Aim:
a)
To determine the effect of HLB surfactant on the emulsion stability.
b)
To investigate the physical and stability effects on the formulation of
emulsion due to the different amount of emulsifying agent.
Introduction:
Emulsion
is a 2 phase system that is not stable thermodynamically. It contains at least
2 immiscible liquids where one of them (internal/dispersed phase) is dispersed
homogenously in another liquid (external/continuous phase). Emulsion can be
categorised into 2 types, oil-in-water emulsion (o/w) and water-in-oil emulsion
(w/o). Emulsion is stabilised by adding emulsifying agent. Emulsifying agent
can be divided into 3 types:
1) hydrophilic
colloid
2) finely
divided solid particles
3) surface
active agent or surfactant.
The HLB method
(hydrophilic-lipophilic balance) is used to determine the quantity and type of
surfactant that is needed to prepare a stable emulsion. Each 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 of
surfactant 1)(HLB surfactant 1) + (quantity of surfactant 2)(HLB surfactant 2)
Quantity of surfactant 1 + Quantity of surfactant 2
Quantity of surfactant 1 + Quantity of surfactant 2
In
this experiment, were using Span 20 and Tween 80 as the suefactants. The HLB
value for Span 20 is 8.6, while for Tween 80 is 15.
Apparatus:
8
test tubes, 50 ml measuring cylinder,2 sets of
pasture pipette, droppers, Vortex mixing device, weighing boat, mortar and
pestle, light microscope, microscope slides, 5ml pipette and pipette-bulb, 50ml
beaker, centrifugation tube 15ml, Coulter counter device, centrifugator,
viscometer, water bath 45oC and refrigerator (4oC)
Materials:
Arachis
oil, distilled water, Span 20, Tween 80, Sudan III solution (0.5 %) and ISOTON
solution III, Mineral oil, acacia, syrup, vanillin and alcohol.
Procedures:
1.
Eight test tubes were labeled and a straight line was drawn 1cm from bottom of
each test tube.
2.
4ml of Arachis oil and 4ml distilled water were mixed in the test tubes.
3.
Span 20 and Tween 80 were added into the test tubes (refer to the table below).
The test tubes were closed and mixed by using Vortex mixer for 45 seconds. Time
taken for the separation phase to achieve 1cm was recorded. The HLB value for
each sample was determined.
Tub
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 were added into 1g of emulsion formed in a
weighing boat and mixed well. The colour spreading of the samples were
explained and compared. A thin layer of sample was spreaded in a glass slide
and observed under light microscope. The shape and globule size formed was
explained.
5.
Using wet gum method, a formulation of Mineral Oil Emulsion (50g) was prepared
using the following formula:
Mineral
Oil
|
20
ml
|
Acacia
|
6.25
g
|
Syrup
|
5
ml
|
Vanillin
|
2
g
|
Alcohol
|
3
ml
|
Distilled
water qs
|
50
ml
|
6.
40g of emulsion formed was put into a 50 ml beaker and homogenization process
was done for 2 minutes using homogenizer device.
7.
2 g of emulsion formed (before and after homogenization) were put into weighing
boats and labeled. A few drops of Sudan III solution was added and mixed well.
The texture, consistency and degree of oily form and the spreading of sample
colour observed under light microscope were explained and compared.
8.
The viscosity of emulsion (15 in a 50 ml beaker) formed after homogenization was determined using
viscometer that has been calibrated using “Spindle” type LV-4. The sample was
then exposed to 45oC (Water bath) for 30 minutes and next at 4oC
(refrigerator) for another 30 minutes. The viscosity of the emulsion was
determined after the exposure to these temperature has finished and the
emulsion reached room temperature (10-15minutes).
Readings
|
Viscosity (cP)
|
Average + SD
|
||
1
|
2
|
3
|
||
Before Temperature cycle
|
740
|
820
|
920
|
730
+ 121.66
|
After temperature cycle
|
900
|
920
|
960
|
613.33
+
343.78
|
Difference (%)
|
15.98%
|
|||
Mineral
Oil(ml)
|
Ratio
of separation phase
|
Average
|
(
|
||||
20
|
Group
1
|
0.6122
|
Group
5
|
0.7291
|
0.6707
|
0.0585±0.6707
|
|
25
|
Group
2
|
0.7826
|
0.5128
|
0.6477
|
0.1349±0.6477
|
||
30
|
Group
3
|
0.7000
|
Group7
|
0.7400
|
0.7200
|
0.0200±0.7200
|
|
35
|
Group
4
|
0.5800
|
Group8
|
0.6667
|
0.6234
|
0.0434±0.6234
|
|
9. 5 g emulsion which has been homogenized was inserted into a centrifugation tube and centrifuged (4500 rpm, 10 minutes, 25oC). The height of separation formed was measured and the ratio was 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
|
|
Phase separation time (min)
|
Group 1
|
8
|
8
|
65
|
38
|
62
|
61
|
40
|
1
|
Group 5
|
8.28
|
73.11
|
69.03
|
13.39
|
15.16
|
18.38
|
4.13
|
0.1
|
|
Average
|
8.14
|
40.55
|
67.02
|
25.70
|
38.58
|
39.69
|
44.13
|
0.55
|
|
Stability
|
Less Stable
|
More stable
|
Most stable
|
Intermediate
|
Stable
|
Stable
|
More stable
|
Least stable
|
|
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
|
|
Phase separation time (min)
|
Group 2
|
*
|
11
|
*
|
27
|
39
|
50
|
15
|
2
|
Group 6
|
*
|
*
|
*
|
72
|
69
|
63
|
49
|
43
|
|
Average
|
*
|
65.5
|
*
|
49.5
|
53
|
56.5
|
32
|
22.5
|
|
Stability
|
Most stable
|
More stable
|
Most stable
|
Intermediate
|
Intermediate
|
Intermediate
|
Less Stable
|
Least stable
|
|
**To find the average for the time taken, if the interphase did not reach 1cm after 120 minutes, the time taken is just assumed to be 120 minutes.*Interphase did not reach 1cm after 120 minutes
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 time (min)
|
Group 3
|
4.52
|
4.47
|
4.30
|
4.15
|
4.10
|
3.48
|
3.47
|
3.35
|
Group 7
|
46
|
52
|
60
|
38
|
40
|
35
|
20
|
15
|
|
Average
|
25.26
|
28.24
|
32.15
|
21.08
|
22.05
|
19.24
|
11.74
|
9.18
|
|
Stability
|
Stable
|
More stable
|
Most stable
|
Intermediate
|
Stable
|
Less stable
|
Less stable
|
Least stable
|
|
Mineral 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 time (min)
|
Group 4
|
68
|
62
|
60
|
59
|
25
|
13
|
7.70
|
0.67
|
Group 8
|
70
|
68
|
60
|
59
|
30
|
16
|
8
|
1
|
|
Average
|
69
|
65
|
60
|
59
|
27.5
|
14.5
|
7.85
|
0.835
|
|
Stability
|
Most stable
|
More stable
|
Most stable
|
Intermediate
|
Intermediate
|
Intermediate
|
Less Stable
|
Least stable
|
|
Colour
Spreading
Figure 1
According to figure 1,the spreading of orange-red colour of Sudan III getting less evenly from weighing boat 1 to weighing boat 8. At weighing boat 1, Sudan III mixed homogenously with the emulsion and the colour appeared to be even. At weighing boat 8, Sudan III did not mix very well with the emulsion. The emulsion colour was not even and looked a bit reddish.
Viscosity
of Emulsion I (20ml palm oil)
Reading
|
Viscosity (cP)
|
Mean ± SD
|
||
1
|
2
|
3
|
||
Before temperature cycle
|
10
|
15
|
13
|
12.67 ±2.05
|
After temperature cycle
|
31
|
40
|
37
|
36±3.741
|
Difference (%)
|
184.41%
|
|||
Viscosity
of Emulsion II (25ml arachis oil)
Reading
|
Viscosity (cP)
|
Mean ± SD
|
||
1
|
2
|
3
|
||
Before temperature cycle
|
740
|
820
|
920
|
730 ± 121.66
|
After temperature cycle
|
900
|
920
|
960
|
613.33±
343.78
|
Difference (%)
|
15.98%
|
|||
Viscosity
of Emulsion III (30ml olive oil)
Reading
|
Viscosity (cP)
|
Mean ± SD
|
||
1
|
2
|
3
|
||
Before temperature cycle
|
40
|
30
|
30
|
33.333 ± 5.773
|
After temperature cycle
|
186
|
217
|
233
|
212 ± 23.895
|
Difference (%)
|
40.85%
|
|||
Viscosity
of Emulsion IV (35ml mineral oil)
Reading
|
Viscosity (cP)
|
Mean ± SD
|
||
1
|
2
|
3
|
||
Before temperature cycle
|
240
|
100
|
180
|
243.571
|
After temperature cycle
|
320
|
240
|
340
|
352.915
|
Difference (%)
|
30.983%
|
|||
DISCUSSION:
1. What
are the values of HLB that will yield a stable emulsion? Discuss.
However, the HLB value required
to produce stable emulsion for each oil may not be as accurate as there may
have errors that occur during experiment. For example inaccurate amount of
water and oil being measured, inaccurate amount of surfactant added, errors
when observing the phase separation in the test tube and time taken for separation
to occur.
According to result obtained, we
could say that different oil needs different HLB value in order to produce
stable emulsion. We found out that the optimum HLBvalue for arachis oil to
prepare the most stable emulsion is 9.67. This is shown by phase separation
time which is longest for Tube 1 compare to other tubes. Furthermore the colour
of Sudan III solution and almost similar size globules are evenly distributed throughout
emulsion.
Same
situation appear on arachis oil. When we add 15 drops of Span 20 and 3 drops of
Tween 80 to make up HLB 9.67, the emulsion exist at the most stable state where
the colour dispersion of Sudan III solution is even, small round globules are dispersed
evenly throughout the emulsion. Phase separation time for this tube is longer than
other tube.
However
for olive oil, it exists in the most stable form at different HLB value than previous
2 oils. It is most stable at HLB 11.17 where the phase separation time is longest
and small round globules with colour of Sudan III solution is evenly dispersed throughout
the emulsion.
For
turpentine oil, the optimum HLB value is 11.34. When the HLB value is 11.34,
turpentine oil droplets appear in small, evenly dispersed throughout the emulsion.
Longest time is needed to separate the phase.
Emusion
of each oil will separate into two phases within the shortest time when there
is neither Span 20 nor Tween 80 is added to the emulsion. This is shown by Tube
8 of each oil, shortest time is required to achieve phase separation. No
surfactant is added to Tube 8. This cause emulsion formed is least stable,
easily be separated out to form 2 immisible phase. By this scene, we could
conclude that surfactant is needed to produce a stable emulsion.
A
combination of different amount of surfactant could produce different HLB value
to stabilise oil droplets in the emulsion. This statement can be observed in
Tube 7 where only one surfactant (Tween 80) is used. The emulsion formed is not
stable compare to other tubes that have combination of surfactant. A good
combination of surfactants is needed to produce a stable emulsion.
Phase
separation time will be longer by adding sufficient surfactant to achieve optimal
HLB value. Surfactant is used to stabilise 2 immisible layer by incorporating its
hydrophilic head in aqueous phase and hydrophobic tail with hydrophobic drug particles.
At certain concentration of surfactant added, micelle will be formed and it will
try to keep the hydrophobic drug particles or lipid globules in the core with
tail pointing inward center while the head will remain in aqueous phase. This
will lower the interfacial tension thus, stability and emulsification is
enhanced.
2. Compare the physical appearance
of the mineral oil emulsions produced and give your comments. What is Sudan III
test? Compare the colour dispersion in the emulsions produced and give your
comments.
Magnification
(40x10)
|
Physical
appearance
|
Colour
spreading
|
Test Tube 1
 |
Most stable form, great dispersion
|
More uniform
|
Test Tube 2
 |
Smaller globules are formed,
|
More uniform of colour spreading
|
Test Tube 3
 |
Globules are getting coalescence.
|
Getting less uniform
|
Test Tube 4
 |
Globules start to coalescence and forming larger
molecule
|
Less uniform
|
Test Tube 5
 |
Some large granules are formed. Other globules are
merging as well.
|
Uniformity decreasing
|
Test Tube 6
 |
More and more large globules are formed
|
Uniformity start to be unseen
|
Test Tube 7
 |
Less small globules are seen.
Larger globules are formed
|
Colour is non-uniform
|
Test Tube 8
 |
Only few small globules.
Mostly are large globules
|
Colour is non-uniform.
|
Before homogenization
|
After
homogenization
|
|
Texture
|
Smooth,
non-homogenous
|
Smoother ,
homogenous
|
Consistency
|
Bad
|
Better
|
Physical oily
degree
|
More oily
|
Less oily
|
Globule size
|
Big
|
Small
|
Color dispersion
|
less uniform
|
Uniform
|
Sudan III solution is prepared by
adding a certain amount of Sudan III (86% dye) to a designated volume of ethyl
alcohol (95%v/v) which was allowed to stand for 24 hours, then filtered. Sudan
III itself is a lysochrome (fat-soluble dye)
diazo dye used for staining of triglycerides in frozen
sections, and some protein bound lipids and lipoproteins on paraffin. A Sudan III test
is performed used to clarify the shape and physical characteristics of an oily
emulsion. It shows the emulsion whether is oil-in-water emulsion or
water-in-oil emulsion by comparing the amount of the globules stained in red
and the colorless portions.
From
this experiment, we could see the obvious changes on an emulsion before and
after homogenization. The preparation will have a greasy (or oily) texture due
to the oily continuous phase in the emulsion system. The globules, apparently
are large.In fact, due to the mutually insoluble phasing condition, the colour dispersion of the Sudan III
solution would be less uniform instead.
After
homogenization, apparently the oily phase is broken down into smaller globules.
The texture of the mineral oil occurs in a smoother and homogenous state.
Hence, the color dispersion, is more uniform. This shows that the globule is
evenly dispersed in the system. We may conclude that the homogenization process
makes the oily globules more stable in the aqueous phase.
3. Plot and discuss:
a. Graph of sample viscosity before and after
the temperature cycle vs. the content of mineral oil.
Type of Oil
|
Amount of Oil (ml)
|
Viscosity average
(cP)
(x ± SD)
|
Difference in viscosity (%)
|
|
Before
|
After
|
|||
Palm Oil
|
20
|
100 ± 14.14
|
120.02 ± 23.42
|
20.02%
|
Arachis Oil
|
25
|
409.9 ± 15.49
|
775.37 ± 72.98
|
89.16%
|
Olive Oil
|
30
|
136.65 ± 91.99
|
254.95 ± 229.53
|
86.57%
|
Mineral Oil
|
35
|
730 ± 121.66
|
613.33 ± 343.78
|
15.98%
|
This
experiment carried out in wrong method. We use different types of oil with
different types of amount. Theoretically, this experiment has to carry out
using the same type of oil with different amount of oil. From the graph, the
viscosity of the emulsion at room temperature and after subjected to
temperature cycle is different according to each type of oil and each amount.
For palm oil, arachis oil and olive oil, the viscosity after subjected to
temperature cycle increased due to took longer time to let the emulsion become
room temperature again. Whereas the viscosity of mineral oil decreased after
subjected to temperature cycle due to it turns to room temperature faster.
Theoretically,
using the same type of oil, the viscosity of emulsion sample will increase when
put in the water bath at 450c for 30 minutes in the temperature
cycle. An increased temperature will cause a fall in apparent viscosity of the
continuous phase and increased kinetic motion of the disperse droplets and the
emulsifying agent at o/w interface. Subsequently, it is put into freezer at 40c
for 30 minutes. At low temperature (40c), kinetic energy of the
system is reduced and this will increase the viscosity of the continuous phase.
This will decrease the rate of migration of the globules in the disperse phase.
Thus, the viscosity of the emulsion will increase after the temperature cycle.
b. Graph
of difference of viscosity (%) vsamount of oil (ml).
Due
to the error in this experiment, from the graph, we can see that the difference
of viscosity increase and then decrease by increasing the amount of oil.
Different type of oil have different type of viscosity, hence we cannot get the
correct graph.The arachis oil and olive oil showing more viscous than palm oil
and mineral oil from the graph.
Theoretically,
using the same type of oil, the higher amount of oil globules in the continuous
phase will increases the viscosity of the emulsion. The graph will show
directly proportional graph, which the difference of viscosity is directly
proportional to the amount of oil.
4. Plot
graph of separated phase ratio formed from the centrifugation process versus
the different amount of Oil. Explain.
The above table and graph
indicate the stability of the emulsion. The preparation of good emulsion need
the well stability, not easily coalesce and undergo phase separation to form two layer. However, there
is no ideal emulsion. A high ratio of phase separation shows that is unstable
emulsion. Unstable emulsion will give the detrimental effect such as increase
tendency of inappropriate dose deliver to the patient. As a result, the desired
outcome of the therapeutics cannot be observed.
In this experiment, 25 ml of Arachis oil
mix with 6.25g of acacia and other excipients show the highest ratio of phase
separation followed by 30 ml of olive oil, 20ml of palm oil and 35ml of themineral
oil. Theoretically, the separation phase ratio should be increasing with the
increasing of the mineral oil contain in the formulation.
The inaccuracy of the data
obtained may be due to some error in the process which the experiment is
carried out. The main reason is due to the different types of oil used in this
experiment. Different types of oil needs different amount of acacia to obtain a
good emulsion. Besides, this may be due to the homogenous process was not done
properly. Inaccurate measurement of the highly viscous surfactant that is to be
added into the formulation is also one of the reasons. In addition, the height
of the separated phase might not be measure accurately too.
5. What are the functions of each ingredient used? How these different ingredients affect the physical characteristics and stability of an emulsion formulation?
Mineral oil act as oily phase in the emulsion. It is
either dispersed phase or continuous phase depend on the type of emulsion
formed. In o/w emulsion, it is the dispersed phase. Acacia act as emulsifying
agent which increase viscosity of the emulsion formed, maintain separation of
droplets in dispersed phase. As acacia is a natural polysaccharides and
susceptible to microbial attack, thus alcohol is added to emulsion as
preservative to prevent growth of microorganisms. Syrup in this emulsion is act as
sweetening agent to improve the taste of emulsion and increase viscosity of emulsion
formed. Vanillin is used as flavouring agent to give vanilla odour in order to increase
tastiness of emulsion. Distilled water is the aqueous phase in emulsion. For o/w
emulsion, it is the continuous phase.
Amount of
mineral oil (dispersed phase) and distilled water (continuous phase) added to
form an emulsion is important in determining the type and stability of emulsion
formed. The volume of dispersed phase should not be more than the volume of the
continuous phase. The stability of emulsion formed will decrease if the volume of
dispersed phase exceeds 50% of the emulsion. Phase inversion tends to occur for
emulsions containing more than about 70% dispersed phase.
Acacia
which is the emulsifying agent is important in determining the uniformity of
oily droplets dispersed throughout the emulsion. It should be used in
appropriate amount based on HLB value. If the acacia amount used is less than
required amount, the oil globules will be less evenly dispersed throughout
emulsion. This cause instability of emulsion and phase inversion is more likely
to occur.
Syrup
will affect the viscosity of the emulsion formed. Higher concentration of syrup
used will produce emulsion with higher viscosity. Thus suitable amount of syrup
should be added to emulsion to give a suitable viscosity. This is important to
ensure appropriateness of emulsion viscosity. Emulsion that has syrup in higher
concentration or amount will face rheological problem such as difficulty in
pouring out the emulsion.
Alcohol
which act as preservative, is added to emulsion to ensure chemical stability
and prevent microbiological contamination of the emulsion. The emulsion will have
stable physicochemical properties for longer duration. However the amount of alcohol
that can be added to emulsion should not be exceeded as it produce toxicity in larger
amount.
CONCLUSION:
Span – 20 in larger volume compare to Tween 80 gives a stable emulsion, this has proven the emulsion formed from four types of oil is oil in water emulsion. Palm oil, Arachis oil, and olive oil increase the viscosity of emulsion Arachis oil and olive oil give an unstable emulsion compare to mineral oil and palm oil as the change in viscosity is high.
REFERENCES:
http://global.britannica.com/EBchecked/topic/186307/emulsion (accessed 25 May 2015)
http://www.slideshare.net/PallaviKurra/pharmaceutical-suspensions-and-emulsions (accessed 23 May 2015)www.firp.ula.ve/archivos/cuadernos/00_Book_Salager_Chap3.pdf
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