OBJECTIVES:
To determine:
1. The effects
of HLB surfactant on the stability of the emulsion.
2. The effects
of different oil phase used in the formulation on the physical characteristics
and stability of the emulsion.
INTRODUCTION:
Emulsion is a
disperse system consisting of two immiscible liquids where one of them is
dispersed homogeneously in another liquid .Liquid droplets are known as
internal phase whereas the liquid in which they are dispersed are known as
external or continuous phase. Emulsion can be classified into different
emulsion phase .They are water-in-oil phase, oil-in-water phase and multiple
phase emulsion. High surface tension of emulsion leads to the instability of
emulsion. Thus, emulsifying agent is used to stabilise the emulsion. In this
experiment, Tween 80 and Span 20 are the emulsifying agent. Emulsifying
agents has hydrophilic part and
lipophilic part in which they will
adsorb onto the oil and water interface to lower down the surface tension
.Emulsifying agent can prevent coalescence of droplet .Hydrophile-lipophile
balance (HLB) is a value to select the appropriate emulsifier to produce a
stable emulsion. Every surfactant is given a number in the HLB scale, that is,
from 1 (lipophilic) to 20 (hydrophilic).A stable emulsion can be formed by
combining two emulsifying agent.HLB value for a combination of emulsifying
agent can be determined by using the following formula:
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
APPARATUS AND MATERIALS:
a. Apparatus
8 test tubes
50 ml measuring cylinder
2 sets of pasture pipettes and droppers
Vortex mixer
Weighing boat
1
set mortar and pestle
Light microscope
Microscope slide
1 set of 5 ml pipette
and bulb
1 150ml beaker
A 15 ml
centrifugation tube
Centrifugation apparatus
Viscometer
Water bath (45⁰C)
Refrigerator (4⁰C)
b.Materials
Palm oil
Arachis oil
Olive oil
Mineral oil
Distilled water
Span 20
Tween 80
Sudan
III solution (0.5 %)
PROCEDURES:
1. Each test
tube was labelled and 1cm was marked from the base of the test tube.
2. 4 ml of oil (according
to Table 1) and 4 ml of distilled water into the test tube.
Group
|
Oil
|
1,5
|
Palm oil
|
2,6
|
Arachis oil
|
3,7
|
Olive oil
|
4,8
|
Mineral oil
|
Table 1
3. Span 20 and Tween 80 were added into the mixture of
oil and water (refer Table 2) .The test tube was closed and its content was
mixed using vortex mixer for 45 seconds. The time needed for the interface to
reach 1 cm was recorded. The HLB value for each sample was determined .Steps
1-3 were repeated in order to obtain an average value of a duplicate.
Tube no.
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
Span 20
(drops)
|
15
|
12
|
12
|
6
|
6
|
3
|
0
|
0
|
Teen 80
(drops)
|
3
|
6
|
9
|
9
|
15
|
18
|
15
|
0
|
Table 2
4.
A few drops of Sudan III solution were added to (1 g) emulsion formed in
weighing boat and it was mixed homogeneously. The spread in the colour in the
sample was compared. Some of the sample was spread on a microscope slide and it
was observed under a light microscope. The appearance and globule size formed
was drawn and described.
5.
A Mineral Oil Emulsion (50g) was prepared from the formulation below by using
wet gum method according to Table 3a & 3b:
Mineral
oil
|
(refer
table 3b)
|
Acacia
|
6.25g
|
Syrup
|
5ml
|
Vanillin
|
2g
|
Alcohol
|
3ml
|
Distilled
water qs
|
50ml
|
Table 3a
Emulsion
|
Group
|
Mineral Oil (ml)
|
I
|
1,5
|
20
|
II
|
2,6
|
25
|
III
|
3,7
|
30
|
IV
|
4,8
|
35
|
Table 3b
6.
40g of emulsion was placed into a 50ml beaker and the emulsion was homogenised for
2 minutes using a vortex mixer.
7.
2g of emulsion (before and after homogenization) was taken and placed into a
weighing boat and labelled. A few drops of Sudan III solution was added and
mixed .The texture, consistency, degree of oily appearance and the spreading of
colour in the sample under the light microscope were stated and compared.
8.
The viscosity of the emulsion formed after homogenization (15g in 50 ml beaker)
was determined using a viscometer that was calibrated with “Spindle” type LV-4
.The sample was exposed to 45⁰C (water bath) for 15 minutes and then 4 ⁰C(refrigerator) for another 15 minutes. The viscosity
of the emulsion was determined after the exposure to the temperature cycle was
finished and the emulsion had reached room temperature (10-15 minutes).Step 8 was
repeated again and an average value was obtained.
CALCULATIONS:
HLB value:
RESULT:
1) PALM OIL
Tube No.
|
Span 20
(Drops)
|
Tween 80
(Drops)
|
HLB Value
|
Time For Phase Separation (min)
|
Spreading of Sudan III solution
|
||
T1
|
T2
|
Average
|
|||||
1
|
15
|
3
|
9.66
|
47.38
|
45.34
|
46.36
|
Sudan III colour dispersed in the emulsion.
|
2
|
12
|
6
|
10.73
|
45.36
|
44.34
|
45.25
|
Sudan III colour dispersed in the emulsion.
|
3
|
12
|
9
|
11.34
|
34.16
|
29.54
|
32.25
|
Sudan III colour dispersed in the emulsion.
|
4
|
6
|
9
|
12.44
|
37.15
|
39.00
|
38.08
|
Sudan III colour dispersed in the emulsion,
|
5
|
6
|
15
|
13.17
|
30.45
|
33.27
|
32.26
|
Sudan III colour dispersed in the emulsion.
|
6
|
3
|
18
|
14.08
|
23.01
|
20.47
|
23.54
|
Sudan III colour dispersed in the emulsion.
|
7
|
0
|
15
|
15.00
|
10.10
|
12.22
|
11.16
|
Sudan III colour dispersed in the emulsion
|
8
|
0
|
0
|
0.00
|
3.40
|
5.12
|
4.26
|
Sudan III colour dispersed in the emulsion
|
2) ARACHIS OIL
Tube No.
|
Span 20
(Drops)
|
Tween 80
(Drops)
|
HLB Value
|
Time For Phase Separation (min)
|
Spreading of Sudan III solution
|
||
T1
|
T2
|
Average
|
|||||
1
|
15
|
3
|
9.66
|
47.22
|
43.18
|
45.20
|
Spreading of Sudan III
is not uniform. Large globules are seen.
|
2
|
12
|
6
|
10.73
|
41.58
|
36.96
|
39.27
|
Sudan III spread rapidly but the spreading is uneven.
|
3
|
12
|
9
|
11.34
|
40.61
|
34.49
|
37.55
|
Sudan III spread slowly and evenly.
|
4
|
6
|
9
|
12.44
|
28.47
|
29.11
|
28.79
|
Sudan III spread uniformly. Large globules are seen.
|
5
|
6
|
15
|
13.17
|
19.38
|
19.00
|
19.19
|
Sudan III spread evenly. Globule sizes are relatively uniform.
|
6
|
3
|
18
|
14.08
|
10.69
|
8.25
|
9.47
|
Sudan III spread unevenly. Globules are mostly large in size.
|
7
|
0
|
15
|
15.00
|
7.34
|
5.16
|
6.25
|
Spreading is quick. Various sizes of globules unevenly
distributed.
|
8
|
0
|
0
|
0.00
|
5.12
|
4.94
|
5.03
|
Sudan III did not disperse in the emulsion.
|
3) OLIVE OIL
Tube No.
|
Span 20
(Drops)
|
Tween 80
(Drops)
|
HLB Value
|
Time For Phase Separation (min)
|
Spreading of Sudan III solution
|
||
T1
|
T2
|
Average
|
|||||
1
|
15
|
3
|
9.66
|
Interphase does not reach 1 cm after 150 minutes
|
Interphase does not reach 1 cm after 150 minutes
|
-
|
Spread very slowly
|
2
|
12
|
6
|
10.73
|
Interphase does not reach 1 cm after 150 minutes
|
Interphase does not reach 1 cm after 150 minutes
|
-
|
Spread very slowly
|
3
|
12
|
9
|
11.34
|
58.27
|
65.3
|
61.79
|
Spread moderately
|
4
|
6
|
9
|
12.44
|
15.29
|
23.45
|
19.37
|
Spread moderately
|
5
|
6
|
15
|
13.17
|
87.2
|
79.34
|
83.27
|
Spread moderately
|
6
|
3
|
18
|
14.08
|
62.29
|
58.1
|
60.2
|
Spread moderately
|
7
|
0
|
15
|
15.00
|
32.17
|
40.55
|
36.36
|
Spread moderately
|
8
|
0
|
0
|
0.00
|
1.2
|
2.4
|
1.8
|
Spread immediately
|
4) MINERAL OIL
Tube No.
|
Span 20
(Drops)
|
Tween 80
(Drops)
|
HLB Value
|
Time For Phase Separation (min)
|
Spreading of Sudan III solution
|
||
T1
|
T2
|
Average
|
|||||
1
|
15
|
3
|
9.66
|
Interphase does not reach 1 cm after 150 minutes
|
Interphase does not reach 1 cm after 150 minutes
|
-
|
Spread very slowly
|
2
|
12
|
6
|
10.73
|
Interphase does not reach 1 cm after 150 minutes
|
Interphase does not reach 1 cm after 150 minutes
|
-
|
Spread very slowly
|
3
|
12
|
9
|
11.34
|
110
|
115
|
112.5
|
Spread slowly
|
4
|
6
|
9
|
12.44
|
101.2
|
104.3
|
102.75
|
Spread slowly
|
5
|
6
|
15
|
13.17
|
87.4
|
89.4
|
88.4
|
Spread moderately
|
6
|
3
|
18
|
14.08
|
69.2
|
70.2
|
69.7
|
Spread moderately
|
7
|
0
|
15
|
15.00
|
19.6
|
51.7
|
35.65
|
Spread slowly
|
8
|
0
|
0
|
0.00
|
5.12
|
1.55
|
3.3
|
Spread immediately
|
Test tube
|
Image under microscope
|
Physical Appearance
|
Colour distribution
|
1
|
 |
There were different sizes of globules and were not
in a proper distribution.
|
The Sudan III solution were well dispersed in the
emulsion. The colour of emulsion was light orange.
|
2
|
 |
Most of the globules were large in size. However,
there were also some in smaller size than the rest.
|
The Sudan III solution were well dispersed in the
emulsion. The colour of emulsion was light orange.
|
3
|
 |
Globules were irregular in sizes and packed to each
other.
|
The Sudan III solution were well dispersed in the
emulsion. The colour of emulsion was light orange.
|
4
|
 |
Globules were irregular in sizes and very packed to
each other.
|
The Sudan III solution were well dispersed in the
emulsion. The colour of emulsion was a bit bright orange.
|
5
|
 |
Size of globules were larger and the water droplets
did not dispersed well in the oil.
|
The Sudan III solution dispersed well in the emulsion. The colour was light orange.
|
6
|
 |
The size of globules were small and packed. The
water droplets were not evenly distributed in the oil.
|
The Sudan III solution dispersed well in the
emulsion. The colour was very light orange, almost white.
|
7
|
 |
There were large and small size of globules. There
was also, a big globule with black ring outside of it. The globules were
packed to each other.
|
The Sudan III solution dispersed well in the
emulsion. The colour of the emulsion was orange.
|
8
|
 |
The globules were very irregular and different in
sizes. The water droplets were not distributed well in the oil.
|
The Sudan III solution dispersed well in the
emulsion. The colour of emulsion was light orange.
|
RESULT PART 2:
Comparation of
emulsion before and after homogenization
Before
homogenization
|
After
homogenization
|
|
Texture
|
Coarse
|
Smooth
|
Consistency
|
Less consistency
|
More consistency
|
Degree of oily appearance
|
Oily
|
Less oily
|
Spreading of color in the sample under
light microscope
|
 |
 |
Viscosity of
Emulsion
Readings
|
Average
Viscosity (Cp)
|
|||
Emulsion I
|
Emulsion II
|
Emulsion III
|
Emulsion IV
|
|
Before Temperature Cycle
|
1826
|
1311.5
|
1312
|
768
|
After Temperature Cycle
|
4689
|
1309
|
1450
|
452
|
Difference
(%)
|
61.06
|
2.5
|
10.52
|
69.91
|
Phase of
Separation
Mineral Oil
(mL)
|
Distance of separation phase (cm), y
|
Average
|
Ration of separation phase= y/x
|
20
|
 2.7 2.9 |
2.80
|
2.80/4.6=1.61
|
25
|
2.6 4.0
|
3.3
|
3.3/5
=0.66
|
30
|
3.0 2.0
|
2.50
|
2.50/4=0.63
|
35
|
3.7 3.2
|
3.45
|
3.45/5=0.69
|
x= distance
before separation
y=distance after
separation
DISCUSSION:
HLB VALUES
A physically stable emulsion is one in
which the dispersed phase retains its initial character, (e.g. size) and
remains uniformly dispersed. There are a few instabilities in emulsion that
include flocculation and creaming, coalescence and breaking, and Ostwald
ripening. To overcome these physical instabilities, knowledge on Stoke’s Law is
applied, for example produce emulsion with small droplet size, control
viscosity of continuous phase, control phase volume ratio and reduce density
difference between phases.
A suitable surfactant can maintain the stability of
emulsion so that the size of droplets does not change significantly with time. HLB
(Hydrophile-Lipophile Balance) is an empirical expression for the relationship
of the hydrophilic ("water-loving") and hydrophobic
("water-hating") groups of a surfactant. The higher the HLB value of
a surfactant, the more water-soluble the surfactant is. Water-in-oil emulsions
(w/o) require low HLB surfactants. Oil-in-water (o/w) emulsions often require
higher HLB surfactants. All emulsifiers consist of a molecule that combines
both hydrophilic and lipophilic groups. The HLB number which is higher than 10
is considered to be more hydrophilic and the surfactant is more polar. On the
other hand, the HLB number which is lower than 10 is considered to be more
lipophilic and the surfactant is less polar.
Surfactant selection for an o/w emulsion
can be simplified if the HLB system is applied. Oils have required HLB numbers
that identify the HLB necessary to give good o/w emulsification. Not all
surfactants having the same HLB value may be acceptable for an o/w emulsion.
HLB values for surfactants can be calculated and they are additive. For
example, if two different surfactants or oils are present, the HLB value can be
calculated by using the formula:
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
Stable emulsions can be
obtained by adding surfactant with lower HLB values and thereby, retard the
rate of creaming. The stable emulsion is said to take longest time for the
phase separation to occur. Based on the experiment, the time taken for the
emulsion to separate into two phases and for the interface to reach 1cm is
longer in mixture of surfactants with lower HLB value, meaning they are more
lipophilic. From the result, we can observe that arachis oil, olive oil,
mineral oil and palm oil show the best stability in emulsion when the HLB value
of surfactant is the 9.66, meaning the lowest value not counting tube 8 without
surfactant. Besides, the emulsions from tube 7 and tube 8 took the shortest time
to separate. This is due to the absence of any surfactant in tube 8 while in
tube 7 only Tween 80 present, with a high HLB value. This proves that the
emulsion will be unstable if only a single surfactant is used in the emulsion and
a suitable combination of surfactants is needed to produce a stable emulsion. Therefore,
the lower the HLB value of the combination of surfactant used, the more stable
the water in oil emulsion is formed.
From the result collected, majority of them
seem to prove this statement right. The inconsistency may be due to errors made
while carrying the experiment, for example when mixing procedures and measuring
the amount of surfactant used. Type of oil used also plays an important role in
determining the stability and viscosity of the emulsion. From the result
obtained, the emulsion using mineral oil as the oil phase is the most stable.
As a conclusion, the more stable an emulsion is, the longer the time taken for
phase separation to occur.
RATIO OF SEPARATION PHASE
Emulsion is a mixture
of aqueous (water) phase and oil phase and it needs an emulsifying agent for
the emulsion to stay in one phase and not separated during after the process of
manufacturing until it is marketed and used.
The optimum stability
of emulsions (Oil in water) are stabilized by 1:1 molar ratios of emulsifying
agent (Spans and Tweens). This is due to association between the emulsifier
molecules adsorbed at the oil-water interface. Thus, stability of the emulsion
can be determined by investigating the rate of sedimentation.
The
method used is centrifugation. Centrifugation refers to the process which
involves the application of the centripetal force for the sedimentation of
heterogeneous mixtures with a centrifuge, and is often used in industrial and
laboratory settings.
This experiment uses
centrifugation method to separate the emulsion into 2 phases which are
immiscible. The fast separation of the emulsion occurs as a result of centripetal
acceleration force. With this, we are able to determine the stability of the
emulsion by calculating the ratio of separation phase. Ratio of separation
phase refers to the ratio of height of separated phase to the height of the
original emulsion. The larger the ratio of separation phase, the more unstable
emulsion in the experiment is.
The stability of the
dispersed system can be determined based on the ratio of separation phase. In the results, the emulsion with 30 ml of
mineral oil shows the least ratio of separation, hence it has the greatest
stability among the other emulsions. However, this result may be inaccurate as
the evaluation procedures may results in the damage of emulsion structure.
Emulsifying agents or
better known as surfactants are compounds that lower the surface tension (or
interfacial tension) between two liquids or between a liquid and a solid. Surfactants
are added to stabilize the emulsion prepared. From this experiment, we get to
know that acacia is one of the important emulsifying agent used in the
preparation of the emulsion. It will stabilized the emulsion by forming a
stable, mechanically strong film of emulsifiers around the globules of
dispersed oil across the aqueous phase in the emulsion. This helps to prevent
the coalescence of oil globules by hindering the drainage of water between
globules with the hydration of films. The increasing volume of the oil phase with
constant amount of acacia will lead to the production of unstable emulsion as a
result of coalescence and breaking of emulsion. This is because there is lesser
capacity of the acacia to form a stable mechanically strong film for the
mineral oil across the water phase in the emulsion. Thus, we can conclude that
the increase the volume of the mineral oil will results in the greater
separation of the emulsion after centrifugation.
VICOSITY OF THE EMULSION
Theoretically,
the higher the content of oil, the more viscous the emulsion is. This is because
the oil always has higher viscosity compared to water. Four emulsions are made
with the content of mineral oil: 20ml, 25ml, 30ml and 35ml respectively.
We can expect
that emulsion with 35ml of mineral oil would have the highest viscosity in
comparison with other emulsion with lower amount of mineral oil. However, in
real experiment, both the viscosities measured on the emulsions before and after
temperature cycles show deviation from the theory stated.
For example, 20ml
mineral oil emulsion shows greater viscosity (1826 cP) compared to 35ml mineral
oil emulsion (768 cP) before the temperature cycle. Next, temperature cycles
are carried out and then respective viscosities are obtained again.
Theoretically, when we are using the same type of oil (mineral oil), the
viscosity of the particular emulsion will increase after the temperature cycle
and shows higher viscosity (cP) value than the one before temperature cycle.
Emulsion is put into the water bath (45°C) for 15 minute in the first stage of
temperature circle and then placed into the freezer (40°C) for 15 minutes. At
low temperature, kinetic energy of the system is reduced and this will increase
the viscosity of the continuous phase. This will decrease the motion of the
globules in the disperse phase. However, deviation of viscosity value once
again appears. For instance, emulsion with 35ml mineral oil shows reduction in viscosity
after passing through the temperature cycle, from 768cP to 452cP. Hence, there are
errors when the experiment is being carried out which cause the deviation to
occur.
These errors include the incorrect measuring technique causing
parallax error and the usage of an unsuitable viscometer spindle. Besides, the
errors may arise during the preparation of emulsion, for example inaccurate
weighing of excipients and more importantly, the active ingredients, especially
oil.
THE CHARACTERISTICS OF THE EMULSION
In this experiment, 8
emulsions are prepared in different test tubes.
Each emulsions is prepared based on different proportion volume of Span
20 and Tween 80 where both of them act as surfactant in the emulsions.
Based on the
observation, Emulsion 1 has both large and small globules which are loosely
packed. Meanwhile, Emulsion 2 is similar to Emulsion 1 at which small and large
globules are present and packed in a loose manner. However, Emulsion 3 has
irregular shape and size of globules but the globules are closely packed.
Emulsion 4 has several bigger molecule which surrounded by small globule around
it and the globules either large or small are regular in shape. Both Emulsion 5
and 6 have a large globules that are arranged closely with small globules
surround it. There are many large globules present in Emulsion 7 compare to
other emulsion. For Emulsion 8, there are few regular sized globules as this
emulsion contain mineral oil and distilled water only.
Sudan dyes is a solvent
dyes often called lysochromes. It is not soluble in water, however it is
oil-soluble. Thus, it is used to colour non-polar substances. Sudan dyes have
high affinity to fats and therefore they are used to demonstrate triglycerides,
lipids, and lipoproteins. The Sudan III solution is red in colour and dissolves
readily in oil. It is added into the emulsion to indicate the position of oily
globules in the emulsion. It will dissolve in oil phase and stain the oil
globule red in colour while the aqueous
phase will not be stained and remain colourless. Hence, Sudan III solution is
used as indicator to determine the types of emulsion whether they are
oil-in-water (o/w) emulsion or water-in-oil (w/o) emulsion.
HLB
(Hydrophile-Lipophile Balance) is an empirical expression for the relationship
of the hydrophilic ("water-loving") and hydrophobic
("water-hating") groups of a surfactant. Low HLB value is required in
preparing a water in oil emulsion, whereas for oil in water emulsion, it requires
high HLB value. The higher the HLB value, the more water-soluble the
surfactant. Thus, the emulsion gets more hydrophilic when the value of HLB
increases. This explained the reason why the intensity of colour decrease from
Emulsion 1 to Emulsion 7 (HLB value increases from Emulsion 1 to Emulsion 7). The
HLB value is for Emulsion 8 is 0. This emulsion is said to be lipophilic.
Therefore, the Sudan III dye is readily dissolve and has the highest intensity
colour of reddish brown.
CONCLUSION:
Combining two surfactants will give a stable emulsion. The higher the volume of oil, the higher the viscosity of the emulsion and the distance of separation of emulsion is higher.
1. Griffin, W. C. Classification of Surface Active Agents by HLB. J. Soc. Cosmet. Chem. 1949, 1, 311-326.
3. Griffin, W. C. Calculation of HLB valuess of Nonionic Surfactants, J. Soc. Cosmet. Chem. 1954, 5, 249-256.
