Sunday, November 8, 2015

Lab 2 EMULSION

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
An emulsion is a thermodynamically unstable two-phase system consisting of at least two immiscible liquids. It contains at least two immiscible liquid where one of them is dispersed and dispersion medium. The dispersed liquid is known as the internal or discontinuous phase, whereas the dispersion medium is known as the external or continuous phase. In general, emulsion can be categorized into 2 types, oil in water emulsion (o/w) and water-in-oil emulsion (w/o). . An o/w emulsion is generally formed if the aqueous phase constitutes > 45% of the total weight, and a hydrophilic emulsifier is used. Conversely, where water or aqueous solutions are dispersed in an oleaginous medium, the system is known as a water-in-oil (w/o) emulsion. W/O emulsions are generally formed if the aqueous phase constitutes < 45% of the total weight and an lipophilic emulsifier is used.
The HLB method (hydrophilic-lipophilic balance) 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 form 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.
The consistency of emulsions varies from easily pourable liquids to semisolid creams. Thus, emulsions are used in many routes of administration. Most commonly, emulsions are used for topical administration. Emulsions are also used as an ointment bases and intravenously administered as part of parenteral nutrition therapy.

Apparatus and materials

a. Apparatus

8 test tubes, a 50ml 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 5ml pipette and bulb, 1 50ml beaker, a 15ml centrifugation tube, centrifugation apparatus, viscometer, water bath (45 celsius) and refrigerator  (4 celsius).

b. Materials
Palm oil, Arachis oil, Olive oil, Mineral oil, Distilled water, Span 20, Tween 80, and Sudan III solution (0.5%)

Procedures:
1. Each test tube was labelled and marked 1 cm from the base of the test tube.
2. 4ml of oil was mixed and 4ml of distilled water into the test tube.
Group
Oil
1,5
Palm oil
2,6
Arachis oil
3,7
Olive oil
4,8
Mineral oil

3. Span 20 and Tween 80 were added into the mixture of oil and water (refer Table 2). The test tube was closed and its content was mixed with vortex mixer for 45 seconds. The time needed for the interface to reach 1cm was recorded. The HLB value for each sample was recorded. Steps 1-3 were recorded in order to obtain an average HLB value of a duplicator.

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
4. A few drops of Sudan III solution were added to (1g) emulsion formed in a weighing boat and mixed homogenously. The spread of the colour in the sample was compared. Some of the sample was spreaded on a microscope slide and observed under light microscope. The appearance and globule size formed was described and drawn.
5. Mineral Oil Emulsion (50 ml) was prepared from the formulation below by using wet gum method according to Table 3a and 3b.
Mineral Oil
(refer Table 3b)
Acacia
6.25g
Syrup
5ml
Alcohol
3ml
Distilled water qs
50ml
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
6. 40g of emulsion was placed intoa 50ml beaker and homogenized for 2 minutes using a vortex mixer.

7. 2g of emulsion (before and after homogenization) was taken and placed into a weighing boat and labelled. 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 under the light microscope was stated and compared.

8. The viscosity of the emulsion formed after homogenization (15g in 50ml) was determined using viscometer that is calibrated with “Spindle” type LV-4. The sample was exposed to 45 celsius (water bath) for 15 minutes and then to 4 celsius (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 emulsion is determined. Step 8 was repeated again and an average value was obtained.


Readings
Viscosity(cP)
Average
1
2
3
4
5
6

Before
Temperature
Cycle













After Temperature Cycle













Difference
      (%)







Table 4
9. 5g of homogenized emulsion was placed into a centrifugation tube was placed and centrifuged (4500 rpm, 10 minutes, 25 celsius). The height of the separation formed was measured and the ratio of the height separation was determined.
Mineral Oil (ml)
Ratio of separation
Average
Ratio of separation phases
20






25






30






35














Result

 For Palm Oil:

 Palm oil
Tube number
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
Time of phase separation (min)
---
---
86
60
  80
113
85
  15

--- = Time taken greater than 130 minutes To find the average, 130 minutes used to represent the time taken for interphase to reach 1cm for ---



For Olive Oil:

 Olive oil
Tube number
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
Time of phase separation (min)
---
---
57
11
  32
14
37
   0
--- = Time taken greater than 130 minutes To find the average, 130 minutes used to represent the time taken for interphase to reach 1cm for ---






For Mineral Oil:


 Mineral oil
Tube number
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
Time of phase separation (min)
---
---
69
61
  56
46
0
   0
--- = Time taken greater than 130 minutes To find the average, 130 minutes used to  represent the time taken for interphase to reach 1cm for ---



For arachis  oil (Group 2 B):

Group 2( Arachis oil)
Tube number
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
Time of phase separation (min)
---
79
75
35
27
40
20
0


HLB value =
[(quantity of surfactant 1)(HLB of surfactant 1) + (quantity of surfactant 2)                              
(HLB of surfactant 2)]
quantity of  surfactant 1 + quantity of  surfactant 2


Calculation of HLB values:
HLB value for Span 20    = 8.6
HLB value for Tween 80  = 15.0
Therefore, example of calculation:

HLB value for Tube 1 = (15 x 8.6) + (3 x 15.0)  
                         (15 + 3)
                  = 9.67

HLB value for Tube 2 = (12 x 8.6) + (6 x 15.0)  
                          (12 + 6)
                  = 10.73

HLB value for Tube 3 = (12 x 8.6) + (9 x 15.0)  
                          (12 + 9)
                  = 11.34



HLB value for Tube 4 = (6 x 8.6) + (9 x 15.0)  
                         (6 + 9)
                  = 12.44

HLB value for Tube 5 = (6 x 8.6) + (15 x 15.0)  
                         (6 + 15)
                  = 13.17

HLB value for Tube 6 = (3 x 8.6) + (18 x 15.0)  
                         (3 + 18)
                  = 14.09

HLB value for Tube 7 = (0 x 8.6) + (15 x 15.0)  
                          (0 + 15)
                  = 15.00

HLB value for Tube 8 = (0 x 8.6) + (0 x 15.0)  
                           (0 + 0)
                  = 0.00



For arachis oil: 

Appearance and globule size under light microscope






Test tube
Shape
Globule size
Spread of colour
1
Round globule
Small
Not Even
2
Round globule
Big
Even
3
Round globule
Tiny
Even
4
Round globule
Small
Not Even
5
Round globule
Tiny
Even
6
Round globule
Small
Even
7
Round globule
Tiny
Not Even
8
Round globule
   Tiny
Not Even


2g of emulsion before and after homogenization 

For 20ml mineral oil
Before homogenization
After homogenization
Texture
Non homogenous (spacious)
Homogenous (packed)
Consistency
Less consistent-crystal clump together
More consistent-crystals dispersed
Degree oily appearance
More greasy
Less greasy
Spreading of colour
 Less spreading
More spreading

For 25 ml mineral oil
Before homogenization
After homogenization
Texture
Non homogenous
Clear and homogenous
Consistency
Less consistent
More consistent
Degree oily appearance
More greasy
Less greasy
Spreading of colour
Less even distribution
Better even distribution

For 30ml mineral oil
Before homogenization
After homogenization
Texture
Course and not homogenous
Smooth and homogenous
Consistency
Not consistent,less viscous
Consistent
Degree oily appearance
More greasy,spherical globules
Les greasy and aspherical globules
Spreading of colour
Unevenly spreading
Evenly spreading


For 35ml mineral oil
Before homogenization
After homogenization
Texture
More watery and non viscous
Less watery and more viscous
Consistency
Not consistent and non viscous
Consistent and mor viscous
Degree oily appearance
More greasy and more globules
Less greasy and globules
Spreading of colour
Unevenly distribution
Evenly distribution



For Viscosity Of Emulsion:

20 ml of mineral oil
Readings
Viscosity (cP)
Average
1
2
3
4
5
6
Before Temperature Cycle
42
42
36
24
30
24
33
After Temperature Cycle
48
45
40
30
38
36
39.5
Difference(%)
19.7











25ml of mineral oil
Readings
Viscosity (cP)
Average
1
2
3
4
5
6
Before Temperature Cycle
30
30
30
30
30
30
30
After Temperature Cycle
44
46
46
48
45
46
45.8
Difference(%)
52.7










30ml of mineral oil
Readings
Viscosity (cP)
Average
1
2
3
4
5
6
Before Temperature Cycle
60
48
54
48
48
54
52
After Temperature Cycle
83
70.4
80.1
72.4
70.4
80.1
76.1
Difference(%)
46.3









35ml of mineral oil
Readings
Viscosity (cP)
Average
1
2
3
4
5
6
Before Temperature Cycle
3.6
3.9
6.9
3.9
18.3
17.1
9.0
After Temperature Cycle
14.4
19.2
10.8
8.4
7.2
8.4
11.4
Difference(%)
26.7






For phase separation after the centrifuge of emulsion:


Mineral Oil (ml)
Ratio of separation phase
       Average
Ratio of separation phase
20
4.00
3.50
4.00
3.83
0.77
25
3.00
3.30
3.50
3.27
0.66
30
2.86
2.70
2.86
2.81
0.56
35
2.25
2.25
2.15
2.22
0.44



Discussion

Stable emulsion means longer time for phase separation to occur. For palm oil, the HLB value that brings to stable emulsion is 11.34. This means that the stable emulsion of palm oil can be prepared by adding 12 drops of Span 20 and 9 drops of Tween 80. For arachis oil, the HLB value that can give stable emulsion is 11.34. For olive oil, the HLB value that can give stable emulsion is 11.34. For mineral oil, the HLB value that can give stable emulsion is 10.70. These show that different oils with different HLB values require different combination of surfactants in order to produce a stable emulsion.
             
From the experiment for arachis oil which is done by our group, it was found that emulsion from test tube 1, 2 and 3 where the HLB values are 9.67, 10.73 and 11.34 respectively give the most stable emulsions. Their phase separation time is very long if compared to the other test tubes. Thus, the corresponding HLB value of 9-13 gives the most effective surfactant for the oils used in this experiment. Appropriate HLB value is important in determining the stability of the emulsion. Meanwhile, emulsions from tube 7 and 8 give the lowest stability where the phase separation time is the shortest. This is because the absence of surfactant as an emulsifying agent in tube 7 and 8. Surfactant enhances the distribution of oily phase into the aqueous phase (o/w emulsion) or the distribution of aqueous phase into the oily phase (w/o emulsion).

A low HLB value have more hydrophobic group while the high HLB value have more hydrophilic group. Span is hydrophobic and is used to make w/o emulsion. Meanwhile, tween is hydrophilic and is used to form o/w emulsion. In the stabilization of oil globules, it is essential that there is a degree of hydrophilicity to confer an enthalpic stabilizing force and a degree of hydrophobicity to secure adsorption at the interface. So, a combination of both offers a suitable HLB value which matches with the system and produces a stable emulsion. The HLB value of the surfactant that fulfills the required HLB value of the emulsion will form the stable emulsion and vice versa. The emulsion is formed by reducing the surface tension of the two immiscible liquid through the presence of surfactant at the interface. Generally, a combination of surfactant is better than a single surfactant, which produces more stable emulsion.

During this experiment , we did mixing the emulsion homogenously.Before homogenization, the globules are not in uniform size and are coarse. There has a combination of small, intermediate and large size globules. However, the size of globules becomes uniform after homogenization and all globules are in smaller size. About the greasiness, the emulsion is greasy and less viscous before homogenization due to the reason of unemulsified oil. However, the emulsion becomes smoother and more viscous after homogenization. This is because the sample tube is spun in the high rate and breaks the globules into smaller sizes. Besides that, the emulsion is less consistent before homogenization. However, the consistency of the emulsion is increased and the degree of oily are decreased after homogenization because during homogenization, forces are applied to the emulsion and thus it causes a better emulsifying effect to take place. After adding Sudan test III solution, the colour of the emulsions becomes milky. It shows good colour dispersion in the emulsions.


Sudan test is a group of azo compound used as biological stains for fat. It is used to show the shape and physical characteristic of oily emulsion. It can differentiate which emulsion is oil-in-water emulsion or water-in-oil emulsion by determining the amount of globules in red colour and the colourless globules. Sudan solution is a red colour solution. It is dissolved in oily phase of the emulsion. So, it will cause the oily globules stain in red colour. The colour dispersion of the emulsions before homogenization is not consistent. However after the homogenization, the colour of dispersion is more consistent. Thus, the emulsion formed is considered as o/w emulsion.



For viscosity of emulsion after homogenization



Graph 1.
Based on the graph 1, the percentage difference of viscosity (%) increases as the amount of mineral oil used increases. But percentage difference of viscosity (%) started to decrease when using 30 and 35 ml of mineral oil. All type of emulsion is exposed to the same temperature and they have the same volume, but different proportion of emulsifying agent, oil and water proportions. The percentage difference is the difference between the viscosity before and after the temperature cycle. For 20 mL of mineral oil,  the percentage difference of viscosity is small which is 19.70% because the emulsion is not too viscous. For 25 mL of mineral oil, the percentage difference of viscosity is 52.67% which is much higher than mineral oil of 20 mL and that is because the emulsion is getting more viscous when adding more mineral oil into it. For 30 and 35 mL of mineral oil used, the percentage difference is 46.30% and 26.70% which is lower than 25 mL of mineral oil used. So this might be due to some errors occurs during handling the experiment because the percentage difference should be higher than 25 mL of mineral oil used. The percentage difference of viscosity (%) should increase with increasing amount of mineral oil used. 

Viscosity means a quantity expressing the magnitude of internal friction , as measured by the force per unit area resisting a flow in which parallel layers unit distance apart have unit speed relative to one another . In simple term , it can be explain as a measurement of a fluid’s internal resistance to flow.
Besides, in this experiment, the other objective is to test the physical effects and stability on the emulsion formulation due to different amount of emulsifying agent. Thus it will be tested at different temperature , and can also effect the emulsifying agent. Emulsifying agent will prevent coalescence of droplets and maintain the individual droplets in the continuous phase. It also acts by adsorb onto the oil-in-water interface and lower the surface interfacial tension. In addition, emulsifying agent tends to promote dispersion of the phase in which they do not dissolve very well.
Increase in amount of mineral oil used in an emulsion will increase amount of acacia that will be use as an emulsifying agent . This is due to the ratio 4:2:1. The 4 part represent oil part , 2 part for the water while 1 part is for the emulsifying agent.
Since acacia does not dissolves in oil, so this will form oil-in-water emulsion. Increase in temperature will also increase droplet size and reduce viscosity. The increasing of droplet size will make the emulsion difficult to disperse and easily separate to two layers . While in cold temperature , the emulsion will become thicker and viscous and difficult to be separate in two layer.
The value of emulsion after the temperature cycle is in greater value than before the temperature cycle. This happen due to loss of some proportion of the volume during heating process. During heating process the molecule will vibrate but interact less. Thus the viscosity value is low.




For the ratio of separation phase
Centrifugation of emulsion is used to separate the oil phase from the aqueous phase. From the result obtained , it proved that the phase of separation ratio will be lower if the more volume of mineral used in the formulation. Oil is less dense than water thus it will be form at top layer after the centrifugation.
If the oil phase is at high level , thus the separation phase will be shorter and result in lower phase separation value.
Throughout this experiment , there were some errors that occur. So , there are some precaution step that we need to take as example we must make sure that the room temperature of the laboratory is constant as it will affect the changing properties of surfactants. Besides , we need to be aware of the parallax error that might occur .
Since the experiment of different volume of mineral use were done by different group in the laboratory thus the result obtained might differ a bit than the theoretical result.


Conclusion

1)   Combination of surfactants form a more stable emulsion than a single surfactant. Different oily phase need a different value of HLB surfactant so that the most stable emulsion can be formed.    
     2) Homogenization process is applied in this mineral oil emulsion to reduce the size of droplets in liquid-liquid dispersions. Before homogenization, the texture of emulsion is non-homogenous,  less consistency, more greasy in degree of oily appearance and less spreading of colour. After homogenization, the texture of emulsion is homogenous, more consistent, non greasy in degree of oily appearance and more evenly spreading.
     3)   In oil-in-water emulsion, decrease in temperature may cause high viscosity of an emulsion and vice versa. High viscosity will increase the time for the separation of emulsion into two layers.
      4)    As more volume of mineral oil is used in formulation, a shorter separation phase will exhibit and this indicates a lower phase separation value.
      5)   The higher the amount of mineral oil used, the higher the amount of emulsifying agent (acacia) needed to lower the surface interfacial tension.

6)  The emulsion stability increases as the HLB value increase because the droplet size decreases.

jReferences
 1. http://chemistscorner.com/hlb-the-easiest-way-to-create-an-emulsion/
2. http://journal.scconline.org/pdf/cc1968/cc019n10/p00683-p00697.pdf
3. http://www.particlesciences.com/news/technical-briefs/2011/emulsion-stability-and-testing.html
4. Florence A.T. 1998. Physicochemical Principle of Pharmacy, 3rd Edition
5. Aulton, M.E. Pharmaceutics: The science of dosage form design. Edinburgh: Churchill Livingstone.

6. http://medical-dictionary.thefreedictionary.com/Sudan+stain+test