~ Laboratory Report ~ (NFNF 2263 PHARMACEUTICAL TECHNOLOGY II) GROUP A7: ASSAY ON THE EFFECTS OF DIFFERENCE IN CONTENT ON THE CHARACTERISTICS OF A EMULSION FORMULATION

Viscosity (cP)

Before heat exposure

After heat exposure

Difference (%)

NFNF2263 TEKNOLOGI FARMASEUTIKAL II

Group Members:

Mohamad Amirul Bin Mohd Yunus A136301
Bazlin Sorfina Binti Sulaiman A139704
Cheong Jia Li A140239
Ong Ching Yao A140288
Puteri Annisa Adhwa Binti Megat Baharudin A140042
Siti Amirah Binti Abdul Wahab A140181

Experiment 1

Title:

ASSAY ON THE EFFECTS OF DIFFERENCE IN CONTENT ON THE CHARACTERISTICS OF A EMULSION FORMULATION

Introduction:

Emulsion is a two-phase system which is not stable in thermodynamic. It contains at least 2 liquids which are not miscible to each other (internal/dispersed phase) dispersed homogeneously in another liquid (external/continuous phase). Emulsions can be classified into 2 types: oil in water emulsion (o/w) and water in oil emulsion (w/o). Emulsions become stable with the addition of emulsifying agents. Emulsifying agents can be classified into 3 types; they are (1) hydrophilic colloid, (2) finely divided solid, (3) surface active agents or surfactants.

HLB system (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 HLB scale from 1 (lipophilic) to 10 (hydrophilic). Commonly, combination of 2 emulsifying agents is used to produce a more stable emulsion preparation. HLB value for combination of emulsifying agents can be determined using the formula below:

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)

Objective:

1. To determine the effect of HLB of surfactants on the stability of the emulsions.

2. To study the effects of physical and stability on the emulsion formulation using different content of emulsifying agents.

Apparatus:

8 test tubes

1 50 ml measuring cylinder

2 sets of pasture pipettes and droppers

Vortex mixer

Weighing balance

1 set of mortar & pestle

Light microscope

Microscope slide

1 set of 5 ml pipette and bulb

1 50 ml beaker

Coulter counter machine

Centrifuge machine

Viscometer

Water bath (45°C)

Fridge (4ºC)

Materials:

Palm oil

Arachis oil

Olive oil

Mineral oil

Distilled water

Span 20

Tween 80

Sudan III solution (0.5%)

ISOTON III solution

Procedures:

1. Each test tube was labeled and 1 straight line was sketched 1 cm from the bottom of the test tubes.

2. 4 ml oil was mixed with 4 ml distilled water in the test tubes.

Table 1

Group	Oil
1, 2	Palm oil
3, 4	Arachis oil
5, 6	Olive oil
7, 8	Mineral oil

3. For mixture of oil and water, a few drops of Span 20 and Tween 80 was added (refer to Table 2). The test tubes were closed and mixed with vortex mixer for 45 seconds. The time needed for the interface to reach the 1 cm line was recorded. The HLB value for each sample was determined.

Table 2

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
HLB value
Time of phase separation (min)
Stability

4. A few drops of Sudan III solution were added to 1 g of emulsion formed in a weighing bot and was flattened. The spreading of colour in the sample was described and compared. Some sample was spread on a microscope slide and was observes under light microscope. The shapes and sizes of globules formed were drawn, described and compared.

5. By using wet gum method, a formulation of Mineral Oil Emulsion (50 g) was prepared using the formula below:

Mineral oil refer Table 3

Acacia 6.25 g

Syrup 5 ml

Vanillin 2 g

Alcohol 3 ml

Distilled water 50 ml

Table 3

Emulsion	Group	Mineral oil (ml)
I	1, 2	20
II	3, 4	25
III	5, 6	30
IV	7, 8	35

6. 40 g emulsion formed was placed in a 50 ml beaker and homogeneous process was carried out for 2 minutes with homogeneous machine.

7. 2 g emulsion formed was taken (before and after homogeneous process) in a weighing bot and was labeled. A few drops of Sudan solution were added and flattened. The textures, consistency, oily shape degree and spreading of colour of the sample were observed and described under light microscope.

8. Viscosity of emulsion (15 g in 50 ml beaker) form after homogeneous process was determined using viscometer calibrated using “Spindle” type LV-4. The sample was exposed to 45°C (water bath) for 30 minutes and then to 4 ºC (fridge) for 30 minutes. Viscosity of emulsions was determined after heat exposure and the emulsion had achieved room temperature (10 - 15 minutes)

9. 5 g emulsion that was homogenized was placed in a centrifuge tube and was centrifuged (4500 rpm. 10 minutes, 25°C). The height of the separation formed was measured and the ration was determined.

Results
For procedure 4:

(In test tube 1, the color dispersion is more difficult to spread and the size of droplets is almost similar, near to each others and distributed evenly.)

(In test tube 2, the color dispersion is easier to spread compare to test tube 1. The droplet's size is almost similar and slightly apart from each others.)

(For test tube 3, the color dispersion is still difficult to spread. The droplets' size is slightly not similar but still near to each others.)

(For test tube 4, the color dispersion is still difficult to spread. The size of droplets is different and slightly near to each others.)

(For test tube 5, the color dispersion is easier to spread. The droplets are slightly different in size and near to each others.)

(For test tube 6, the color dispersion is easy to spread. The size of droplets is slightly different. Some of the droplets are slightly far apart.)

(For test tube 7, the color dispersion is more easier to spread. The size of droplets is not same and some of them are bigger in size.)

(For test tube 8, the color dispersion is very easy to spread. The size of droplets varies, not regular and the droplets are not evenly distributed.)

Table II

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	9.667	10.733	11.343	12.44	13.171	14.086	15	0
Phase separation time (min)	-	-	14	-	69	-	55	10 sec
Stability	Stable	Stable	Not table	Stable	Not Stable	Stable	Not Stable	Not stable

Comparison Of Phase Separation Time Between Different Test Tubes (Minutes)

OIL	Olive oil
Tube 1	-
Tube 2	-
Tube 3	14 min
Tube 4	-
Tube 5	69 min
Tube 6	-
Tube 7	55 min
Tube 8	10 sec

Examination On Olive Oil Emulsion

Test Tube	Physical Characteristics
1	Naked eye: · Colour dispersed Microscopic: · Globules with similar sizes · Mostly round shape of globules were seen It is oil in water emulsion
2	Naked eye: · Colour dispersed Microscopic: · Medium and small size globules · All round shape of globules It is oil in water emulsion
3	Naked eye: · Colour dispersed Microscopic: · Small and large size globules · All round shape of globules It is oil in water emulsion
4	Naked eye: · Colour dispersed Microscopic: · Small and large globules sizes · Some globules are not in uniform shape It is oil in water emulsion
5	Naked eye: · Colour dispersed Microscopic: · Small and large globules near each other · Mostly round globules were seen It is oil in water emulsion
6	Naked eye: · Colour dispersed Microscopic: · Small and large globules near each other · Mostly round globules were seen It is oil in water emulsion
7	Naked eye: · Colour dispersed Microscopic: · Small and large globules near each other · Round globules were seen mostly It is oil in water emulsion
8	Naked eye: · Colour did not dispersed Microscopic: · Small and large globules · Different shape of globules Emulsion will not formed without surfactant, phase separation occur very fast.

Examination On Mineral Oil Emulsion (35ml Mineral Oil)

	Description
Before homogenization	Texture: smooth and cloudy Consistency: not consistent Degree of greasiness: very greasy Shape: Spherical globules Size: not uniform with both big and small size Color dispersion: unevenly dispersed, less red spot Viscosity: less viscous
After homogenization	Texture: smooth and milky Consistency: consistent Degree of greasiness: less greasy Shape: Spherical globules Size: uniform size Color dispersion: evenly dispersed, more red spot Viscosity: more viscous

CALCULATIONS:

Difference = (Average of after temperature cycle - Average of before temperature cycle) X 100 %

Average of before temperature cycle

SD =

Viscosity Of Mineral Oil Emulsion (35ml Mineral Oil)

Readings	Group		Viscosity (cP)			Average ± SD
Readings	Group		1	2	3	Average ± SD
Before temperature cycle(˚C)		7	5610	5970	5850	5810 ± 149.67
After temperature cycle(˚C)		7	11220	12120	12060	11800 ± 410.85
Difference (%)		17%

Separation Height

Height ratio = Seperation phase

Emulsion phase

Mineral Oil (ml)	Group	Separation phase (mm)	Initial emulsion (mm)	Ratio of Separation Phase	Average Ratio (Average ± SD)
Emulsion I (20mL)	1	1.8	4.4	0.41	0.49 ± 0.08
Emulsion I (20mL)	2	2.6	4.6	0.57	0.49 ± 0.08
Emulsion II (25mL)	3	3.4	5.0	0.68	0.61 ± 0.07
Emulsion II (25mL)	4	2.7	5	0.54	0.61 ± 0.07
Emulsion III (30mL)	5	1.5	7	0.21	0.375 ± 0.165
Emulsion III (30mL)	6	27	50	0.54	0.375 ± 0.165
Emulsion IV (35mL)	7	12.6	43	0.29	0.295 ± 0.005
Emulsion IV (35mL)	8	14	46	0.30	0.295 ± 0.005

DISCUSSION

1. What are the HLB values that will produce a stable emulsion? Discuss.

Hydrophilic-Lipophilic Balance (HLB) value is a ratio of polar and non-polar group in the surfactant. In other words, HLB value is the balance of oil soluble substance and water soluble substance in a surface active agent. In this experiment, surfactants were used to create emulsion. Then the emulsion will be observed in macroscopic level and microscopic level. If the emulsion is stable, he emulsion will not separate while if the emulsion is not stable, they will form 2 distinct layers. In this experiment, eight emulsions were formed which each emulsion either combination of Span 20 and Tween 80 or a surfactant was used alone. In test tube 1, 15 drops of Span 20 mixed with 3 drops of Tween 80. In test tube 1, 15 drops of Span 20 mixed with 3 drops of Tween 80. In test tube 2, 12 drops of Span 20 mixed with 6 drops of Tween 80. In test tube 3, 12 drops of Span 20 mixed with 9 drops of Tween 80. In test tube 4, 6 drops of Span 20 mixed with 9 drops of Tween 80. In test tube 5, 6 drops of Span 20 mixed with 15 drops of Tween 80. In test tube 6, 3 drops of Span 20 mixed with 18 drops of Tween 80. In test tube 7, only 15 drop of Tween 80 used. In test tube 8, no surfactant was added and acts as control. Our result shows that the stable emulsion form on test tube 1, 2, 4 and 6 where there are no separation occurs. Since the result is not consistent one to other, there might some errors when we done the experiment.

By using,

HLB ( A + B ) = ( Ax + By ) / ( x + y )

The HLB number of a mixture composed of x% of surfactants of HLB A and y% of surfactants of HLB B is obtained by the following formula. According to our result, the HLB values that produce stable emulsion are 9-11, 12-13 and 14. The HLB of an emulsifier is related to its solubility. Thus, an emulsifier having a low HLB will tend to be oil-soluble, and one having a high HLB will tend to be water-soluble, although two emulsifiers may have the same HLB and yet exhibit quite different solubility characteristics. The SPAN emulsifiers are usually lipophilic and the TWEEN products are usually hydrophilic

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.

In this experiment, Emulsion I contains 20ml of mineral oil, Emulsion II contains 25 ml of mineral oil, Emulsion III contains 30ml of mineral oil while Emulsion IV contains 35ml of mineral oil.

The objective of the homogenization process is to mix the oil and other substances by using external stimuli. The effect of homogenization on the emulsion is the changes in the viscosity of the emulsion. The viscosity of all the emulsion after the homogenization is much greater than before homogenization. Both before and after homogenization, the texture of the mineral oil is smooth and cloudy. Size of the mineral oil droplet was not consistent before the homogenization due to the mixture is not well mix when there is no force. But, after the homogenization, the size of the droplets becomes more consistent. The colour of the mixture before the homogenization was not evenly dispersed while after the homogenization the colour more evenly dispersed.

Sudan test or called Sudan red test is use to detect the lipid or fat present in some substances such as blood and faeces. The Sudan dye will attach to the lipid substances and colour it. In this experiment, the Sudan dye is used to show the position of the lipid in the mixture. Since the mixture is not an ionic mixture, the Sudan dye can work appropriately. By using this experiment, the function of Sudan dye can be explained. The red stain of Sudan dye in Emulsions Ι, ΙΙ, ΙΙΙ and ΙV is uneven before homogenization. So, we can say that these emulsions formed are water-in-oil emulsion. However, after homogenization, the red stain is even. Red globules have been seen in uniform dispersion on a colourless background. The size of the globules is small. Hence, oil in water emulsion is formed after homogenization. Here, phase inversion occurred.

3. Plot and give comments on:

(a) Graph of the sample’s viscosity before and after the temperature cycle against the different amount of oil content.

Amount of mineral oil (ml)	Average Viscosity (cP) (mean±SD)		Difference of viscosity (%) (mean±SD)
Amount of mineral oil (ml)	Before temperature cycle	After temperature cycle	Difference of viscosity (%) (mean±SD)
20	18.93 ± 2.0422	19.30 ± 3.9800	1.95 ± 64.36
25	1360 ± 519.81	1180 ± 61.64	14.17 ± 157.60
30	3020 ± 96.44	6250 ± 984.53	69.69 ± 164.31
35	5810 ± 149.67	11800 ± 410.85	17.00 ± 23.30

In the experiment, 4 different emulsions were prepared by varying the amount of mineral oil which is 20, 25, 30 and 35 ml. Emulsifying agent used in the formulation was acacia. Acacia had a HLB value of 8 which renders them as an oil in water emulsifier. Before the temperature cycle, the viscosity of the sample increases as the proportion of the mineral oil increases. oil has higher viscosity compared to water. Thus, sample with higher amount of oil will give a higher value of the average viscosity as shown in the graph above. The trend is comparable with the plots after the temperature cycle despite that the values are much higher. The emulsion shows a higher viscosity if compared to the value before the temperature cycle except for sample with 25ml of mineral oil where there is a decrease in viscosity after the temperature cycle.

The purpose of treating the emulsion with exaggeration of the temperature fluctuations is to compare the physical instabilities of the emulsion. Increase in temperature of emulsion will cause decrease in viscosity of continuous phase and will increase the kinetic motion of dispersed phase. As the kinetic energy of dispersed phase (oil phase) increases, the globules gain energy and collide with each other more frequently, causing the breakdown of the emulsifier thus resulting in coalescence of globules. At low temperature, crystallization of some of the water causes the globules to be forced closer together, thereby promoting globule-globule interactions. The temperature cycle will disrupt the adsorbed layer of emulsifying agent at the oil/water interphase and hence affect the stability of the emulsion.

Besides that, an oil-in-water emulsion that is stabilized by non-ionic emulsifying agent such as acacia will undergo phase inversion into water-in-oil emulsion upon heating. This is because as the temperature increases, the HLB value of a non-ionic emulsifying agent will decrease as it becomes more hydrophobic. At the phase inversion temperature (temperature at which the emulsifier has equal hydrophilic and hydrophobic tendencies), the emulsion will be inverted. As the emulsion had been converted into a water-in-oil emulsion, the contribution of oil as the continuous phase made the viscosity become higher after the temperature cycle.

As stated before, sample with 25ml of mineral oil had shown a decrease in viscosity after the temperature cycle. This result differs from the theory in which it could be arose from several errors encountered during the experiment. One of the errors could be from unproper rinsing of the viscometer spindle that might affect the concentration of the emulsion components. Besides that, the sample might have not being exposed to the temperature according to the duration stated in the procedure. Due to shorter time of exposure or shorter time of leaving the sample to room temperature, the viscosity of the sample was affected.

(b) Graph of the viscosity difference (%) against the different amount of oil content.

Amount of mineral oil (ml)	Average Viscosity (cP) (mean±SD)		Difference of viscosity (%) (mean±SD)
Amount of mineral oil (ml)	Before temperature cycle	After temperature cycle	Difference of viscosity (%) (mean±SD)
20	18.93 ± 2.0422	19.30 ± 3.9800	1.95 ± 64.36
25	1360 ± 519.81	1180 ± 61.64	14.17 ± 157.60
30	3020 ± 96.44	6250 ± 984.53	69.69 ± 164.31
35	5810 ± 149.67	11800 ± 410.85	17.00 ± 23.30

From the graph above, the viscosity differences before and after the temperature cycle among each sample shows a clear different percentage which are 1.95%, 14.17%, 69.69% and 17% for emulsions prepared from 20 mL, 25 mL, 30 mL and 35 mL of mineral oil respectively. Large viscosity difference was seen in sample III with 30ml mineral oil. By theory, larger viscosity difference indicates a weaker and less stable emulsion. As the volume of dispersed phase (oil phase) increases, the stability of the emulsion decreases and phase inversion may occur. The small viscosity differences seen in sample I might due to that the emulsion is quite stable and does not undergo phase inversion. In order to ensure a more accurate, several precautionary measures should be taken such as the mineral oil emulsion must be stirred first before running the viscometer for each reading. Next, never forget to rinse the viscometer spindle before a new emulsion is going to be examined. Besides, do make sure that the practitioner truly understand how to set up the viscometer machine so that the sample’s viscosity is being determined under the required condition.

4. Plot the graph of the ratio of separation phase after centrifugation process against the different contents of mineral oil. Give your comments.

Mineral Oil (ml)	Ratio of Separation Phase % (Average ± SD)
Emulsion I (20mL)	0.49 ± 0.08

Emulsion II (25mL)	0.61 ± 0.07

Emulsion III (30mL)	0.375 ± 0.165

Emulsion IV (35mL)	0.295 ± 0.005

Theoretically, a stable emulsion will have a minimum ratio of phase separation. This is because the tendency for the emulsion to become separated after centrifugation process is lower when the composition of the oil, water and emulsifier are in appropriate amount. An increase in amount of the dispersed phase led to an unstable emulsion thus the ratio of separation phase is higher. However, from the graph above, the ratio of separation phase increase from sample I to II and decreases from sample II to III and IV. The highest ratio is in sample with 25ml mineral oil and the lowest is in sample with 35ml mineral oil. The result deviates from the theory.

The deviation might be due to errors arose at the beginning level which is the preparation of the emulsion. The amount of each ingredient might had not been measured accurately. Besides, practitioner might have prepared it by using method other than wet gum method.

5. What are the functions of every substance used in this emulsion preparation? How the different contents of substances can affect the physical characteristics and stability in the formulation of an emulsion?

The most important ingredients to prepare basic emulsion are oil, water and emulsifying agent. The ingredients that involve in this preparation of the emulsion are mineral oil, distilled water, acacia, syrup, Vanillin, and alcohol.

Type of oil that we used in this experiment is the mineral oil. We can other than mineral oil such as olive oil. In this experiment, the oil involve in the formation of the emulsion which is the oil phase or dispersed phase in oil-in-water emulsion. The amount of the oil used must not more than 70%. If the amount is more than that, the emulsion produced is water-in-oil emulsion.

Besides, water also needed to form emulsion. Distilled water was used in this experiment. More than 70% of distilled water was use to form oil-in-water emulsion. Distilled water was use instead of tap water is due to we want to prevent the contamination of microbial in the emulsion. Tap water have higher chance to be contaminated with the microorganism.

Next is the emulsifying agent which is acacia. The emulsifying agent is use to stabilized the mixture of oil and water. The emulsifier agent wills located on the surface of oil droplet and form a protective layer that prevent the physical contact between the water and oil. Since oil and water immiscible, the emulsifier properties of the acacia are important to stabilize the mixture. Besides, it also use as thickening agent.

Other ingredient that involve in the preparation of emulsion is syrup. It is one of the excipient that widely used. The main function of involving the syrup in this emulsion is to use it to mask the badly oil taste. Syrup is a sweetening agent. Simple syrup contains sucrose and purified water. The importance of the syrup can be observed from medication that formulated for children. Syrup also has the viscosity properties that are important for emulsion viscous properties. The amount of syrup must be precisely calculated to prevent the emulsion from having flow difficulties. The use of syrup is strictly prohibited for diabetic patient.

Vanillin also being use to mask the bad taste of oil. Vanillin now accepted as one of the main favorite flavor in the world. The main function is as a flavoring agent. It can help the problem of non-compliance among patients.

The last ingredient is the most important thing as safety profile of the emulsion. Alcohol is use as preservative in this emulsion. It prevents the growth of the microorganism. This emulsion have good environment for bacteria growth due to involvement of syrup and water. The amount of the alcohol must be considered according to the amount of the emulsion.

Furthermore, physical characteristics can also be affected in this case. Some of the oils used have different colours and this will produce emulsion with different colors. In addition, palm oil has antioxidant property and this can improve the stability of the emulsion formed. The differences discussed above will produce an emulsion with different physical characteristics and chemical stability. For chemical characteristics, the usage of the ingredient must not interact with each other. If there are interactions, the chemical stability can be interrupted.

Conclusion

The HLB value should be between 8-18 to form a stable oil-in-water emulsion and it should be in 3-6 to form a stable water-in-oil emulsion. Besides the HLB value of the surfactants, the types of surfactant that used are important too. To stabilize an emulsion, we usually use a combination of surfactant because it gives more benefits. Apart from that, minimum seperation phase is essential for a stable emulsion. The composition of surfactant and volume of oily phase used are important factors to determine the physical characteristics and stability of the emulsions. The viscosity of emulsion before temperature cycle should be lower than the viscosity after temperature cycle.

Appendix