Experiment 3
Evaluation
of the effect of different composition in the formulation of emulsion
Objective :
1) To
determine the effects of surfactant’s HLB value on the emulsion stability
2) to
determine the different physical effects and stability of the emulsion
formulation by using different emulsifying agent.
Introduction:
Emulsion refers to the two-phase-system
which is thermodynamically unstable. It contains of at least two immiscible
liquids in which one of them (internal/dispersed phase) is dispersed
homogenously in the other liquid (external/continuous phase).There are two type
of emulsion as which is oil-in-water emulsion (o/w) and water-in-oil emulsion
(w/o). The emulsifying agent is used to stabilize the emulsion and it can
divide into 4 type which is hydrophilic colloid, finely divided solid particle,
and surface-active-agent or surfactant.
Besides that , the quantity and the
type of surfactant needed to prepare a stable emulsion can be determined by HLB
system (hydrophilic-lipophilic balance).In general , we combine two emulsifying
agents in order to produce more stable emulsion as each surfactant is ranged
from 1 (lipophilic) to 20 (hydrophilic). Hence , the HLB value for the
emulsifying agent combination can be determined by using the formula:
HLB
value = (quantity of surfactant 1)(HLB of surfactant 1) + (quantity of
surfactant 2)(HLB surfactant 2)
quantity of surfactant 1+ quantity
of surfactant 2
MATERIAL AND
APPARATUS
Apparatus:
8 test tube , 50 ml measuring cylinder , 2
sets of pipette pasture , droppers Vortex
mixing device ,Weighing boat , Mortar and pestle , Light microscope , Microscope
slides , 1 set of pipette (5 ml) and
pipette-bulb, 50 ml beaker,1 centrifugation tube 15 ml ,Coulter counter device
Centrifugator
, Viscometer ,Water-bath (45°C) ,Refrigerator (4°C)
Ingredients:
Palm oil, arachis oil
, olive oil , turpentine oil , distilled water , Span 20, Tween 80 , Sudan III
solution (0.5%) , ISOTON solution III
Procedures :
1) Each
test tube were labeled and a straight line 1cm from the bottom of the test tube
were drawn.
2) 4mL
of oil and distilled water were mixed in the test tubes based on Table 1.
Group
|
Type of
oil
|
1,5
|
Palm oil
|
2,6
|
Arachis
oil
|
3,7
|
Olive oil
|
4,8
|
Mineral
oil
|
3) Drops
of Span 20 and Tween 80 were added into the water-oil mixture based on Table 2.
Test tubes were closed and vortex mixer was added for 45 seconds. Time taken
for the interphase to reach the 1cm line mark were recorded. HLB value were
determined for each sample.
4) A
few drops of Sudan III were added to 1g of emulsion that were formed in a
weighing boat. The colour distribution of the sample were explained and
compared. Some of the sample were spread on a microscope slide and observed
under light microscope. The shape and size of globule were drawn, explained and
compared.
5) A
mineral oil emulsion preparation were prepared using wet gum method following
these formula :
Mineral
oil
Acacia
Syrup
Vanillin
Alcohol
Distilled
water, qs
|
(Refer
table 3 )
6.25g
5ml
2g
3ml
50ml
|
Table 3
Emulsion
|
Groups
|
Oil(ml)
|
I
|
1,5
|
20
|
II
|
2,6
|
25
|
III
|
3,7
|
30
|
IV
|
4,8
|
35
|
6) 40g
of emulsion produced were added in 50mL beaker and homogenization process were
done for 2 minutes using the homogenizer.
7) 2g
of emulsion (before and after homogenization) were added into weighing boat and
labeled. A few drops of Sudan III were added. The texture, consistency and
colour distribution under the light microscope were explained and compared.
8) The
viscosity of 15g emulsion in 50mL beaker were determined after the
homogenization process using viscometer. The sample was the exposed to 45 celcius (water bath) for 30 minutes and at 4 celcius(refrigerator) for 30 minutes. The viscosity
were determined after the sample were exposed to room temperature for 15 minutes.
9) 5g
of emulsion that were homogenized were added into a centrifuge tube and were
centrifuged for 10 minutes. The height of the interface formed were measured
and the ratio were determined.
Results :
Palm 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
|
|
Time needed for phase
separation to reach 1cm (min)
|
Group 1
|
Phase separation did not reach 1cm after 120 minutes.
|
58.00
|
61.00
|
45.00
|
25.00
|
0.50
|
||
Group 5
|
Phase separation did not reach 1cm after 120 minutes.
|
16.00
|
30.00
|
39.00
|
16.00
|
7.00
|
|||
Average
|
-
|
37.00
|
45.50
|
42.00
|
20.50
|
3.75
|
|||
Stability
|
Yes
|
Yes
|
Yes
|
No
|
No
|
No
|
No
|
No
|
|
Arachis 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
|
||
Time needed for phase
separation to reach 1cm (min)
|
Group 2
|
Phase separation did not reach 1cm after 120 minutes.
|
27.00
|
40.00
|
55.00
|
19.00
|
9.00
|
|||
Group 6
|
Phase separation did not reach 1cm after 120 minutes.
|
38.00
|
49.00
|
61.00
|
19.00
|
25.00
|
||||
Average
|
-
|
-
|
-
|
32.50
|
44.50
|
58.00
|
19.00
|
17.00
|
||
Stability
|
Yes
|
Yes
|
Yes
|
No
|
No
|
No
|
No
|
No
|
||
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
|
||
Time needed for phase
separation to reach 1cm (min)
|
Group 3
|
Phase separation did
not reach 1cm after 120 minutes.
|
8.19
|
14.48
|
87.35
|
58.35
|
19.49
|
0.33
|
||
Group 7
|
Phase separation did not reach 1cm after 120 minutes.
|
63.00
|
Interphase did not
reach 1cm after 120 minutes.
|
45.00
|
2.50
|
|||||
Average
|
-
|
75.18
|
-
|
32.25
|
1.42
|
|||||
Stability
|
Yes
|
Yes
|
No
|
No
|
No
|
No
|
No
|
No
|
||
Mineral oil
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
|
||
Time needed for phase
separation to reach 1cm (min)
|
Group 4
|
119.00
|
114.00
|
108.00
|
94.00
|
80.00
|
34.00
|
8.00
|
0.50
|
|
Group 8
|
Phase separation did
not reach 1cm after 120 minutes.
|
|
24.00
|
28.00
|
29.00
|
15.00
|
18.00
|
0.50
|
||
Average
|
-
|
o
|
66.00
|
61.00
|
54.5
|
24.50
|
13.00
|
0.50
|
||
Stability
|
Yes
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
||
The shapes and globule
sizes formed (view under microscope) of mixtures of oil and water. Tubes 1 til
8 observations are as follow :
Tube
|
Emulsion
|
Tube
|
Emulsion
|
1
|
Light red droplets well distributed in white colour solution.
|
5
|
 |
2
|
Very small light red droplets distributed evenly in white
solution.
|
6
|
Light red droplets with average size well distributed in
white solution.
|
3.
|
Light red droplets with different sizes distributed evenly
in white colour solution.
|
7
|
Light red droplets with larger in size distributed unevenly
in the solution.
|
4
|
 The light red droplets with larger in size distributed evenly in the solution. |
8
|
Comparison of image of
emulsion under light microscope :
Before
 |
After
 |
Before
|
After
|
|
Texture
|
Less smoth
|
Smoother
looking
|
Consistency
|
Less viscous
|
Viscous
|
Degree of oily surface
|
Less oil
globule
|
More oil
globule
|
Distribution of color sample under the
light microscope
|
Less even
colour distribution
|
Even colour
distribution
|
Comparison of the
viscosity of emulsion of different oil type :
Types of Oil
|
Readings
|
Viscosity (cP) 1
|
Viscosity (cP) 2
|
Viscosity (cP)3
|
Average ±STD
|
Palm oil
|
Before temperature cycle
|
100
|
110
|
120
|
110 ± 10
|
After temperature cycle
|
130
|
140
|
140
|
136.7 ± 5.8
|
|
Difference (%)
|
24.3 ±42
|
||||
Arachis Oil
|
Before temperature cycle
|
389.9
|
419.9
|
419.9
|
409.9 ± 17.3
|
After temperature cycle
|
779.8
|
779.8
|
659.9
|
739.8 ± 69.2
|
|
Difference (%)
|
80.5 ± 300
|
||||
Olive Oil
|
Before temperature cycle
|
239.9
|
210.0
|
210.0
|
220 ± 17.3
|
After temperature cycle
|
539.9
|
449.9
|
389.9
|
459.9 ± 75.5
|
|
Difference (%)
|
109 ± 336.4
|
||||
Mineral Oil
|
Before temperature cycle
|
650
|
650
|
600
|
633.3 ± 28.9
|
After temperature cycle
|
300
|
300
|
300
|
300 ± 0
|
|
Difference (%)
|
52.6 ± 100
|
||||
Height of separation :
Height (mm)
|
|
Separation Phase
|
35 mm
|
Original emulsion
|
50 mm
|
Ratio of height
|
7 : 10
|
Discussion :
1．
What are the HLB values to form a stable emulsion?
Discuss.
In this experiment, emulsions with the greatest stability are defined according to the time needed for phase separation to reach 1cm. The longer the time taken for 1cm of the phase separation to form, the greater the stability of the emulsions. Based on this hypothesis, when the palm oil was used, emulsions tube1-3 give the greatest stability where the HLB value is 9.67-11.34. For the arachis oil, emulsions tube1-3 also give the greatest stability where the HLB value is 9.67-11.34. For the olive oil, emulsions tube 1 and 2 give the greatest stability where the HLB value is 9.67 and 10.73 respectively. For the mineral oil, emulsion from tube 1 gives the greatest stability where the HLB value is 9.67. Generally, for all type of oils used in this experiment, emulsions from the tube 1 - 3 give the greatest stability. Therefore, the most ideal HLB value in this experiment is in the range of 9.67-11.34.
The ideal emulsion consists of the following characteristic: the smallest spheral globule size, the closest packing between globules, and the largest separation distance or the most consistent colour dispersion. An emulsion is considered to be in the stable state if all these criteria are fulfilled. Surfactants act by enhancing the distribution of oily phase into the aqueous phase (o/w emulsion) or the distribution of aqueous phase into the oily phase (w/o emulsion). It is often we have a mixture of surfactants such as oil soluble (low HLB) and a water soluble (high HLB) surfactant to produce the desired HLB. The combination of oil soluble surfactant Span 20 (HLB=8.6) and water soluble surfactant Tween 80 (HLB=15.0) give an optimal effect in emulsifying the dispersed oily phase in the aqueous continuous phase. The hydrophobic hydrocarbon tails of surfactants are in the oil phase while the hydrophilic head groups are in the aqueous phase. The surfactants (emulsifying agents) function to serve as a bridge between the two totally immiscible phases and mixed up them. To stabilize the oil globules, it is ideally that certain degree of hydrophilicity is achieved to confer an enthalpic stabilizing force and also a degree of hydrophobicity to secure adsorption at the interface. Thus, the combination of surfactants that achieve the desired HLB value of emulsion will produce a stable emulsion.
Comparing all the test tube in the experiment, it is believed that test tube 8 has the lowest stability as the phase separation time is the shortest. This is mainly because of the absence of surfactant as an emulsifying agent that stabilizes the emulsion system. Also, some errors may be carried out when the experiment was conducted and lead to the inaccurate results. The emulsion might not be mixed properly when subjected to the vortex mixer. The time used for the observation for phase separation to reach 1cm was limited to 120 minutes. However, some of the emulsions may require more time to reach that 1cm. A longer time for observation may be needed to complete the phase separation so that comparison can be done easily.
2. Compare the physical structures for the mineral oil emulsions formed and explain. What is the Sudan III Solution? Compare the colour dispersion in the emulsions formed and explain.
Before homogenization,
the texture of the emulsions is generally coarse and less spherical. From the
observation under the microscope, the sizes of globules are not uniform, some
of them are big and some are small. The emulsions are less consistent and high
degree of oily. After adding Sudan III solution, it shows poor colour
dispersion in the emulsions. After the homogenization process, when the
emulsions are observed, the textures generally smooth and more spherical. The
sizes of globules are smaller and more uniform in size when observed under
microscope. The emulsions are more consistent and the degree of oily are
decreased. After adding Sudan III solution, it shows good colour dispersion in
the emulsions and the colour of the emulsions become milky in colour.
Sudan III solution is a red solution
which can dissolve in oil and give red colour to the globules formed. It is used to determine whether an emulsion
formed is an oil-in-water emulsion or a water-in-oil emulsion. If oil is the
external phase, the red dye color of Sudan solution gradually spreads
throughout the emulsion. But if water is the external phase the color does not
spread but is confined to the oil with which it comes in contact on the
surface. In this experiment, Sudan III solution stains the globules red and the
continuous phase remains colorless. This indicates that the globules are oil
phase, and thus it is an oil-in-water emulsion.
The colour dispersion is actually
due to the dissolubility of the Sudan III solution in the oil phase. The colour dispersion of the
emulsions before homogenization is not consistent and the red colour is covered
more than the background. Whereas after homogenization, the colour of
dispersion is more consistent and the red colour spread evenly with the
background. Inconsistence of the colour is due to the emulsifying agents
are unable to emulsify the emulsion.
3. Table 1: Average viscosity of
emulsion before and after temperature cycle and viscosity
difference (%).
Types of Oils
|
Average Viscosity (cP)
|
Viscosity Difference
(%)
(Mean ± STD)
|
|
Before Temperature cycle
|
After Temperature Cycle
|
||
Palm Oil
|
110
|
136.7
|
24.3 ± 42
|
Arachis Oil
|
409.9
|
739.8
|
80.5 ± 300
|
Olive Oil
|
220
|
459.9
|
109 ± 336.4
|
Mineral Oil
|
633.3
|
300
|
52.6 ± 100
|
In this experiment, we are measuring the viscosity of emulsion containing different types of oil, before and after temperature cycle. Those 4 types of oil are palm oil, arachis oil, olive oil and mineral oil. Viscometer is used to measure the viscosity of the emulsion. After homogenization process, the viscosity of each emulsion sample is determined before exposing it to temperature cycle. The homogenization process aims to reduce the mean globule diameter, thereby increasing the apparent viscosity of an emulsion.The temperature cycles consist of exposing the sample to water bath of 45oC for 30 minutes, followed by freezing the sample at temperature of 4oC in the refrigerator for another30 minutes, in order to ensure complete oil and water crystallization. Later, the sample is left aside for it to reach room temperature for about 15 minutes, to melt any crystalline fat, before finally introducing it to viscometer for another viscosity measurement.
From graph 1, we can see that all the emulsion undergo
viscosity changes after temperature cycle. Emulsion containing palm oil,
arachis oil and olive oil experienced increase in average viscosity after
temperature cycle, whereas decrease in average viscosity only occurred in
mineral oil emulsion. The viscosity of the emulsion changes because the
temperature cycle has altered the physicochemical properties of the emulsion
which leads to a considerable decrease in the stability. Nature of each of the
vegetable oils; palm oil, arachis oil and olive oil influences the speed of
demulsification and emulsion stability. As for the mineral oil, it is made up
of mixtureof alkanes in the C15 to C40 range from a mineral source, particularly
a by-product of petroleum distillation; thereby its nature is different from
the other 3 vegetable oils. Viscosity index of mineral oil is determined
primarily by its hydrocarbon composition. The hydrocarbons in the mineral oil
range of molecular weight are very complex. This might explanation the decrease
in the average viscosity in the mineral oil after temperature cycle.
In the temperature cycle process, an increased in temperature
to 45oC will increase kinetic motion of both, the dispersed oil
droplets and emulsifying agent at the oil/water interface. The increased in
kinetic motion leads to increase in numbers of collision between oil globules.
The increase in the motion of emulsifying agent will result in a more expanded
monolayer, thereby coalescence is more likely. Freezing the sample to
temperature of 4oC will alsoresult in coalescence. Freezing causes
water to crystallize which force the dispersed oil droplets to concentrate in
non-frozen region remaining in the water phase causing the dispersed oil
droplets to come into closer contact with each other. When free water phase
becoming so little that it cannot fully hydrate the oil droplets surfaces, the
droplet-droplet interactions become stronger and the droplets become closer
together until coalescence occurred.
Besides that, ice crystals formed during freezing may exert
unusual pressure on the oil droplets and penetrated into the oil droplets which
result in disruption of their interfacial membranes. This allowed the oil
droplets more prone to coalescence between them. In addition, freezing also may
have caused some of the fat in the oil droplets to crystallize and the fat
crystals may stick out into the water phase resulting in partial coalescence.
Partial coalescence occurs when the fat crystals come into contact with nearby
droplets, penetrate through the film of another droplet and lead to coalescence
of the oil droplets.The presence of fat crystals in the dispersed oil droplets
causes a considerable decrease in the stability. Partial coalescence of oil
droplets causes the viscosity of the emulsion to increase, and it may even lead
to phase inversion.The amount of crystals form depends mainly on the
composition of oil and temperature. Emulsions consisting of polydispersed
droplets will tend to exhibit a lower viscosity than a monodispersed system,
due to differences in electrical double-layer size and thus in the energy
interaction curve.
The result of the viscosity differences (%) for the four
different types of oils are represented in graph 2. Graph 2 shows that olive
oil has the highest viscosity difference (%), whereas palm oil produces less
viscosity difference (%) among the oils studied. Different oils have different
nature and different hydrocarbon composition, hence showing a vary viscosity.Besides
that, emulsion prepared using different stirring intensity and emulsion that is
stirred using different RPM during viscosity measurement also shows a vary
range of viscosity. Olive oil is stirred with RPM 20 using the viscometer,
whereas others oils are stirred with RPM 12, thus greater force are exerted on
the olive oil emulsion. The two factorsare likely to be the reason for the high
viscosity difference (%) in olive oil.
In this experiment, error may arise from either human error
of technical error in handling the viscometer. While using the viscometer, we
must make sure that viscometer spindle did not touch bottom of the beaker. For
accurate reading of viscosity, the spindle must be dipped into the emulsion
deep enough, which is until the emulsion reaches the mark on the spindle.
Suitable and appropriate size of spindle should be used as different spindle
account for different degree of viscosity. We should start with the smallest
size of spindle, LV-4 before changing to greater size of spindle if error is
obtained in the viscosity reading.
4. Plot a graph of ratio of separation phase against
different amount of mineral oil. Discuss.
Mineral Oil (mL)
|
Ratio of separation phase (x ± SD)
|
20
|
0.67±0.085
|
25
|
0.65 ± 0.19
|
30
|
0.72±0.028
|
35
|
0.63±0.064
|
Based on the
graph above, the mean and standard deviation indicate the stability of the
emulsion. Emulsion with a high ratio of separation phase is not stable while the
emulsion with the lowest ratio of separation phase is the most stable.In this
experiment, Arachis oil show the highest ratio of phase separation followed by
of palm oil, olive oil and mineral oil.Means, arachis oil showed the least
stable of emulsion,while mineral oil showed the most stable emulsion.
5. What are the functions of each ingredient used? How these different ingredients affect the physical characteristics and stability of an emulsion formulation?
In this experiment, oil-in-water emulsions are produced. An emulsion is defined as a thermodynamically unstable two-phase system consisting of at least two immiscible liquids. The palm oil, arachis oil, olive oil or mineral oil functions to form the dispersed phase/internal phase in these oil-in-water emulsions. Distilled water acts as the dispersion medium which form the continuous phase/external phase in which the oil is homogenously dispersed with the help of the surfactants.
Span 20 and Tween 80 are surfactants that act as the emulsifiers in the emulsion system. They can lower the interfacial tension of liquids, thus decrease the possibility that collisions between droplets that would lead to coalescence and also increase the kinetic stability of the dispersion (less thermodynamically unstable). They are unique in their structure as they contain the polar head group(hydrophilic) and also the non polar tails (hydrophobic). The hydrophobic tails are inserted into the oily phase while the hydrophilic head group is inserted into the aqueous phase, thus the surfactants form a bridge between the two immiscible liquids, stabilizing the whole structure.
Sudan III solution (0.5%) is a dye used for Sudan staining. It is used to color nonpolar substances like oils and also separates the lipids from the material being tested. ISOTON III solution acts as isotonic buffered diluents in the experiment. Acacia is hydrophilic colloids emulsifying agents that can stabilise emulsion by forming thick multimolecular layers. Syrup is viscous solution which contains high percentage of sugar, thus functions as a sweetening agent in the formulation. Vanillin is a flavoring agent used to mask unpleasant taste of the emulsion. Since there is a high proportion of water present in the oil-in-water emulsion, alcohol is added as a preservative in this formulation to make it less susceptible to microbial contamination.
The proportion of distilled water (aqueous phase) and the (oily phase) in emulsion may have great impact in the physical characteristics and stability of each emulsion formed. The phase volume ratio must be maintained at a certain value such as emulsion should contain less than 25% of the dispersed phase. In other words, there should be less than 25% of the oil in the dispersion medium (water) in this experiment. If there is too much oily phase in the o/w emulsion (more than approximately 70%), the phase inversion is likely to occur as the dispersed phase becomes the continuous phase and it is converted to water in oil emulsion.
The selection of different oil phases also produce emulsions with slightly different physical appearance including odour, colour, texture, consistency, etc. Emulsions with different stability may also be produced. Oxidation of certain components of emulsion can result in unpleasant odour and taste that destabilized the system. Palm oil may form a more stable emulsion as it contains antioxidant: tocopherol. The emulsion formed is less prone to oxidation than other emulsion with other types of oil.
Suitable combination of emulsifying agents with desired HLB value should be selected in order to produce a stable emulsion. Unsuitable surfactants produce emulsions with different physical properties such as globule size, texture, consistency, oily phase dispersion, etc. These may affect the therapeutic effects of the emulsion.
The components of the formulation such as acacia, syrup, water provide a suitable culture medium for many types of microorganisms. So, oil-in-water emulsions are very susceptible to microbial attacks. Thus, a suitable preservative should be added to avoid microbial growth that would contribute to instability of the system.
CONCLUSION
The stability of the emulsion
can be increased by adding of emulsifying agent such as surfactant. HLB value is
used to select the appropriate emulsifier to produce a stable emulsion.
Emulsion formed from different oil required surfactant with different HLB value
in order to stabilize the emulsion formed. Combination of surfactants, such as
Span and Tween will form a more stable emulsion than a single surfactant.
REFERENCE:
1. Aulton.
M. E. (2007). Aulton’s Pharmaceutics: The Design and Manufacture of Medicines. Churchill
Livingstone Elsevier.
No comments:
Post a Comment