Introduction
Emulsion consists of two
different phases that is not thermodynamically stable. It contains two
immiscible liquids. One of them is dispersed as globules (dispersed
phase=internal phase), the other one is continuous phase (external
phase). It can be classified as oil-in-water (o/w) emulsion or
water-in-oil(w/o) emulsion. The droplet size
affects the distribution of dispersed phase of emulsion. For micro emulsion,
the droplets size range 0.01 to 0.1 mm. For macro emulsion, the droplets size
range approximately 5 mm. Emulsion can be stabilized by adding stabilizing
agents. Emulsifying agents can be divided into 3 types, which are hydrophilic
colloid, finely divided solid particles, and surface active agents or surfactants.
HLB method
(hydrophilic-lipophilic balance) was used to determine the quantity and type of
surfactants that will needed to prepare a stable emulsion. Each of surfactants,
will be given a number in HLB scale which is from 1 (lipophilic) until 20 (hydrophlilic).
Normally, combination of two emulsifying agents used to produce an emulsion
that is more stable. HLB value for combination of emulsifying agents can be
determined by using the formula as followed:
HLB value = (Amount of surfactant 1) (HLB surfactant 1) + (Amount of surfactant 2) (HLB surfactant 2)
--------------------------------------------------------------------------------------------------------------
Amount of surfactant 1 + Amount of surfactant 2
Objectives
1. Determine the effects of HLB surfactants to the stabilized emulsion.
2. To study the physical effects and stabilizing effects to the formulation of emulsion due to usage of different emulsifiers.
Apparatus
8
test
tubes
1 set of 5ml pipette and bulb
1
measuring cylinder
50ml
50ml beaker
2
set of pasture pipette and
droppers
15ml centrifuge
Vortex
mixing
tools
Centrifuge machine
Weighing
boat
Coulter
counter
1
set of mortar and
pestle
Viscometer
Light
microscope
water bath (45°c)
Slides
for
microscope
refrigerator (4°c)
Materials
Palm
oil
Span 20
Arachis
oil
Tween 80
Olive
oil
Sudan
III solution (0.5%)
Mineral
oil
ISOTON III solution
Distilled
water
Procedures
1. Each test tube is labeled and draws a line
about 1cm from bottom of the test tube.
2. Mix 4ml of oil and 4ml of distilled water into
the test tubes.
3. Drop Span 20 and Tween 80 into each test tube
(refer table II). Close the test tubes and mix it with vortex mixing machine
for 45 seconds. Record the time needed to reach 1cm line. Determine HLB value
for each sample.
4. Drops a few of Sudan III solution into a few
(1g) emulsions that formed in the weighing boat. Describe and compare color
spread in the sample. Take a little sample onto the microscope slide and
observe the sample with using light microscope. Draw, describe and compare the
appearance and globule size that formed.
5. With using wet gum method, prepare an
formulation of Mineral oil Emulsion (50g) by using formula as followed:
6. Put 40g emulsion that formed into 50 ml beaker
and homogenization for 2 minutes by using homogenizing machine.
7. Take a little (2 g) emulsion that formed
(before and after homogenization) into weighing boat and label. Drops a few of
Sudan III solution and smooth it. Describe and compare the textures,
consistency, degrees of oily appearance and color spread of the sample by using
light microscope.
8. Determine the viscosity of emulsion (15 g in
50 ml beaker) that formed after homogenization by using viscometer that
calibrated with “Spindle” type LV-4. The sample is exposed at temperature about
45°C (water bath) for 30 minutes and latter about 4°C (refrigerator) for 30
minutes. Determine the viscosity of emulsion after temperature cycle exposure
done and emulsion reach room temperature (10-15 minutes).
9. Put 5 g emulsion that homogenized into the
centrifuge tube and spin it (4500 rpm, 10 minutes, and 25°C). Measure the
distance of separation formed and determines the ratio of degree of separation.
Results:
Procedure step 2: Table II
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(min)
|
Interphase
did not reach 1cm after 120 minutes.
|
58.00
|
61.00
|
45.00
|
25.00
|
00.30
|
||
|
Stability
|
Yes
|
Yes
|
Yes
|
No
|
No
|
No
|
No
|
No
|
Procedure step 4: Sudan III are added
to emulsion.
Observation under microscope:
Tube 1:
Tube 2:
Tube 3:
Tube 4:
Tube 5:
Tube 6:
Tube 7:
Tube 8:
Procedure
step 8:
Procedure
step 9:
Discussion
1. What are the HLB values to form a stable
emulsion? Discuss.
|
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(min)
|
Interphase
did not reach 1cm after 120 minutes.
|
58.00
|
61.00
|
45.00
|
25.00
|
00.30
|
||
|
Stability
|
Yes
|
Yes
|
Yes
|
No
|
No
|
No
|
No
|
No
|
Table above shows group 5 results on palm oil.
Optimum HLB values are achieved differently
according to what type of oil we used.
Our group experiment used palm oil to carry out the test. We found that
optimum HLB values for palm oil to produce the most stable emulsion are 9.67, 10.73
and 11.34. This is because the time taken for phase separation of these three
values is the longest. The addition of surfactant such as Span 20 and Tween 80
can reduce the surface tension of water and stabilize the oil & aqueous
phase. Surfactant should be used in low concentration and the adsorption of the
surfactant between the oil and aqueous phase will stabilize the emulsion and
thus increase the time for phase separation. From the table, we found that tube
8 gives the lowest stability of emulsion. Its HLB value is zero with no
surfactant is added. As a result, the time for phase separation is just 0.30
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. In
short, surfactant can increase the stability of emulsion and a combination of
surfactants will further increase the time for phase separation.
2. Compare the physical state of mineral oil emulsion formed.
What is Sudan test III ? Compare the color dispersion in emulsion formed.
Observation of Emulsion III (before and after
homogenization) :
Generally, globules are
not uniform in size, less consistent as crystals tend to clump together,
non-homogenous where it appears to be more spacious, and are greasier before
homogenisation. After homogenisation, globules are relatively uniform in size,
more consistent as crystals are well dispersed, homogenous whereby it looks
packed, and are less greasy than the ones before homogenisation.
Sudan III solution is a
fat-soluble dye that is used in Sudan III test. As it is lipid soluble, it does
not mix with water. It is used for staining the sudanopilic substances usually
lipids in this test. The dye in Sudan III test will mix with oil and colour it
reddish brown. In this experiment, Sudan
III was used as an indicator to stain the oil phase to be observed under the
light microscope. Sudan III test can also be used to determine the
types of emulsion formed, whether it is oil-in-water (o/w) or water-in-oil
(w/o) emulsions. If the oil is dyed red, a
red background with globules indicates a w/o emulsion whereas if red globules
in a clear background indicates an o/w emulsion.
Since the Sudan III test
forms reddish brown crystals by staining the oils, the more
greasy form of the emulsion before homogenisation should appear reddish brown
in colour. However, maybe due to lighting or different colour interpretation,
the colour spread of the emulsion was observed to be yellowish. On the other hand, the
colour spread of the less greasy emulsion after homogenisation was observed to
be white. Therefore, the emulsion is oil-in-water (o/w) emulsion.
3. Graph
of Viscosity
4. Graph
of Separation height
|
Mineral Oil (ml)
|
Group
|
Separation phase(mm)
|
Initial emulsion (mm)
|
Ratio of Separation Phase
|
Average Ratio
(Average ± SD)
|
|
Emulsion I (20mL)
|
1
|
1.8
|
4.4
|
0.41
|
0.49 ± 0.08
|
|
2
|
2.6
|
4.6
|
0.57
|
||
|
Emulsion II (25mL)
|
3
|
3.4
|
5.0
|
0.68
|
0.61 ± 0.07
|
|
4
|
2.7
|
5
|
0.54
|
||
|
Emulsion III (30mL)
|
5
|
1.5
|
7
|
0.21
|
0.375 ± 0.165
|
|
6
|
27
|
50
|
0.54
|
||
|
Emulsion IV (35mL)
|
7
|
12.6
|
43
|
0.29
|
0.295 ± 0.005
|
|
8
|
14
|
46
|
0.30
|
Phase separation ratio is used to indicate the
stability of an emulsion. A high ratio of phase separation will result in
unstable emulsion and form two distinct phases. The presence of two distinct
phases shows that the emulsion possesses inadequate stability. Centrifuges
accelerate the phase separation processes in the emulsion by enhancing the
specific gravity differences. The concept of the phase separation by
centrifugal is based on density difference of the oil and water phase in an
emulsion, either oil-in-water emulsion or water-in-oil emulsion. After the
process, phase separation will occur where the water and oil phase will
separate into two significant layers. Since the oil has lower density than
water, it will rise upward and appear at the upper layer while water is at the
bottom layer. There is not much difference between densities of oil being used.
Hence, the type of oil did not give much effect to the result. According to the
theory, the separated phase ratio will also increase follow by the increasing
amount of the oil. This is because the amount of oil added in emulsion is
beyond the amount of oil required in which a stable emulsion can be formed.
Thus, phase separation will occur at a faster rate. However, according to the
graph above, phase separation ratio decreases when the amount of oil increases.
This shows that the result did not follow the theory exactly. This is because
some errors occurred during the experiment. For instance, the presence of
contaminant in the emulsion has influence on the accuracy of the result.
Besides that, the incorrect amount of oil also affects the accuracy of results
being obtained. Parallax error may also occur when measuring the height of
separation phase. There is also possibility that some groups measured the
separation phase by using height of water phase instead of oil phase.
Therefore, many problems arise during the experiment due to the variety of
workforce and inaccurate measurements of readings. Ingredient that is use in
this emulsion is Acacia, Syrup, Vanillin, Alcohol, Olive oil and distilled
water. Olive oil and distilled water is used as a basic ingredient in the
making of emulsion. The distilled water used as vehicle and can function as
aqueous phase (continuous phase) in oil-in-water emulsion whereas the oil as
oil phase in oil in water emulsion. Acacia are emulsifying agents used to
emulsify two immiscible liquid which are liquid and oil into a miscible form
called emulsion. The hydrophobic tails will be in contact with the oily phase
while the hydrophilic head group will be in contact with the aqueous phase.
This lower the surface tension of water molecule and provide an evenly mixing
between oil and water molecule. This make the emulsion more stable. However, it
promotes the growth of microorganism, hence antimicrobial agents should be
added to prevent the growth of the microorganisms. The antimicrobial
preservative use is alcohol. Different type of oil will have different
viscosity. The more viscous the oil , the more stable the emulsion. Different
composition of oil and water will determine the type of emulsion either oil in
water or water in oil emulsion. If there is too much oily phase in an o/w
emulsion, the emulsion will become very unstable, and phase inversion will
occur where it is converted into w/o emulsion. Hence, suitable emulsifying
agents with suitable HLB value should be selected in order to produce a stable
emulsion. Different proportion of emulsifying agents will give different
stability and emulsifying effect. Sometimes, a combination of the surfactant
can be used to improve the stability of the emulsion. Unsuitable surfactants
will produces 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 use of different type of mineral oil
will affect the physical characteristics and chemical stability of emulsion.
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.
5. What
are the functions of every type of substances used in the emulsion preparation?
How the different content of substances is affects the physical characteristics
and stability in the formulation of an emulsion?
In this emulsion
preparation, mineral oil which is inert is used as the oil phase. It can be
either continuous phase or dispersed phase based on its amount. The stable
range for disperse phase is thirty to sixty percent. If more than that, the
formulation becomes unstable. Besides that, it acts as laxative to treat
constipation.
In this experiment, acacia acts as emulsifying agent to
prevent droplets from coalescence and maintain the individual droplets in the
continuous phase. It acts by adsorbing onto the oil and water interface to
lower the surface interfacial tension. Consequently, the free energy of the
system is lowered hence stabilizes the emulsion.
Besides that, syrup plays the role as sweetening agent to
mask the non-palatable taste of the mineral oil. Syrup increase the viscosity
of the emulsion and ease of pour ability.
Vanillin is dissolved in alcohol and it is function as
flavoring agent. Alcohol is the preservativeadded to inhibit the growth of
microorganisms because in the presence of water which may provide a suitable
environment for bacteria groth.
Distilled
water is used in the emulsion as the aqueous phase, acting as a continuous
phase in which the oily phase will be homogenously dispersed, by the aid of the
surfactants.
In
this emulsion preparation, the distilled water acts as the aqueous phase while
the mineral oil acts as oily phase. Both have great influence in the physical
characteristics and stability of certain emulsion formulation.
The
amount of each phase or the volume ratio in certain emulsion is determined by
the desired type of emulsion, either be o/w or w/o emulsion. If there is too
much oily phase in an o/w emulsion, the emulsion will become very unstable, and
phase inversion will occur where it is converted into w/o emulsion.
Suitable
emulsifying agents with suitable HLB value should be selected in order to
produce a stable emulsion. This is because a combination of the surfactants
improves the stability of the emulsion. Unsuitable surfactants will produce
emulsions with different physical properties such as globule size, texture,
consistency, and oily phase dispersion which may affect the therapeutic effects
of the emulsion.
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:
--Mixture of distilled water , mineral
oil, Span 20 and Tween 80 into each test tube (refer table II).
--Close the test tubes and mix it with
vortex mixing machine.
--Preparation of a formulation of Mineral oil
Emulsion (50g) using wet gum method.
--Emulsion with Sudan III dye added.
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