ratio of separation phase
|
Average ratio ( Average ± SD)
|
20
|
32
|
49
|
0.6531
|
0.6535 ± 0.0005
|
34
|
52
|
0.6538
| ||
25
|
36
|
54
|
0.6667
|
0.6636 ± 0.0045
|
35
|
53
|
0.6604
| ||
30
|
38
|
55
|
0.6909
|
0.6881 ± 0.0040
|
37
|
54
|
0.6852
|
According to the theory, as the amount of oil increases, the separated phase ratio increases. This is because the added amount of oily phase in emulsion has exceeded the oil amount at which the stable emulsion is formed. Separation will occur in a faster rate.
Centrifugation 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. Compare with a non-homogenous emulsion, a homogenous emulsion will not separate easily. To produce a stable and homogenous emulsion, the phase separation ratio must always be kept at minimum level so that the drug can be dispersed uniformly in the emulsion and the administration of accurate dose can be achieved.
5. What is the function of each type of substances used in the
emulsion preparation? How does the physical characteristics and stability of an
emulsion affected by the contents of substances?
Ingredient
|
Function
|
Mineral Oil
|
As oil phase in this formulation whereby mineral oil
is dispersed phase. 40%-60% oil is used to produce stable emulsion. Also give an emollient effect to the end
product.
|
Acacia
|
Emulsifying agents or stabilizers. To minimize or
prevent droplet-droplet contact and subsequent coalescence.
|
Syrup
|
Sweeten the emulsion and to mask the bad taste. The
viscosity of the emulsion increase hence will reduce the contact between the
taste bud and the emulsion.
|
Vanillin
|
Flavoring agent and increase patient acceptability.
|
Alcohol
|
Prevent the microorganism growth in the emulsion.
|
Distilled water
|
As water phase in this formulation in which oil in
water emulsion (continuous phase).
|
Different ingredient used to formulate
an emulsion will change the physical characteristics and stability of the
product. This is because different type of oil has different viscosity. Moreover, different percentage of water and
oil used to formulate can change the physical and chemical composition of the
emulsion. That is why we need to make sure we want to produce either oil in
water or water in oil emulsion. If the percentage of water is more than the oil
phase, then it will produce oil in water emulsion and if the percentage of oil
is more than the water, then it will produce water in oil emulsion.
Furthermore, the types of mineral oil involve in chemical and physical
characteristic. For example, palm
oil has anti-oxidant properties which increase the chemical stability of the
emulsion. This type of emulsion will be less prone to oxidation than using
other types of oil. Next, emulsifying agent with suitable HLB
should be choosing in order to produce a stable emulsion. The amount of syrup
will increase the viscosity hence can prevent the emulsion become in contact to
the taste buds and bad taste can be masked.
Conclusion:
Low HLB values, 3-11 are suitable to be used in water in oil emulsion while high value of HLB will be suitable for oil in water emulsion. The best way is to use combination of surfactants such as Tween and Span in order to produce a more stable emulsion with a longer phase separation time. Furthermore, optimum HLB values are achieved differently according to what type of oil we used.
References :
1) Collett, D.M. & Aulton, M.E. 1990.
Pharmaceutical practice. Ed. ke-3. Edinburgh: Churchill Livingstone.
2) Aulton, M.E. 1998. Pharmaceutics: The science of
dosage form design.Edinburgh: Churchill Livingstone.
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