Title: Effects of different component to the formulation of emulsion
Objectives:
(1) To determine effects of HLB in stability of emulsion
(2) To identify effects of different surfactant to the surfactant
in term of physical changes and stability.
Introduction:
Emulsion is a thermodynamically instable two phase system. It
consisted of at least two liquids which immiscible in one another being
dispersed homogeneously in another liquid. Emulsion can be classified into two:
oil in water emulsion and water in oil emulsion. In an oil in water emulsion,
oil is the dispersed phase being dispersed in water (continuous phase) while
water is being dispersed in oil phase for water in oil emulsion. Multiple emulsions are available also,
including a “water-in-oil-in-water” emulsion or an “oil-in-water-in-oil’
emulsion. An unstable emulsion tends to separate into oil and water phase when
there are no energy inputs such as stirring, shaking or homogenizing. A stable
emulsion can be achieved by adding emulsifier. Emulsifier can be classified
into four: hydrocolloid, fine solid particles and surface active agent or
surfactant. In this experiment, Tween 80 and Span 20 were used in combination
at different ratio to determine the effects of different quantity of surfactant
towards the stability of emulsion formulation. Tween and Span are both
non-ionic surfactant which stabilise emulsion by positioning themselves on the
interfaces between oil and water. They are stable in mild acids, alkalis and
electrolytes and do not react with ionic ingredients.
HLB (hydrophilic-lipophilic balance) has been used to determine the
quantity and type of surfactant to prepare a stable emulsion. Each surfactant
has a HLB value ranged from 1(most lipophilic) to 20(most hydrophilic).
Normally, combinations of 2 surfactants produce a more stable emulsion. In this
experiment, Tween 80 with HLB value 15 and Span20 with HLB value 8.5 were used.
Combination of Tween 80 and Span 20in different ratio and their effects of the
combination in different formulation of emulsion were investigated. HLB value
of surfactant combination can be calculated using the formula below:
Chemical and apparatus:
(a)
Apparatus:
8
test tubes
1
50ml measuring cylinder
2
set pipette and droppers
Vortex
Weighing
boat
Light
microscope
Microscope
slide
1
set 5ml pipette and bulb
1
50ml beaker
1
15ml centrifuge tube
Coulter
Counter
Viscometer
Water
bath (45˚C )
Refrigerator
(4˚C)
(b)
Chemicals
Palm
oil
Arachis
oil
Olive
oil
Mineral
oil
Distilled
water
Span
20
Tween
80
Sudan
III (0.5%)
ISOTON
III
Procedure:
1.
Each
test tube was labelled and a clear line was drew 1cm from the bottom of each
test tube.
2.
4ml
oil and 4ml water were mixed in test tube according to table 1.
3. Span
20 and Tween 80 was added to the mixture according to the volume stated in
Table 2. Test tubes were closed and mixed well by using the Vortex for 45
seconds. Time taken for the interface to reach 1cm mark was recorded. HLB value
for each samples were determined.
4. Few
drops of Sudan III were added to 1g of emulsion spread in weighing boat.
Spreading pattern of the emulsion was recorded and compared. A little sample was spread on microscope
slide and observed under light microscope. The shape and size of globule formed
was drew, recorded and compared.
5. By
using wet gum method, mineral oil emulsion (50g) was prepared using the formula
below:
6. 40g
emulsion was poured into 50ml beaker and homogenized using the homogenizer for
2 minutes.
7. 2g
of emulsion (before and after homogenized) was put into weighing boat and
labelled. A few drops of Sudan III were added. The texture, consistency, oily
degree and colour spread ability under light microscope.
8. Viscosity
of the emulsion formed after homogenized (15g in 50ml beaker) was determined using Viscometer (calibrated with LV-4 Spindle). After that, sample was left in water
bath (45˚C) for 30 minutes and then refrigerated (4˚C) for 30 minutes as well.
The viscosity of emulsion after fluctuation of temperature and viscosity of
emulsion achieved room temperature (10-15minutes) were determined.
9. 5g
homogenized emulsion was poured in centrifuging tube and centrifuged (4500rpm,
10minutes, 25˚C The height of emulsion separation produced was measured and
the height ratio were determined.
Result
Comparison
Of Phase Separation Time Between Different Test Tubes
Examination
On Palm Oil Emulsion
Test tube 1: The spherical shaped globules are differing
in size. The smaller globules are arranged in long chain and tend to stick
around the edge of bigger globules. There are also some red-coloured globules
disperse unevenly in the sample.
Test tube 2: The shapes of globules are relatively
irregular and loosely packed（not in spherical shape）. The smaller globules tend to stick around the bigger
globules. The difference in size of
globules is relatively large also. Red-coloured globules dispersed in the
continuous phase.
Test tube 3: The spherical globules formed are relatively
uniform but smaller in size. They are most closely packed compared with other
formulation and tend to stick together.
Test tube 4: The spherical globules are arranged loosely
and similar to that in test tube 1. The sizes of the globules are not uniform
and the smaller one tends to stick on the bigger globules. The globules are
stained red and dispersed in continuous phase with flocculation.
Test tube 5: The globules formed are spherical in shape.
The size of globules is different. .
Flocculation occurred between small globules as the small globules dispersed in
the continuous phase with some stick with other globules
Test tube 6: There are globules in spherical and also
irregular shape. They are varies in size and loosely packed. Some of the
globules stained red while some do not but they sticked together.
Test tube 7: The spherical shape globules have a great
different in size. The globules are not closely packed also. Only small and
medium size globules are stained red.
Test tube 8: A few of globules formed in this formulation.
The globules formed are irregular in shape and scattered apart in the
continuous phase. The size of the globules form n test tube 8 is the smallest
among these test tubes. The continuous
phase is in red colour with small globules dispersed in it. There is no
flocculation because the globules do not stick to each other.
Calculations:
Viscosity
of Mineral Oil Emulsion (20ml of Mineral Oil)
Discussion
1.
What is
the HLB value that produces stable emulsion? Discuss.
Hydrophilic-Lipophilic Balance (HLB) value is a ratio of polar and
non-polar group in the surface active agent or surfactant. In other words, HLB
value is the balance of oil soluble substance and water soluble substance in a
surfactant. HLB has been used to determine the quantity and type of surfactant
to prepare a stable emulsion. Generally, a
surfactant is defined as a
material that can greatly reduce the surface tension of water when used in very
low concentrations. Surfactant is an important emulsifying agent used in order
to produce a stable emulsion. Surfactant is used to stabilize both the
oil and aqueous phase which are immiscible. The micelles formation will aid in
the trapping of drug particles which are usually hydrophobic or oil globules.
The adsorption of the surfactant between the oil and aqueous phase will reduce
the surface tension and this will stabilized the emulsion formed. A stable
emulsion will remain in dispersion form longer as the phase separation occurs
more slowly. Therefore, the slowest the phase separation time, the greater the
stability of emulsion formed.
Normally, combinations of two surfactants produce a more stable
emulsion. In the experiment, Tween 80 with HLB value 15 and Span20 with HLB
value 8.5 were used. Different types of oil used to prepare an emulsion will
need different optimum HLB values. Our result shows that the optimum HLB value
for Palm Oil to produce the most stable emulsion is 11.29 where the time for
phase separation to occur is the longest (80 minutes). The longer phase
separation time, the more stable the emulsion. Besides, HLB values of 10.67 and
14.07 also produce stable emulsion as the time for separation phase to occur
are 68 minutes and 67 minutes respectively.
From the experiment, tube 8 gives the lowest stability of emulsion
produced with HLB value of 0. This is because, there is neither Span 20 nor
Tween 80 is used as surfactant in this tube to aid the dispersion of oil phase
into aqueous phase (oil in water emulsion) or dispersion of aqueous phase into
oil phase (water in oil emulsion). The phase separation occurs in the shortest
time that is 10 minutes only. Besides, use of only one type of surfactant will
affect the stability of emulsion. In tube 7, only Tween 80 is used and the
phase separation occurs in shorter time (24 minutes) which indicates not stable
emulsion is produced.
Although a very useful tool, the HLB system does
have some limitations. For example additional water phase ingredients are not
considered but still may impact the stability. The method also does not provide
information as to how much surfactant is needed, but 2 to 4% surfactant is a
good starting point to begin further optimization for stability.
2.
Compare
the physical appearance for the mineral oil
emulsions formed and explain. What is the Sudan III Solution? Compare the
colour dispersion in the emulsions formed and explain.
The physical appearance is compared in terms of globule shape and
size, texture, greasiness and consistency; before and after homogenization.
Before homogenization, droplets are of a combination of
intermediate and smaller sizes. After homogenization, smaller spherical
droplets are formed. During the compounding of
emulsion, vigorous mixing is incorporated. This low-energy mixing reduces
extremely large droplets to a medium size range. Homogenizer, however, provides
high energy which reduces the medium-size droplets to a small-size range. Since
higher energy is needed to reduce a smaller droplet than that needed to reduce
a larger droplet, premix is done before subjecting the sample to homogenizer so
that the energy of homogenizer is not expended on reducing extremely large
droplets to a medium size range, when this can be done with low energy mixing.
The
emulsion before homogenization is greasy, most probably due to non-uniform
sizes of intermediate and smaller oil droplets in water during premix stage. The
emulsion is less greasy after homogenization as the droplets achieve uniform
smaller sizes. The texture of the emulsion before homogenization is more
slippery than that after homogenization, due to the presence of larger droplets
of dispersed phase.
The
droplets are more consistent in size and arrangement after homogenization.
Before homogenization, low forces are applied to disrupt the emulsion. During
homogenization, large forces are applied to the emulsion, disrupt and deform
the droplets into smaller sizes. Rapid adsorption of surfactant leads to small
stable droplets. (J. Weiss, 2008) Cavitation theory (APX, 2009) explains that
due to large pressure drop in valve of homogenizer which produces high velocity
of emulsion flow, the vapour pressure of the liquid exceeds the ambient
pressure causing formation of vapour bubbles or cavities in the liquid. When
the cavitation bubbles implode, shock waves are generated in the liquid. These
shock waves break apart the dispersed droplets. Hence, smaller consistent
droplets are formed after homogenization.
Sudan
III solution is a lysochrome (fat-soluble dye) diazo dye. It is used to
determine whether an emulsion formed is an oil-in-water emulsion or a
water-in-oil emulsion. This can be done by its ability to dissolve in oil
phase. When it dissolves in oil phase, it stains them red, which can be easily
detected and distinguished from colourless phase. In this experiment, Sudan III
solution stains the droplets red and the continuous phase remains colouless.
This indicates that the droplets are oil phase, and this is an oil-in-water
emulsion.
The
colour dispersion is more consistent in the emulsion containing 20ml of mineral oil, as the
ideal volume ratio is achieved. The colour dispersion becomes more inconsistent
as the volume of mineral
oil increases, due to the emulsifying agents are gradually less able to
emulsify the emulsion. Besides, as the volume of mineral oil increases,
the droplet size becomes bigger, the emulsion formed is greasier, more slippery
texture, and there are more red droplets observed. A phase inversion may probably occur
when the oil phase is in excess.
3.
Plot
and discuss on:
(a)
Viscosity
before and after cycle of temperature against different volume of mineral oil
graph
In the experiment, 4 different emulsions
were prepared by varying the amount of mineral oil which is 20, 25, 30 and 35
ml. Theoretically, an emulsion produced is more viscous with increasing amount
of mineral oil in its formulation. This is because oil has
higher viscosity compared to water. From the graph above, the viscosity of the
emulsion before the temperature cycle increases consistently as the proportion
of the mineral oil increases. The trend of graph is compared with the plots for
after temperature cycle. Also, the graph shows an increase in the viscosity of
the emulsion as the amount of mineral oil increases. Therefore, it can be
concluded that the emulsion with 20 ml of mineral oil has the lowest viscosity
and emulsion with 35 ml of mineral oil has the highest viscosity for both
before and after temperature cycle.
Besides, after the temperature cycle, all
emulsion should have a higher viscosity compared to those before temperature
cycle. This is because heating of the oil in water emulsion will cause
conversion to the water in oil emulsion. This is referred as reverse emulsion. Usually,
emulsion of water in oil has a higher viscosity compared to the oil in water
emulsion. This phase inversion shows instability of the emulsion. The
experiment shows that instable emulsion can be influenced by temperature cycle
thus affect its viscosity. The purpose of treating the emulsion with
exaggeration of the temperature fluctuations (temperature cycling) is to compare the physical instabilities of
the emulsion. The temperature cycle will disrupt the adsorbed layer
of emulsifying agent at the oil/water interphase and hence affect the stability
of the emulsion. As the result, the emulsion becomes unstable. Thus, its viscosity
increases.
From the result, emulsion with 30 ml and 35 ml of mineral oil shows a
drastic increase in viscosity after temperature cycle compared to before
temperature cycle. This is due to higher instability of the emulsion. In
opposite, 20 ml and 25ml of mineral oil had shown a decrease in viscosity after
the temperature cycle compared to before temperature cycle. This result differs
from the theory in which it could be due to some possible errors encountered
during the experiment. One of the errors could be from improper rinsing of the
viscometer spindle that might affect the concentration of the emulsion
components. Besides, the inaccurate result may also due to the different sizes of spindles that we
used to measure the viscosity of the emulsions. The emulsion that might
have not being exposed to the temperature according to the duration stated in
the procedure also affects its viscosity.
(b)
Difference
of viscosity (%) against different amount of mineral oil (ml) graph
In this experiment, emulsion containing 20ml of
mineral oil shows the greatest difference in the viscosity, that is 171.43%.
The smallest difference in viscosity, which is 23.30%, is shown by the emulsion
containing 35ml of mineral oil.
The higher the difference in viscosity, the less
stable is the emulsion. From the results obtained in this experiment, emulsion
with 20 ml mieneral oil is the most
unstable emulsion while emulsion with 35 ml mineral oil is the most stable one.
This is because when there is a lower oily phase present in an emulsion, the
emulsion is actually becoming more unstable. Therefore, when these different
types of emulsion are subjected to temperature cycling, the amount of ice
crystals formed is usually directly proportional to the extent of the
instability of the emulsion, or the volume of the oil used. The more the ice
crystals are formed, the greater is the reduction in sample viscosity, and thus
the greater is the viscosity difference (%). Therefore, emulsion with 20 ml mineral
oil is not the most stable emulsion.
4.
Plot
a graph of separated phase ratio due to centrifugation against different amount
of turpentine oil. Give comments.
Average ratio of phase separation for different amount
of oil
Phase
separation ratio is used to determine the stability of an emulsion. A high
ratio of phase separation will be resulted in unstable emulsion which it will
have two separated phases. The non-homogenous emulsion is easily separated as
compared with homogenous emulsion. The uniformity of drug in the emulsion will
be altered and inaccurate dosage is being administered.
Based
on the graph plotted, separated phase ratio increase from 20mL mineral oil
emulsion to 25mL mineral oil emulsion, decrease from 25mL mineral oil emulsion
to 30mL mineral oil emulsion and increase from 30mL mineral oil emulsion to
35mL mineral oil emulsion. Emulsion with 25mL mineral oil by using arachis oil
oil has highest separated phase ratio while 30mL olive oil emulsion has lowest
separation phase ratio.
According
to theory, as the amount of oil increase, the separated phase ratio will
increase. This is because the added amount of oily phase in emulsion has
exceeded the oil amount at which stable emulsion is formed. Therefore,
separation will occur in a faster rate.
However,
the results obtained from graph do not follow this theory. This may be due to
several errors that occur during experiment. For example, inaccuracy in
measuring amount of oil before forming the emulsion, insufficient
homogenisation that has been carried out on emulsion or the height of separated
phase is not measured accurately. Besides, if the volume of each test tube is
not equal during centrifuge, the result of centrifuge will be inaccurate. Using
of wrong method of preparation of emulsion, that is, the wet gum method may
affect the result too. If good emulsion is failed to be produced, it will affect
stability of emulsion which will then affect the result of the experiment.
5.
What
are the functions of each ingredient used? How these different ingredients
affect the physical characteristics and stability of an emulsion formulation?
Alcohol
is used as a preservative in this oil-in-water emulsion, because the high
proportion of water present in the emulsion is very susceptible to microbial
contamination and also deterioration of emulsion by other external
environmental factors.
Besides
that, syrup which contain high amount of sugar act as sweetening agent to mask
the unpleasant taste of the mineral oil to increase patient compliance. It can
also be used to increase the viscosity of the emulsion. . However, the amount
of syrup used should be controlled so that the emulsion produced is not too
viscous and form a layer on the side of the container.
Vanillin
acts as flavouring agent which can increase the taste of emulsion because an
emulsion always has a taste not preferred by most of the patients. Thus by
adding vanillin, patient compliance can be increased.
Distilled
water can function as aqueous phase (continuous phase) in oil-in-water
emulsion.
The
type of emulsion, either o/w or w/o emulsion can be determined by the amount of
each phase or the volume ratio. 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. 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. The
quantity of syrup is limited to avoid rheological problem and physical
properties of the emulsion.
Conclusion:
The
more mineral oil in the emulsion, the more viscous the emulsion formed.
References:
1.
APV
(2009). Homogenizer Handbook: Processing of Emulsions and Dispersions. SPX http://www.apv.com/pdf/catalogs/Process_E_D_By_Homog_3005_01_06_2008_US.pdf
2.
J.
Weiss (2008). Emulsion Processing: Homogenization http://people.umass.edu/mcclemen/FoodEmulsions2008/Presentations(PDF)/(5)Emulsion_Formation.pdf
3.
Micheal
E. Aulton, Aulton’s Pharmaceutics – The
Design & Manufacture of Medicines (3rd Ed), Elsevier Inc.
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