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 at least
two immiscible liquids. The
dispersed liquid is known as the internal or
discontinuous phase,
whereas the dispersion medium is known as the external or
continuous phase. Where oils, petroleum hydrocarbons, and/or
waxes are the dispersed phase, and water or an aqueous solution is the
continuous phase, the system is called an oil-in-water (o/w) emulsion. Conversely, where water or aqueous solutions are
dispersed in an oleaginous medium, the system is known as water-in-oil (w/o)
emulsion.
Emulsions are used in many routes of
administration. Oral administration can be used, but patients generally object
to the oily feel of emulsions in the mouth. But sometimes, emulsions are the
formulation of choice to mask the taste of a very bitter drug or when the oral
solubility or bioavailability of a drug is to be dramatically increased.
Emulsions are, by nature, physically
unstable; that is, they tend to separate into two distinct phases or layers
over time. Thus, emulsion is stabilised by adding
emulsifying agent. Griffin (1954) introduced a very useful system for the classification
of surfactants on the basis of their solubility in water. The numerical values
in this system are called hydrophylic-lipophylic balance (HLB) that denotes the
relative affinity of the surfactant for oil and water. (Khan et. Al.,2011). Every
surfactant is given a number in the HLB scale, that is, from 1 (lipophilic) to
20 (hydrophilic). Usually a combination of 2 emulsifying agent is used 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
|
The mechanism of action of emulsifying agents
depends upon the formation of film they form at the interface of two phases.
There are three types of films formed by emulsifying agents (Paul, 2005). These
includes monomolecular film, multimolecular film and solid particle film. These
types od film form influence the properties of emulsifying agent in order to
achieve the stable form of emulsion with different mechanisms.
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 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 was labelled and marked 1cm from the base of the test tube.
2. 4ml
of oil (according to table 1) and 4ml of distilled water were mixed 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 referring to Table
2. The test tube was closed and its content was mix with vortex mixture for 45
seconds. The time needed for the interface to reach 1cm was recorded. The HLB value for each sample was determined.
4. Step
1-3 was repeated in order to obtain an average HLB 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
|
Tween 80 (drops)
|
3
|
6
|
9
|
9
|
15
|
18
|
15
|
0
|
Table 2
5. A
few drops of Sudan III solution was added to 1g emulsion formed in a weighing
boat and mixed homogenously. The spread of the colour in the sample was
observed. Some of the sample were spread on a microscope slide and observed
under light microscope. The appearance and globule size formed was drawn and
described.
6. A
50g Mineral Oil Emulsion was prepared from the formulation below by using wet
gum method according to table 3a&3b :
Mineral Oil
|
(refer Table 3b)
|
Acacia
|
6.25 g
|
Syrup
|
5 ml
|
Vanillin
|
2 g
|
Alcohol
|
3 ml
|
Distilled water qs
|
50 ml
|
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
7. 40g
of emulsion was placed into a 50ml beaker and homogenized for 2 minutes using a
vortex mixer.
8. 2g
of emulsion was taken (before and after homogenization) and placed into
weighing boat and labeled. 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 was stated and compared under the light microscope.
9. By
using 5g of emulsion in 50ml beaker, the viscosity of the emulsion formed was
determined after homogenization using a viscometer that is calibrated with
“Spindle” type LV-4. The sample was exposed to 45°C of water bath for 15
minutes and then to 4°C in refrigerator for another 15 minutes. After the
exposure to the temperature cycle was finished and the emulsion had reached
room temperature, the viscosity of the emulsion was determined.
10. Step
9 was repeated to obtain an average value.
11. 5g
of homogenized emulsion was placed into a centrifugation tube and centrifuged
at 4500rpm for 10 minutes with temperature of 25°C. The height of the
separation formed was measured and the ratio of the height separation was
determined.
Draw and describe the appearance and
globule size formed.
Magnification (40 x 10)
|
Physical appearance
|
Colour distribution
|
 |
Phase separation occurs rapidly.
|
Sudan III is being dispersed. The
emulsion is being stained light orange.
|
 |
The water phase are not properly
dispersed in the oil phase.
|
Sudan III is being dispersed. The
emulsion is being stained light orange.
|
 |
The water phase are not properly
dispersed in the oil phase.
|
Sudan III is being dispersed in
the emulsion which causing the emulsion to be stained light orange.
|
 |
The water phase are being
properly dispersed in the oil phase.
|
Sudan III is being dispersed in
the emulsion which causing the emulsion to be stained light orange.
|
 |
The water phase are being
properly dispersed in the oil phase.
|
Sudan III is being dispersed in
the emulsion which causing the emulsion to be stained light orange.
|
 |
The water phase are being
properly dispersed in the oil phase.
|
Sudan III is being dispersed in
the emulsion which causing the emulsion to be stained light orange.
|
 |
Phase separation occurs rapidly
due to not enough amount of surfactant being added.
|
Sudan III is being dispersed. The
emulsion is being stained light orange.
|
 |
Phase separation occurs very
rapidly due to no surfactant were added into the emulsion.
|
Sudan III is not being dispersed.
Red globules of Sudan III form on the emulsion surface.
|
State and compare the
texture, consistency, degree of oily appearance and the spreading of colour in
the sample under the light microscope.
Emulsion I (Group 1 and 5)
Characteristic
|
Before homogenisation
|
After homogenisation
|
Texture
|
Coarse
|
Smooth
|
Consistency
|
More viscous
|
Less viscous
|
Degree of oily appearance
|
Lower
|
Higher
|
Spreading of colour
|
Spread evenly
|
Spread evenly
|
Emulsion II (Group 2 and 6)
Characteristic
|
Before homogenisation
|
After homogenisation
|
Texture
|
Coarse
|
Smooth
|
Consistency
|
Less consistent
|
More consistent
|
Degree of oily appearance
|
More greasy and spherical globule
|
Less greasy and spherical globule
|
Spreading of colour
|
Spread evenly
|
Spread evenly
|
Emulsion III (Group 3 and 7)
Characteristic
|
Before homogenisation
|
After homogenisation
|
Texture
|
Coarse, not homogenised, not smooth, cloudy
|
Homogenous, smooth, milky
|
Consistency
|
Not consistent, less viscous
|
Consistent and viscous
|
Degree of oily appearance
|
More greasy
|
Less greasy
|
Spreading of colour
|
Unevenly dispersed, less red spots
|
Evenly dispersed, more red spots
|
Emulsion IV (Group 4 and 8)
Characteristic
|
Before homogenisation
|
After homogenisation
|
Texture
|
Absence of bubbles, form a homogenous
emulsion
|
Presence of bigger size of bubbles, form
a smooth and homogenous emulsion
|
Consistency
|
Has a bigger consistency of bubbles size
|
Has a smaller consistency of bubbles
size
|
Degree of oily appearance
|
Less oily
|
More oily
|
Spreading of colour
|
Unevenly distributed pale brown colour
|
Evenly distributed dark brown colour
|
FOR PALM OIL (GROUP 1 AND 5)
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
|
TIME TAKEN (1ST VALUE) (MIN)
|
51:32:00
|
47:20:00
|
40:15:00
|
38:24:00
|
37:51:00
|
36:09:00
|
10:42:00
|
01:15:00
|
TIME TAKEN (2ND VALUE) (MIN)
|
49:31:00
|
48:10:00
|
43:02:00
|
37:17:00
|
32:19:00
|
32:25:00
|
09:07:00
|
00:50:00
|
AVERAGE
|
50:32:00
|
47:45:00
|
41:38:00
|
37:50:00
|
35:05:00
|
34:17:00
|
09:55:00
|
01:02:00
|
FOR ARACHIS OIL
(GROUP 2 AND 6)
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
|
TIME TAKEN (1ST VALUE) (MIN)
|
2:16:40
|
1:58:26
|
1:32:16
|
1:13:21
|
41:18
|
27:38
|
10:19
|
00:06
|
TIME TAKEN (2ND VALUE) (MIN)
|
170
|
113
|
100
|
67
|
42
|
31
|
19
|
1
|
FOR OLIVE OIL (GROUP 3 AND 7)
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
|
TIME TAKEN (1ST VALUE) (MIN)
|
Does not reach interphase after 120 mins
|
Does not reach interphase after 120 mins
|
Does not reach interphase after 120 mins
|
128:00
|
70:00
|
55.00
|
3:00
|
1:00
|
TIME TAKEN (2ND VALUE) (MIN)
|
0:07:43
|
Does not reach interphase after 120 mins
|
Does not reach interphase after 120 mins
|
Does not reach interphase after 120 mins
|
0:36:28
|
0:59:00
|
0:09:58
|
0:03:11
|
AVERAGE
|
Does not reach interphase after 120 mins
|
Does not reach interphase after 120 mins
|
Does not reach interphase after 120 mins
|
Does not reach interphase after 120 mins
|
0:53:14
|
0:57:00
|
0:06:29
|
0:02:05
|
FOR 30 ML OF MINERAL OIL
Readings
|
Viscosity (cP)
|
Average + SD
|
|||||
1
|
2
|
3
|
4
|
5
|
6
|
||
Before Temperature cycle
|
462
|
420
|
396
|
438
|
420
|
408
|
424+22.43
|
After Temperature cycle
|
876
|
876
|
972
|
846
|
792
|
834
|
866+60.56
|
Difference (%)
|
104.25
|
||||||
Difference in viscosity before and
after temperature cycle
Groups
|
Mineral oil (mL)
|
Difference (%)
|
Group 1
|
20
|
49.19
|
Group 2
|
25
|
13.02
|
Group 3
|
30
|
47.62
|
Group 4
|
35
|
24.00
|
Group 5
|
20
|
65.22
|
Group 6
|
25
|
79.57
|
Group 7
|
30
|
104.25
|
Group 8
|
35
|
43.60
|
Ratio of separation phase
Mineral oil (mL)
|
Ratio of separation phase
|
Average
|
Ratio of separation phase
|
|||
20
|
Group 1
|
0.78
|
Group 5
|
0.7
|
0.74
|
|
25
|
Group 2
|
0.52
|
Group 6
|
0.5
|
0.51
|
0.02
|
30
|
Group 3
|
0.58
|
Group 7
|
0.42
|
0.50
|
0.08
|
35
|
Group 4
|
0.89
|
Group 8
|
2.15
|
1.52
|
|
Discussion
HLB
(Hydrophile-Lipophile Balance) is an empirical expression for the relationship
of the hydrophilic and hydrophobic groups of a surfactant. The higher the HLB
value, the more water-soluble the surfactant is. Oils have required HLB numbers
that identify the HLB necessary to give good o/w emulsification. In this
experiment, there are several types of oils used such as palm oil, arachis oil,
olive oil and mineral oil. The surfactants used in this experiment are Tween 80
and Span 20, where the quantity of each surfactant is varied respectively in 8
test tubes, hence varies the HLB values.
For
palm oil emulsion, the result shows that in one hour time, the emulsion in all
the test tubes form separate phase. The most stable palm oil emulsion is in
Tube 1 where the average time taken for the emulsion to form separate phase is
the longest, which is 50 minutes and 32 seconds. In this case, we assume that
the HLB values to form a stable emulsion containing palm oil must be less than
9.67.
For
arachis oil emulsion, the result shows that in one hour time, the emulsion in
Tube 5, 6, 7 and 8 with HLB values 9.67, 14.09, 15.00 and 0.0 respectively form
separate phase while the emulsion in Tube 1, 2, 3 and 4 requires more than one
hour time to get separate. After two hours (120 minutes), only the emulsion in
Tube 1 does not form separate phase, thus we assume that the emulsion in Tube 1
is the most stable and the HLB values to form stable emulsion containing
arachis oil is 9.67.
For
olive oil emulsion, the result shows that after two hours (120 minutes),
emulsion in Tube 1, 2, 3 and 4 still do not reach interphase. Whereas emulsions
in Tube 5, 6, 7 and 8 form separate
phase in average time of less than one hour. In this case, we assume that the
HLB values to form a stable emulsion containing olive oil must be 12.44 and
below.
For
mineral oil emulsion, the result shows that in one hour time, most of the
emulsions in all test tubes have reached interphase, however the average time
taken for emulsion in Tube 1 to form separate phase is almost 66 minutes.
Therefore in this case, the most stable emulsion of mineral oil as compared to
all test tubes is the emulsion in Tube 1. Thus, we assume that the HLB values
to form a stable emulsion containing mineral oil is 9.67 and below.
Based
on the experiment, the most stable emulsion is olive oil emulsion followed by
arachis oil emulsion, mineral oil emulsion and palm oil emulsion respectively.
However, the results may not be true because of possible errors occur during
formulation such as inaccurate quantity of the emulsifiers poured into the test
tubes leading to inconsistency of the results. Next, the apparatus and
materials used may have been contaminated with other substances.
The
combination of 15 drops of Span 20 and 3 drops of Tween 80 as emulsifiers which
gives HLB value of 9.67 formed the most stable emulsion in all types of oil
tested. HLB value is less than 10, thus the emulsions containing these 4 types
of oils respectively are water in oil emulsion.
Sudan
III is a dye used for Sudan staining. It is used to identify the presence of
lipids in liquids. Sudan is red-coloured oil-soluble dye, thus it will stain
only the oil part. In this case, Sudan III test is performed to study the
dispersal pattern of oil phase in the emulsion. Hence, we can determine whether
the emulsion formed is oil-in-water (o/w) or water-in-oil (w/o) emulsion. In
this experiment, all of the emulsions in Tube 1 until 8 are tested with Sudan
III, and the results shows that for the emulsion in Tube 1, 2, 3, 4, 5, 6 and
7, Sudan III is being dispersed. However, the size of oil globules increases
from Tube 1 to Tube 7 respectively. This is because the HLB value is increasing
from Tube 1 to Tube 7. The higher the HLB value, the bigger the oil globules,
which indicates that the emulsion is going to water-in-oil type of emulsion. In
this experiment, emulsions in Tube 1 to 6 are o/w emulsion while Tube 7 is w/o
emulsion. For Tube 8, Sudan III is not being dispersed, red globules of Sudan
III form on the emulsion surface. This is because there is no emulsifier added,
causing the emulsion to experience phase separation very rapidly.
Based on the
experiment,the viscosity of homogenized emulsions at room temperature and after
going through temperature circle are obtained. Theoretically, when we are using
the same type of oil (mineral oil), the viscosity of the particular emulsion
will increase when it is put into the water bath (45°C) for 15 minute in the
first stage of temperature circle. An increased temperature will cause a fall
in viscosity of the continuous phase due to the increase in kinetic motion of
the disperse droplets. When it is put into the freezer (4°C) for 15 minutes. At
low temperature, kinetic energy of the system is at low level and this will
increase the viscosity of the continuous phase due to the decrease in the
motion of the globules in the disperse phase. Finally, the viscosity of the
emulsion will increase after the temperature cycle and shows higher viscosity
(cP) value than the one before temperature cycle. Based on the experiment also,
it is clearly shown that the cP value of the emulsion is higher after passing
the temperature cycle than the before one in which it follow the theory.
Theoritically,
as the amount of oil globules in continuous phase increase, the viscosity of
the emulsion increase. For instant, the viscosity of the emulsion is
propotional to the amount of oil used. However it has been expected that the
emulsion with 35ml mineral oil will have the higest viscosity but the result
obtained from the experiment shows that our expectation was incorrect. The
emulsion with 25ml mineral oil shows the most viscous among the emulsions
produced and this could be due to the errors occured during the experiment was
carried out. One of error suspected is the wrong setting of the viscometer and
the wrong type of spindle used when measuring the viscosity of the emulsions.
Another error suspected is the uneven
temperature of the laboratory due to the air movement can cause the
emulsion to lose or recieve heat from surrounding. Since the viscosity of the
emulsion is very sensitive to current temperature, so it is considered as one
of the error during conducting the experiment.
Based
on theoritical explanation, as the volume of mineral oil increase, the ratio of
separation phase increase. Thus, this means that the emulsion with low
separation phase ratio is a stable emulsion and the emulsion with high ratio of
separation is undesireable and was unable to stay longer in emulsified form and
tend to become physically saparated. From the previous discussion, the higher
the amount of mineral oil, the higher the viscosity of the emulsion and we
already knew that the viscosity is one of the major influence to ratio of
separation. As the viscosity increase the the lower the tendency for the
emulsion to undergo coalescence which then lead to complete saparation. Thus,
our ratio of separation phase is in-line with the respective viscosity of the
emulsions with different amounts of mineral oil.
Theoritically,
the higher the volume of mineral oil the higher the phase separation ratio.
However, based on the observed data, and uneven values obtained once again
which mean some errors occured during the experiment was carried out. As each group only did one emulsion, the minor
differences during the procedure may have resulted in errors.
Conclusion
We must know the HLB values and calculations in
order to obtain a stable emulsion. The type and amount of oil used also plays
an important role in selecting an emulsifier.
References
Ali
Khan, Naveed Akhtar, et.al. 2011. African Journal of Pharmacy and Pharmacology. Basics of Pharmaceutical Emulsion; A Review. Africa. Vol (5) :
2715-2725.
Paul B (2005). Remington the Science and Practice of Pharmacy. Lippincott
Williams and Wilkins. Philadelphia, USA. pp. 325-335, 759-760.
Prepared by:
1.
Prasannah a/p Govindan A153129
2.
Wan Nur Husnul Khatimah
binti Wan Mansor A152352
1.
Nur Shahirah binti Ishak A153147
2.
Nina Athirah binti Hasinin A152865
3.
Tasneem bt Mahayudin A152348
4.
Mohd Faiz bin Abd Latif A153049
