PHARMACEUTICAL
PRODUCT DEVELOPMENT
NFNF2213
SEMESTER 1
SESSION
2016/2017
LAB
REPORT 2
LECTURER
: DR.NG SHIOW FERN
GROUP
A
GROUP
2 MEMBERS :
NISHAAL
KORAN (A153334)
TAN
MIN ANNE (A152663)
MUHAMMAD
ROSMAN BIN JUNAIDI (A153262)
MUHAMAD
HAZWAN BIN HARUN (A152983)
LEOW
PUI MUN (A152593)
TITLE
Characterisation of Emulsion Formulation
OBJECTIVE
The objective of the
investigation is to study the effects of HLB surfactant on the stability of the
emulsion. Besides that, the effects of different oil phases used in the
formulation on the physical characteristics and stability of the emulsion is
studied as well.
INTRODUCTION
Emulsion is a two-phase
system that is not stable thermodynamically. It contains at least two
immiscible liquids where one of them know as internal or disperse phase is
dispersed homogenously in another liquid considered to be the continuous phase.
In general, emulsion can be divided into two types, oil-in-water emulsion (o/w)
and water-in-oil emulsion (w/o). Emulsion is stabilised by adding emulsifying
agent. The hydrophilic-lipophilic balance or in short HLB method is used to
determine the quantity and type of surfactant that is needed to prepare a
stable emulsion. 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.
Therefore, HLB value for
the combination of emulsifying agents can be determined using the formula below
:
APPARATUS
8 test tubes
50 ml measuring cylinder
2 sets of pasture pipettes and droppers
Vortex mixer
Weighing boat
1 set of mortar and pestle
Light microscope
Microscope slides
1 set of 5 ml pipette and bulb
50 ml beaker
15 ml centrifugation tube
Centrifugation apparatus
Viscometer
Water bath (45 degree Celsius)
Refrigerator (4 degree Celsius)
MATERIALS
Palm oil
Arachis oil
Olive oil
Mineral oil
Distilled water
Span 20
Tween 80
Sudan III solution (0.5%)
PRODECURES
1. Each test tube is labelled and 1 cm is marked from
the base of the test tube.
2. 4 ml of oil (according to Table 1) is mixed with 4 ml of
distilled water in the specific test tubes.
TABLE 1
|
Group
|
Oil
|
|
1,5
|
Palm
oil
|
|
2,6
|
Arachis
oil
|
|
3,7
|
Olive
oil
|
|
4,8
|
Mineral
oil
|
3. Span 20 and Tween 80 is added into the mixture of
oil and water (according to Table 2). The test tube is closed and the contents
are mixed with vortex mixer 45 seconds. The time needed for the interface to
reach 1 cm is recorded. The HLB value for each of the sample is determined. Steps
1-3 is repeated 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 is added to 1 g
emulsion formed in a weighing boat and is mixed homogenously. The spread of the
colour in the sample is compared. Some of the sample is spread on a microscope
slide and is observed under light microscope. The appearance and globule size
formed is described.
5. A Mineral Oil Emulsion (50 g) 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. 40 g of emulsion is
placed into a 50 ml beaker and homogenized for 2 minutes using a vortex mixer.
7. 2 g of emulsion is
taken (before and after homogenization) and placed into a weighing boat and
labelled. A few drops of Sudan III solution is added and mixed. The texture,
consistency, degree of oily appearance and the spreading of colour in the
sample under the light microscope is stated and compared.
8. The viscosity of the
emulsion formed after homogenization is determined using a viscometer that is
calibrated with “Spindle” type LV-4. The sample is exposed to 45 degree Celsius
water bath for 15 minutes and then to 4 degree Celsius refrigerator for another
15 minutes. After the exposure to the temperature cycle is finished, the
viscosity of the emulsion is determined when the emulsion has reached room
temperature. Step 8 is repeated again and the average value is obtained.
TABLE 4
|
Readings
|
Viscosity
(cP)
|
Average
|
|||||
|
1
|
2
|
3
|
4
|
5
|
6
|
||
|
Before
Temperature Cycle
|
|
|
|
|
|
|
|
|
After
Temperature Cycle
|
|
|
|
|
|
|
|
|
Difference
(%)
|
|
||||||
9. 5 g of
homogenised emulsion is placed into a centrifugation tube and centrifuged. The
height of the separation formed is measured and the ratio of the height
separation is determined.
|
Mineral
Oil (ml)
|
Ratio
of phase separation
|
Average
|
Ratio
of separation phase
|
|
20
|
|
|
|
|
25
|
|
|
|
|
30
|
|
|
|
|
35
|
|
|
|
RESULTS
|
Test Tube
|
Microscopic image
|
HLB value
|
Observation of Sudan III Test
(naked eye)
|
Observation under microscope
|
Type of emulsion
|
|
1
|
 |
9.67
|
The emulsion is clear.
The sudan III disperse evenly.
|
A few globules are formed.
The size of globules is small
Red dye can be seen on continuous
phase.
|
Water in oil
|
|
2
|
 |
10.70
|
The emulsion is milky but less viscous.
Sudan III disperse evenly but slowly.
|
Large and small globule size are
formed and evenly distributed.
Red dye can be seen on continuous
phase.
|
Water in oil
|
|
3
|
 |
11.34
|
The emulsion is milky and less viscous.
The Sudan III dye disperse evenly.
|
Small and medium size of globules
unevenly distributed.
Red dye can be seen on disperse phase.
|
Oil in water
|
|
4
|
 |
12.44
|
The emulsion is very milky and viscous.
Sudan III disperse unevenly.
Red precipitate shown in the emulsion.
|
A large globule are formed.
The small and medium globules are
semi-distributed.
Red dye can be seen on disperse phase.
|
Oil in water
|
|
5
|
 |
13.17
|
White milky emulsion is formed and the
emulsion is viscous.
The Sudan III disperse evenly but
slowly.
Bubble is observed on top .
|
Medium and small globules are formed
and evenly distributed.
Red dye can be seen on disperse phase.
|
Oil in water
|
|
6
|
 |
14.09
|
The emulsion is a bit milky and less
viscosity.
Sudan III disperse throughout the
emulsion.
Many bubbles is observed.
|
A more uniform, medium size of globule
is formed.
Red dye can be seen on disperse phase.
|
Oil in water
|
|
7
|
 |
15.00
|
Milky emulsion is formed and the
emulsion is less condensed.
Sudan III dye disperse evenly.
Little bubbles is formed
|
Small and medium size of globule is
distributed uniformly.
Red dye can be seen on disperse phase.
|
Oil in water
|
|
8
|
 |
0
|
The emulsion is milky but not so thick.
The Sudan III disperse rapidly in
emulsion.
Little bubbles is formed.
|
Many large globules are formed.
Red dye can be seen on continuous
phase.
|
Water in oil
|
For 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
|
|
Time taken (1st value) (min)
|
51:32
|
47:20
|
40:15
|
38:24
|
37:51
|
36:09
|
10:42
|
01:15
|
|
Time taken (2nd value) (min)
|
49:31
|
48:10
|
43:02
|
37:17
|
32:19
|
32:25
|
09:07
|
00:50
|
|
Average
|
50:32
|
47:45
|
41:38
|
37:50
|
35:05
|
34:17
|
09:55
|
01:02
|
For 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
|
|
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
|
|
Average
|
153
|
115:30
|
96
|
70
|
41:30
|
29
|
14:30
|
0:53
|
For 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
|
|
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
|
|
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)
|
1:23:52
|
1:23:16
|
0:52:49
|
0:55:30
|
0:33:21
|
0:28:06
|
0:20:00
|
0:02:35
|
|
Time taken (2nd value) (min)
|
49:46
|
24:59
|
23:05
|
18:45
|
12:19
|
9:34
|
3:27
|
3:03
|
|
Average
|
66
|
53:30
|
37.5
|
36:30
|
22:30
|
18:30
|
12
|
1:30
|
COMPARISON
BEFORE AND AFTER HOMOGENIZATION
Differences in
viscosity before and after temperature cycle for 25mL oil
|
Reading
|
Viscosity
(cP)
|
Average
|
|||||
|
1
|
2
|
3
|
4
|
5
|
6
|
|
|
|
Before
Temperature cycle
|
42.4
|
41.6
|
39.9
|
41.8
|
44.1
|
42.5
|
42.05
|
|
After
temperature cycle
|
44.2
|
46.3
|
62.0
|
56.4
|
42.4
|
38.8
|
48.35
|
|
Difference
percent
|
48.35-42.05/48.35
x 100 = 13.02%
|
|
|||||
DIFFERENCE IN VISCOSITY BETWEEN GROUPS BEFORE AND AFTER
TEMPERATURE CYCLE
|
Groups
|
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
|
33.60
|
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
|
0.04 + 0.74
|
|
25
|
Group
2
|
0.52
|
Group
6
|
0.5
|
0.51
|
0.01
+ 0.51
|
|
30
|
Group
3
|
0.58
|
Group
7
|
0.42
|
0.50
|
0.08
+ 0.50
|
|
35
|
Group
4
|
0.89
|
Group
8
|
0.47
|
0.68
|
0.21 + 0.68
|
DISCUSSION
In this experiment, we can know the type of certain
emulsion by Sudan III test. This test can indicate the position of oil in the
emulsion. It solution is in red colour and it will dissolve in the oil phase.
It will give a red colour to the oil phase. The aqueous solution will not
stained by this solution and will appear in colourless. We can see the stained
of oil phase by using microscope. Hence, it can be used to determine the type
of emulsion whether it is oil in water (o/w) emulsion or water in oil (w/o)
emulsion. If the emulsion is oil in water (o/w) emulsion, the globules formed
will be stained in red colour while if the emulsion is water in oil (w/o)
emulsion, red background will formed.
From the table for palm oil, we can conclude that tube
1 -3 are more stable emulsion compared to tube 4-8. In
tube number 8, there are no emulsifiers added, therefore the average time taken
for interphase to reach 1 cm are very fast which only takes 1.02 minutes. The palm oil emulsion is not stable
at all at this stage as it separates into distinct layer rapidly. From tube
number 4 until 7, the emulsion also not stable due to the imbalance
distribution of emulsifiers added. In conclusion, the palm oil emulsion is most
stable at HLB value 9.67-11.34 (can
be observe at tube 1-3) provided the emulsifiers are slightly
hydrophilic, hence this is an oil-in-water emulsion.
Arachis oil are mix with water and emulsifying agent are added to make
the two solution miscible. After all solution are added, test tube are placed
on vortex mixer so that all the solution are mix uniformly. Time taken for the
interface to reach 1 cm are recorded. The result are as above. The time taken for the interface to reach 1 cm can be concluded as the
stability of the mixture. From tube number 3 until 7, the
emulsions are not stable because the interphase are separated before or around 120 minutes. The cohesive forces are greater than adhesive
forces. In tube 8, there are no emulsifying agents added. So the emulsion is
unstable and yet the globules tend to coalescence to form big globules. In
short, the arachis oil emulsion is stable at HLB value of 9.67-11.34. Since the
emulsifiers are slightly hydrophilic, we assume this is an oil-in-water emulsion.
From the table, the olive oil
emulsion in tube 1-4 are the most stable because inter phase did not reach 1cm
after 120 minutes. After shearing, emulsifiers such as Span 20 and Tween 80
break the globules into smaller globules to increase the surface area and hence
increase surface free energy. So the HLB values of emulsifiers are in the range
of 9.67-10.37 is the most suitable. Since the emulsifiers also slightly
hydrophilic, we assume this is an oil-in- water emulsion. However, results of
Group 7 showed that in tube 4, the interphase also did not reach 1cm after 120
minutes but this is different from Group
3 results as the interphase is separated at 128 minutes. After taking the
average readings, the emulsion in those tubes are considered unstable. This may
occur due to error occurred during the experiment such as accidentally added
excessive emulsifiers. Error
also occur at tube 1 for group 7, because the emulsions in tube 1 are supposed
to be stable and did not separate but the emulsion separates so quick. Error
that may occur are such as impurities present causing alteration in the
emulsions.
From
the table, mineral
oil emulsion used in tube 1 of both Group 4 and 8 showed the increasing of time taken for inter phase to reach 1 cm
as the HLB values increase. The stability of mineral oil are relatively lower
than other oil because the average time taken for interphase to reach 1 cm are
faster compared to other oil.
Based on the results, it can be concluded that only
tube 1 have the stable emulsion thus the suitable HLB is 9.67.
Temperature
cycle test is used to determine the stability of the emulsions in terms of
stability, storage, shelf life of the emulsions as we never know what will
happen to the emulsion given to a patient if they store it in different
temperature and conditions. Firstly, the viscosity of the emulsions should be
measured before and after the temperature cycle in order to calculate the
difference between the two values. If there is a small difference in the
viscosity before and after the test, it means that the emulsion is a stable emulsion
because the emusion deviates less from the original emulsion. According to the
results, emulsion with 25ml of mineral oil (group 2) has the smallest
difference in viscosity, which should be the most stable emulsion. In this
experiment, there might be certain errors occurred during the experiment like
insensitivity of the viscometer.
Centrifugation of the emulsion is
used in order to test the rate stability of emulsion by calculatng the rate of
sedimentation. The higher the ratio of separation phase, the lower the
stability of the emulsions.In the experiment, the emulsion with 25ml of mineral
oil shows the least ratio of separation which means that it has the greatest
stability among other emulsions, proven in the results that were compared with
other groups. However, this does not justify accurately as there might be some
broken structure of emulsion due to extreme shear of centrifugation.
CONCLUSION
Combination of a few surfactants
will give the accurate HLB value required to form a stable emulsion. Different
types of oil have different viscosity. The higher the amount of oil, the higher
the viscosity and the more the separation phase.
