Practical 2
Title
Characterisation of Emulsion Formulations
Objective
To determine:
1. The effects of HLB surfactant on the stability of the emulsion.
2. The effects of different oil phases used in the formulation on the physical characteristics and stability of the emulsion.
Introduction
Emulsion is a two-phase system that is not stable thermodynamically. It contains of a mixture of aqueous phase and oil or at least two immiscible liquids where one of them (internal/dispersed phase) is dispersed homogenously in another liquid (external/continuous phase). There are 4 types of emulsion which are oil-in-water emulsion (o/w) or water-in-oil emulsion (w/o), multiple emulsion, microemulsion and nanoemulsion. Emulsion is stabilised by adding emulsifying agent. The HLB method (hydrophilic-lipophilic balance) is used to determine the quantity and type of surfactant that is needed to prepare a stable emulsion. The higher the HLB number, the more hydrophilic the surfactant is. Every surfactant is given a number in the HLB scale, that is, from 1 (lipophilic) to 20 (hydrophilic). Usually it is better to have a mixture of 2 emulsifying agents such as oil soluble (low HLB) and a water soluble (high HLB) surfactant to form a more stable emulsion. HLB value for a combination of emulsifying agents can be determined by using the following formula:
HLB value =
|
(quantity surfactant 1)(HLB surfactant 1) +( quantity surfactant 2)(HLB surfactant 2)
|
Quantity surfactant 1 + quantity surfactant 2
|
Apparatus and Material
a. Apparatus
8 Test tubes 1 set of 5ml pipette and bulb
A 50ml measuring cylinder 1 50ml beaker
2 sets of pasture pipettes and droppers A 15ml centrifugation tube
Vortex mixer Centrifugation apparatus
Weighing boat Viscometer
1 set of mortar and pestle Water bath (45°C)
Light microscope Refrigerator (4°C)
Microscope slides
b. Materials
Palm oil Span 20
Arachis oil Tween 80
Olive oil Sudan III solution (0.5%)
Mineral oil
Distilled water
Procedures
1. Each test tube is labelled and marked 1cm from the base of the test tube.
2. 4ml of mineral oil and 4ml of distilled water are mixed into the test tube.
3. Span 20 and Tween 80 are added into the mixture of mineral oil and water (refer Table 2). The test tube is closed and its contents are mixed with vortex mixer for 45 seconds. The time needed for the interface to reach 1cm is recorded. The HLB value for each sample is determined. Step 1-3 are repeated in order to obtain an average HLB value of a duplicate.
Table 2
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
|
4. A few drops of Sudan III solution are added to (1g) emulsion formed in a weighing boat and are mixed homogenously. The spread of the colour in the sample is compared. Some of the sample is spread on a microscope slide and it is observed under light microscope. The appearance and globule size formed are drawn and described.
5. A Mineral Oil Emulsion (50g) is prepared from the formulation below by using wet gum method according to Table 3a & 3b:
Table 3a
Mineral Oil
|
(refer Table 3b)
|
Acacia
|
6.25 g
|
Syrup
|
5 ml
|
Vanillin
|
2 g
|
Alcohol
|
3 ml
|
Distilled water qs
|
50 ml
|
Table 3b
Emulsion
|
Group
|
Mineral Oil (ml)
|
I
|
1,5
|
20
|
II
|
2,6
|
25
|
III
|
3,7
|
30
|
IV
|
4,8
|
35
|
6. 40g of emulsion is placed into a 50ml beaker and it is homogenized for 2 minutes using a vortex mixer.
7. 2g of emulsion (before and after homogenization) is taken and placed into a weighing boat and it is labelled. A few drops of Sudan III solution are added and mixed. The texture, consistency, degree of oily appearance and the spreading of colour in the sample are stated and compared under the light microscope.
8. The viscosity of the emulsion formed is determined after homogenization (15g in 50ml beaker) using a viscometer that is calibrated with “Spindle” type LV-4. Expose the sample to 45°C (water bath) for 15 minutes and then to 4°C (refrigerator) for another 15 minutes. After the exposure to the temperature cycle is finished and the emulsion had reached room temperature (10-15 minutes), the viscosity of the emulsion is determined. Step 8 is repeated again and an average value is obtained.
9. 5g of homogenised emulsion is placed into a centrifugation tube and centrifuged (4500 rpm, 10 minutes, 25°C). The height of the separation formed is measured and the ratio of the height separation is determined.
Result
PALM OIL (GROUP 1 OR 5)
Tube No.
|
Span 20
(Drops)
|
Tween 80
(Drops)
|
HLB Value
|
Time For Phase Separation (min)
| ||
T1
|
T2
|
Average
| ||||
1
|
15
|
3
|
9.66
|
47.38
|
45.34
|
46.36
|
2
|
12
|
6
|
10.73
|
45.36
|
44.34
|
45.25
|
3
|
12
|
9
|
11.34
|
34.16
|
29.54
|
32.25
|
4
|
6
|
9
|
12.44
|
37.15
|
39.00
|
38.08
|
5
|
6
|
15
|
13.17
|
30.45
|
33.27
|
32.26
|
6
|
3
|
18
|
14.08
|
23.01
|
20.47
|
23.54
|
7
|
0
|
15
|
15.00
|
10.10
|
12.22
|
11.16
|
8
|
0
|
0
|
0.00
|
3.40
|
5.12
|
4.26
|
ARACHIS OIL (GROUP 2 OR 6)
Tube No.
|
Span 20
(Drops)
|
Tween 80
(Drops)
|
HLB Value
|
Time For Phase Separation (min)
| ||
T1
|
T2
|
Average
| ||||
1
|
15
|
3
|
9.66
|
47.22
|
43.18
|
45.20
|
2
|
12
|
6
|
10.73
|
41.58
|
36.96
|
39.27
|
3
|
12
|
9
|
11.34
|
40.61
|
34.49
|
37.55
|
4
|
6
|
9
|
12.44
|
28.47
|
29.11
|
28.79
|
5
|
6
|
15
|
13.17
|
19.38
|
19.00
|
19.19
|
6
|
3
|
18
|
14.08
|
10.69
|
8.25
|
9.47
|
7
|
0
|
15
|
15.00
|
7.34
|
5.16
|
6.25
|
8
|
0
|
0
|
0.00
|
5.12
|
4.94
|
5.03
|
OLIVE OIL (GROUP 3 OR 7)
Tube No.
|
Span 20
(Drops)
|
Tween 80
(Drops)
|
HLB Value
|
Time For Phase Separation (min)
| ||
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
|
-
|
2
|
12
|
6
|
10.73
|
Interphase does not reach 1 cm after 150 minutes
|
Interphase does not reach 1 cm after 150 minutes
|
-
|
3
|
12
|
9
|
11.34
|
58.27
|
65.3
|
61.79
|
4
|
6
|
9
|
12.44
|
15.29
|
23.45
|
19.37
|
5
|
6
|
15
|
13.17
|
87.2
|
79.34
|
83.27
|
6
|
3
|
18
|
14.08
|
62.29
|
58.1
|
60.2
|
7
|
0
|
15
|
15.00
|
32.17
|
40.55
|
36.36
|
8
|
0
|
0
|
0.00
|
1.2
|
2.4
|
1.8
|
MINERAL OIL (GROUP 4 & 8)
Tube No.
|
Span 20
(Drops)
|
Tween 80
(Drops)
|
HLB Value
|
Time For Phase Separation (min)
| ||
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
|
-
|
2
|
12
|
6
|
10.73
|
Interphase does not reach 1 cm after 150 minutes
|
Interphase does not reach 1 cm after 150 minutes
|
-
|
3
|
12
|
9
|
11.34
|
110
|
115
|
112.5
|
4
|
6
|
9
|
12.44
|
101.2
|
104.3
|
102.75
|
5
|
6
|
15
|
13.17
|
87.4
|
89.4
|
88.4
|
6
|
3
|
18
|
14.08
|
69.2
|
70.2
|
69.7
|
7
|
0
|
15
|
15.00
|
19.6
|
51.7
|
35.65
|
8
|
0
|
0
|
0.00
|
5.12
|
1.55
|
3.3
|
Palm Oil
Arachis Oil
Olive Oil
Mineral Oil
Emulsion I (group 1 & 5) – 20ml Mineral Oil
Characteristics
|
Before homogenisation
|
After homogenisation
|
Texture
|
Coarse
and not homogenous
|
Smooth
and even looking, homogenous
|
Consistency
|
Less
consistent
|
More
consistent
|
Degree
of oily appearance
|
More
oily and globules
|
Less
oily and globules
|
Spreading
of colour
|
Spread
evenly
|
Spread
evenly
|
Emulsion II (group 2 & 6) – 25ml
Mineral Oil
Characteristics
|
Before homogenisation
|
After homogenisation
|
Texture
|
Coarse
|
Smooth
|
consistency
|
Less
consistent, more dilute
|
More
consistent, more viscous
|
Degree
of oily appearance
|
More
oily, spherical globules
|
Less
oily, spherical globules
|
Spreading
of colour
|
Spread
evenly
|
Spread
evenly
|
Emulsion III (group 3 & 7) – 30ml
Mineral Oil
Characteristics
|
Before homogenisation
|
After homogenisation
|
Texture
|
Coarse and non-homogenous, cloudy
|
Milky, smooth and homogenous
|
Consistency
|
Not consistent, less viscous
|
Consistent, more viscous
|
Degree
of oily appearance
|
Greasier, more globules
|
Less greasy, less globules
|
Spreading
of colour
|
Unevenly dispersed color, less red spot
|
Evenly dispersed color, more red spot
|
Emulsion IV (group 4 and 8) – 35ml Mineral
Oil
Characteristics
|
Before homogenisation
|
After homogenisation
|
Texture
|
Not
homogenous, coarse and less milky
|
Homogenous,
smooth and more milky
|
consistency
|
Not
consistent and less viscous
|
Consistent
and more viscous
|
Degree
of oily appearance
|
More
greasy and more globules
|
Less
greasy and less globules
|
Spreading
of colour
|
Unevenly
distribution
 |
Evenly
distribution
 |
Viscosity
Own
Group (Group 4 – 35ml Mineral Oil)
Table 4
Readings
|
Viscosity (cP)
|
Average
|
|||||
1
|
2
|
3
|
4
|
5
|
6
|
||
Before Temperature
Cycle
|
792
|
786
|
792
|
750
|
708
|
774
|
768
|
After
Temperature
Cycle
|
540
|
474
|
456
|
408
|
432
|
402
|
452
|
Difference (%)
|
 |
||||||
Comparison
with other group:
Readings
|
Average Viscosity
(cP)
|
|||
Emulsion I
(20ml mineral oil)
|
Emulsion II
(25ml mineral oil)
|
Emulsion III
(30ml mineral oil)
|
Emulsion IV
(35ml mineral oil)
|
|
Before
Temperature
Cycle
|
1826
|
1311.5
|
1312
|
768
|
After
Temperature
Cycle
|
4689
|
1309
|
1450
|
452
|
Difference
(%)
|
156.79
|
0.19
|
10.52
|
69.91
|
Phase Separation
Table 5
x= Distance before separation
y= Distance after separation
Determination of HLB value
The
HLB method (hydrophilic-lipophilic balance) is used to determine the quantity
and type of surfactant that is needed to prepare a stable emulsion. The HLB value which is greater than 10 is
hydrophilic while the HLB value which is smaller than 10 is lipophilic. Span 20
and Tween 80 are surfactants that are used in this experiment. Span 20 is
hydrophobic and it is used for w/o emulsion while Tween 80 is hydrophilic and
is used for o/w emulsion. Therefore, a combination of Span 20 and Tween 80
offers an appropriate HLB value.
In this experiment, different
proportions of Span 20 and Tween 80 were used to determine the most appropriate
HLB value of surfactants that can produce the emulsion with greatest stability.
The stability of an emulsion formed is indicated by the time needed for the
interface to reach 1cm. An emulsion is stable and suitable to be used if it
needs a long time for phase separation to occur. Based on the result of the
experiment, the most suitable HLB value for palm oil are 9.66 and 10.73. The
most suitable HLB value for arachis oil are 9.66 and 10.73. The most suitable
HLB value for olive oil are 11.34 and 13.17. The most suitable HLB value for
mineral oil are 11.34, 12.44 and 13.17. Since most of the HLB value of stable
emulsions exceeds 10, it can be deduced that the emulsifiers used are slightly
hydrophilic and the emulsions formed are o/w emulsions. Tube 8 for each type of
oils were prepared to prove the importance of surfactants. Without the
surfactants, the emulsions will separate into two phases in a short time. The
emulsions will also be not stable if only one type of surfactant is used as the
single surfactant may be too hydrophilic or too hydrophobic.
Some experimental errors occur
during this part of experiment. As Span 20 and Tween 80 are added into the
emulsions in drops, the amount of surfactants added into the emulsions may not
be accurate since the size of surfactant droplets are not constant. This may
lead to inaccuracy in the result of the experiment. Besides, parallax error may
occur while determining the 1cm phase separation of the emulsions.
Addition of Sudan III solution and
determination of globule size
This is the molecular
structure of Sudan III. Sudan III is a lysochrome, fat-soluble
dye diazo dye. It is used
for staining of triglycerides in frozen sections and some
protein bound lipids and lipoproteins on paraffin sections. Sudan ΙΙΙ solution
is used in this experiment to indicate the position of oily globules in the
emulsion. Sudan ΙΙΙ solution has a red colour and it will dissolve in the oil
phase to give a red colour to the oily globules. The aqueous phase globules
will remain colourless. Hence,
in this experiment, Sudan III is also used to determine the type of
emulsion formed either oil in water (o/w) emulsion or water in oil (w/o)
emulsion. Oil in water (o/w) emulsion has red globules on a colourless
background while water in oil (w/o) emulsion has colourless globules on a red
background.
Mineral oil is used in
this experiment for our group. Based on the result of the experiment, the
emulsions in tubes 1, 3, 6, 7 and 8 are water in oil emulsions. The emulsions
in tubes 2, 4 and 5 are oil in water emulsions.
The globules of emulsion
in tube 1 are of small size and evenly distributed while the globules of
emulsion in tube 2 are of smallest size. The globules of emulsion in tube 3 are
of biggest size compared to all the globules of emulsion in other tubes. It can
be deduced that the globules in tube 3 have undergone coalescence and the
emulsion in tube 3 are not a preferable emulsion. Emulsions in tube 1 and 2 are
better emulsions.
For the globules in
emulsion from tube 4 to tube 7, the distance between each globules become
closer and closer from tube 4 to tube 7. This occurrence is known as
flocculation.
Starting from tube 5,
more Tween 80 is used as surfactant in terms of the proportions in the
emulsions. This causes the emulsions to be less stable. In tube 4, the globules
are large to small sized. In tube 5, there are also both medium size and small
size globules.
In
tube 7, where only Tween 80 is used as surfactant, the emulsion formed is very
unstable. The globules formed are irregular in shape and size. The globules are
flocculated and coalescence also occurs.
In tube 8, as no surfactants are used, the
Sudan III solution has no medium to disperse but end up forming a layer on the
emulsion. The particles as seen under the microscope are the particles in the
aqueous phases and none of the particles are appeared to be stained by the
Sudan III solution.
Before and after homogenization
From this experiment,
there is a significant change on emulsion before and after homogenization.
Before homogenization, the colour distribution is uneven and the emulsion is
not broken into smaller globules. Large and inconsistent globules are observed
under microscope and the emulsion is greasy. The emulsion formed before
homogenization is considered as water in oil (w/o) emulsion. However, after
homogenization, the colour dispersion is even under light microscope and the
smaller globules are observed. The emulsion is homogenous and milky. It is less
greasy. Hence, oil in water (o/w) emulsion is formed after homogenization. Homogenization
process makes the oily globules more stable in the aqueous phase. It can be
concluded that phase inversion occurred before and after homogenization.
Viscosity
Readings
|
Average Viscosity (cP)
|
|||
Emulsion I
(20ml mineral oil)
|
Emulsion II
(25ml mineral oil)
|
Emulsion III
(30ml mineral oil)
|
Emulsion IV
(35ml mineral oil)
|
|
Before
Temperature
Cycle
|
1826
|
1311.5
|
1312
|
768
|
After
Temperature
Cycle
|
4689
|
1309
|
1450
|
452
|
Difference (%)
|
156.79
|
0.19
|
10.52
|
69.91
|
Graph of viscosity before and after the
temperature cycle vs. the amount of mineral oil.
From the graph, the
viscosity of the emulsion at room temperature and after subjected to
temperature cycle is different according to different amount of oil. Before
temperature cycle, the viscosity of the emulsion with 20 ml of mineral oil is
the highest, which is 1826, followed by 25 ml mineral oil and 30 ml mineral oil.
The lowest value of viscosity before temperature cycle is 35 ml mineral oil.
However, the viscosity for all the emulsion increases after temperature cycle
except for 25 ml mineral oil and 35 ml mineral oil. The highest value is still
the 20 ml mineral oil (4689) and the lowest is the 35 ml mineral oil (452).
Theoretically, the temperature increases, the kinetic energy of the disperse
droplets increases, thus the viscosity decreases. When the emulsions were
placed in the refrigerator at about 4ºC for 30 minutes, the kinetic energy of
the system decreases and thus result in coalescence. This is because the rate
of migration of the globules in the disperse phase decreases. The difference in
viscosity of the emulsion is due to the different amount of mineral oil used.
There are some random errors occur throughout this experiment that cause the
results to appear inaccurately. Hence, several precautions should be taken
throughout this experiment. First, the emulsion should be stirred thoroughly
before getting the viscosity readings from the viscometer. Next, the emulsion
should be placed in the correct position and within the gap of the rotor of the
viscometer. Furthermore, the rotor in the viscometer should be cleaned by
dipping it in the distilled water before taking the following readings.
Graph of viscosity difference (%) against amount of mineral oil added.
From the graph above,
20 ml of mineral oil shows the highest viscosity difference which is 156.79%. The
amount of mineral oil used contribute to the different viscosities. This
experiment should be carried out using the same type of oil. The higher amount
of oil globules in the continuous phase will increases the viscosity of the
emulsion. Theoretically, the graph will show directly proportional graph, which
the difference of viscosity is directly proportional to the amount of oil.
Centrifugation
Graph
of separated phase ratio formed from the centrifugation process versus the
different amount of oil
From the graph shown
above, 35ml mineral oil has higher ratio of phase separation than the other
amount of mineral oil. High ratio of phase separation shows that it is an unstable
emulsion. Unstable emulsion will give the detrimental effect such as increase
tendency of inappropriate dose deliver to the patient. Hence, the phase
separation ratio must be kept at the minimum level all the time to ensure the
emulsion is stable and homogenous, not easily coalesce and undergo phase
separation to form two layer.
There are many
ingredients used in this experiment. Acacia is a natural material that is often
used as an emulsifying agent during the preparation of emulsions. It is used to
increase the viscosity among the interphase of the oily and aqueous phase. A
hydrophilic barrier is formed between the oil and aqueous phases and thus
coalescence is restricted.
Vanillin serves as
vanilla taste flavouring agent. Syrup in this preparation acts as sweetener as
it will help to mask non-palatable taste of the oil in order to increase
patients’ compliance but is not suitable for diabetic patients. Alcohol acts as
a preservative which help to prevent the growth of microorganisms that may be
present in the raw material. The emulsion will have stable physicochemical
properties for longer duration.
Mineral oil form the
dispersed phase in the oil in water emulsion (o/w emulsion). The ratio of the
oil and water in the preparation of emulsion must be specified to prevent the
phase inversion to occur. Emulsion with more than 70% dispersed phase may
undergo phase inversion.
Viscosity of the
emulsion will be affected on the physical stability and the rheological characteristic
of the emulsion. We have to consider the ease of pouring out the emulsion from
the container. Therefore, the viscosity of the emulsion has to be strictly
monitored to avoid the rheological problem.
Conclusion
In conclusion, emulsion stability is affected by
the HLB of the surfactant and the oil/surfactant ratio. The most stable emulsion requires a
combination of surfactants which has larger volume of Span-20 compared to Tween
80. The emulsion produced is not stable when single type of surfactant is used.
This proves that the emulsion formed is oil in water emulsion. Different oils
have different properties and thus different viscosities. Furthermore, higher oil content is desirable in order
to dissolve more drugs hence will increases the viscosity of the
emulsion.
References
