Pharmaceutical Technology Group A: April 2014

Effects of Different Substance used on The Characteristics of an Emulsion.

INTRODUCTION

An emulsion is a dispersion in which the dispersed phase is composed of small globules of a liquid distributed throughout a vehicle in which it is immiscible. In emulsion terminology, the dispersed phase is the internal phase, and the dispersion medium is the external or continuous phase. Emulsions with an oleaginous internal phase and an aqueous external phase are oil-in-water (o/w) emulsions. Conversely, emulsions having an aqueous internal phase and an oleaginous external phase are termed water-in-oil (w/o) emulsions. Because the external phase of an emulsion is continuous, an o/w emulsion may be diluted or extended with water or an aqueous preparation and a w/o emulsion, with an oleaginous or oil-miscible liquid.

Generally, to prepare a stable emulsion, a third phase, an emulsifying agent or surfactant, is necessary. Depending on their constituents, the viscosity of emulsions can vary greatly and pharmaceutical emulsions may be prepared as liquids or semisolids. A good emulsion should be stable itself, chemically inert, nontoxic and cause no irritation upon application, be odourless, tasteless, and colourless and be inexpensive.

Surfactants can be classified using the HLB system established by Griffin. This system provides a scale of surfactant hydrophilicity (HLB value range from 1 to 20 which from most hydrophobicity to hydrophilicity). Generally, 2 emulsifying agents are usually used to form a stable emulsion preparation. The HLB value can be determine using the equation below:

HLB value = (Surfactant quantity1) (HLB surfactant1) + (Surfactant quantity2) (HLB surfactant2)

Surfactant quantity 1 + Surfactant quantity

In this experiment, different composition of Span 20 and Tween 80 are used and the HLB value of the different combination of these two emulsifying agents is determined using formula above. The different volume of emulsifying agents used is to determine the effect of each kind of emulsifying agents. Different HLB value give sifferent emulsifying effect.

Apparatus:

8 test tube, 50 ml measuring cylinder, 2 pipette and dropper, Vortex mixture, Weighing boat, Mortar&pestle, Light microscope, Microscope slide, 50 ml beaker, Coulter counter, Viscometer, Water bath, Refrigerator, Centrifuge, and 15 ml centrifuge tube

Material

Palm oil, arachis oil, Olive oil, Mineral oil, Distilled water, Span 20, Tween 80, Sudan III(0.5%) solution, ISOTON III solution.

Procedure:

1. Each test tube was labeled and a straight line of 1 cm apart from the base of the test tube was sketched.

2. 4 ml of oil (Table 1) was mixed with 4 ml of distilled water in the test tube.

Group	Oil test
1 & 2	Palm oil
3 & 4	Arachis (Peanut) oil
5 & 6	Olive oil
7 & 8	Mineral oil

Table 1

3. Drops of Span 20 and Tween 80 (Refer Table 2) were added to the mixture of oil and water in the test tube. The test tube was sealed and the content was homogenized by using vortex mixer device for 45 seconds. The time taken for the interface to reach the straight line which has been drawn earlier was recorded. The HLB value for each sample was determined.

Test tube number	1	2	3	4	5	6	7	8
Span 20 (Drop)	15	12	12	6	6	3	0	0
Tween 80 (Drop)	3	6	9	9	15	18	15	0
HLB value	9.7	10.7	11.3	12.4	13.2	14.1	15.0	0.0
Time of phase separation (min)
Stability

Table 2

4. Several drops of Sudan III solution were added to a little amount (1 g) of formed emulsion in a weighing boat and the mixture was evenly spread. The color dispersion in the sample was described and compared. A little amount of the sample was put on the microscope slide and was observed under a light microscope. The appearance and globule size formed were drawn, described and compared.

5. By using a wet gum method, a formulation of mineral oil emulsion (50 g) was prepared based on the following formula:

Mineral oil (Refer Table 3)

Acacia 6.25 g

Syrup 5 ml

Vanillin 2 g

Alcohol 3 ml

Distilled water, qs 50 ml

Emulsion	Mineral oil (ml)
I	20
II	25
III	30
IV	35

6. 40 g of the resulting emulsion was transferred into 50 ml beaker and the process of homogenizing was done for 2 minutes by using the homogenizer device.

7. A small amount (2 g) of the emulsion formed for both before and after homogenizing process were taken and placed in a weighing boat and labeled. Drops of Sudan III solution were added and evenly spread. The texture, constitency, degree of oily appearance and color distribution of the sample under the light microscope were described and compared.

8. The viscosity of resulting emulsion (15 g in 50 ml beaker) after homogenizing was determined using viscometer device which has been calibrated by using ‘Spindle’ LV-4 type. The sample was then exposed to a temperature of 45°C (Water bath) for 30 minutes and later at 4°C (refrigerator) for 30 minutes. The viscosity of the emulsion was determined after the temperature cycle exposure has finished and the emulsion has reached room temperature.

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

Result:

Result 1

Mineral oil

Test tube number	1	2	3	4	5	6	7	8
Span 20 (Drop)	15	12	12	6	6	3	0	0
Tween 80 (Drop)	3	6	9	9	15	18	15	0
HLB value	9.7	10.7	11.3	12.4	13.2	14.1	15.0	0.0
Time of phase separation (min)	No phase separation	92	118	84	68	74	17	3 s
Stability	Yes	Yes	Yes	No	No	No	No	No

Result 2

test tube 1

test tube 2

test tube 3

test tube 4

test tube 5

test tube 6

test tube 7

test tube 8

Result 3

A small amount (2 g) of the emulsion formed for both before and after homogenizing process were taken and placed in a weighing boat and labeled. Drops of Sudan III solution were added and evenly spread. The texture, constitency, degree of oily appearance and color distribution of the sample under the light microscope were described and compared.

Before homogenizing

After homogenizing

Result 4

(i) For 20 ml of mineral oil

Reading	Viscosity (cP)			Average + SD
Reading	1	2	3	Average + SD
Before temperature cycle	810	840	840	830 +/- 17.32
After temperature cycle	960	570	570	700 +/- 225.17
Difference (%)	Average: 16.99 SD: 171.43

(ii) For 25 ml of mineral oil

Reading	Viscosity (cP)			Average + SD
Reading	1	2	3	Average + SD
Before temperature cycle	630.0	660.0	690.0	660 +/- 30.00
After temperature cycle	1500	1680	1710	1630 +/- 113.59
Differences (%)	Average: 84.72 SD: 116.43

(iii) For 30 ml of mineral oil

Reading	Viscosity (cP)			Average + SD
Reading	1	2	3	Average + SD
Before temperature cycle	440	550	570	520+/- 70
After temperature cycle	2800	2900	2250	2650+/- 350
Difference (⁰/₀)	Average: 134.38 SD: 66.67

(iv) For 35 ml of mineral oil

Reading	Viscosity (cP)			Average + SD
Reading	1	2	3	Average + SD
Before temperature cycle	5450	5700	4900	5350 +/- 409.27
After temperature cycle	7170	6230	5720	6373.33 +/- 735.55
Differences (%)	Average: 17.46 SD: 57.00

Result 5

Mineral Oil (ml)	Interface (mm)	Initial emulsion (mm)	Ratio of Separation Phase	Average Ratio of separation (Average ± SD)
Emulsion I (20mL)	35	55	0.64	0.61 ± 0.05
Emulsion I (20mL)	26	46	0.57	0.61 ± 0.05
Emulsion II (25mL)	27	50	0.53	0.61 ± 0.11
Emulsion II (25mL)	34	50	0.68	0.61 ± 0.11
Emulsion III (30mL)	34	50	0.68	0.49 ± 0.27
Emulsion III (30mL)	18	60	0.30	0.49 ± 0.27
Emulsion IV (35mL)	14	46	0.30	0.27 ± 0.04
Emulsion IV (35mL)	12	50	0.24	0.27 ± 0.04

Discussions:

1. What are the HLB values needed to produce a stable emulsion? Discuss.

(i) This is the results for our group ( mineral oil )

Test tube number	1	2	3	4	5	6	7	8
Span 20 (Drop)	15	12	12	6	6	3	0	0
Tween 80 (Drop)	3	6	9	9	15	18	15	0
HLB value	9.7	10.7	11.3	12.4	13.2	14.1	15.0	0.0
Time of phase separation (min)	No phase separation	92	118	84	68	74	17	3 s
Stability	Yes	Yes	Yes	No	No	No	No	No

Different types of oil used to prepare an emulsion will need different optimum HLB values. From our experiment, we found out that the optimum HLB values for Mineral Oil to produce the most stable emulsion is 9.7 where it did not cause phase separation to occur. The longer phase separation time, the more stable the emulsion. When no phase separation occur, it simply means that the emulsion is stable.

Generally, a surfactant is briefly 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. Micelles consist of a hydrophobic tail and a hydrophilic head. The hydrophilic head of the surfactant will be located inside the aqueous phase while the hydrophobic tail will surround the hydrophobic drug particles. The adsorption of the surfactant between the oil and aqueous phase will reduce the surface tension and this will stabilized the emulsion formed. Hence, the phase separation will occur more slowly. Therefore, the slowest the phase separation time, the greater the stability of emulsion formed.

From the experiment, we also found that Tube 8 gives the lowest stability of emulsion produced with HLB value of 0. This is because, there is no use of surfactant (Span 20 or Tween 80) 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) through the formation of micelles. Hence, phase separation occurs in the shortest time in Tube 8 of 3 seconds only.

Besides that, to produce the most stable emulsion, we need a combination of surfactants like we use in this experiment (Span 20 and Tween 80). When only a single type of surfactant is used in the emulsion, the emulsion produced is not stable. For example, Tube 7 only utilizes Tween 80 in its formulation with HLB value of 15. As the result, the emulsion formed is quite unstable. A good combination of surfactants is needed to produce a stable emulsion.

2 . Compare the physical features of mineral oil emulsion and give explanation. What is Sudan III Test? Compare dispersion of color in the emulsion formed and give explanation.

There are 8 emulsion produced in this experiment. All emulsion has different proportion volume of Span 20 and Tween 80. Emulsion 4 has big and intermediate size globules not adhering to each other while emulsion 5 and emulsion 6 have a few large globules that arranged closely with small globules surrounding it. For emulsion 7, it has a few big globules together with small globules but are separating from each other. For emulsion 8, it has a few droplets of big sizes accumulation since it contains only mineral oil and distilled water.

Sudan III solution is oil or lipid soluble. So, it would not mix with water. Thus, the purpose of this test is to stain the sudanopilic substances usually lipids. The oil will not be on top of the Sudan. The Sudan III solution is red in colour and dissolves in oil. It will give red colour to the oil globules when it is dissolved in oily phase. It is used to determine the type of emulsion formed. We can classify the emulsion into oil-in-water emulsion (o/w emulsion) or water-in-oil (w/o emulsion) through the Sudan test.

For all the emulsion, the reddish brown colour is spread in the emulsion. The difference is the intensity of the colour is differing. As the HLB value increase, the more hydrophilic its nature. So, the intensity of colour decrease for emulsion 4 to 7 as the HLB value keeps increasing for the emulsion. In the emulsion 8, the HLB value is 0. Thus, the property of the emulsion is lipophilic. So, the Sudan III solution is mix well in the emulsion and give the high intensity colour of reddish brown colour.

3. Plot and explain:

(i) Graph of the sample’s viscosity before and after the temperature cycle against different contents of mineral oil.

Theoretically, an emulsion produced is more viscous with increasing amount of mineral oil in its formulation. From the graph above, the viscosity before temperature cycle decreases as the amount of mineral oil increases until 30mL of mineral oil is used. That shows that in this experiment, the viscosity of emulsion is low with the composition of mineral oil of 20mL, 25mL and 30mL in emulsions. After that, the viscosity increases in the emulsion that contains 35mL of mineral oil, emulsion IV. For those emulsion after temperature cycle, the same theory is applied, whereby an increase in the amount of mineral oil will produce a more viscous emulsion. In this experiment, the viscosity after temperature cycle did increase as the amount of mineral oil increased. The emulsion that contains 35mL of mineral oil has the highest viscosity while emulsion that contains 20mL of mineral oil has the lowest viscosity.

Besides, after the temperature cycle, all emulsion should have a higher viscosity compared to those before temperature cycle. The reason of this theory is on heating the oil in water emulsion, there will be a conversion to the water in oil emulsion. This is referred as reverse emulsion. Generally, emulsion of water in oil has a higher viscosity compared to the oil in water emulsion. This phase inversion is a phenomenon that shows instability. The experiment shows that instable emulsion can be influenced by temperature cycle thus affect its viscosity. Therefore, it is true to say that the more unstable the emulsion, the higher the viscosity of emulsion. The purpose of treating the emulsion with exaggeration of the temperature fluctuations (temperature cycling) is to compare the physical instabilities of the emulsion. When the emulsion is heated and frozen, the continual formation of the small ice crystals disrupts the adsorbed layer of the emulsifying agent at the oil-water interface. As a result, the emulsion becomes unstable. Hence, its viscosity increases.

The inaccurate results above may be due to the errors that occur during the experiment. Actually all the group members had done a mistake where we used different volumes of different types of oils to prepare the emulsions for procedures 5 to 9. In fact, we should use the same type of oil, which is the mineral oil, but in different volumes to investigate the effect of the amount of oil on the physical characteristics and the stability of the emulsion formed. However, this mistake was only discovered after the practical session and therefore we could not do any correction. This leads to the inaccurate result in this experiment.

On top of this, for the measurement of the viscosity, the difference of the results from theory may also due to the different sizes of spindles that we used to measure the viscosity of the emulsions. We were not sure of the suitable spindle size to measure the viscosity of our emulsions. The viscosity measurements of the emulsion might not be accurate because we did not use the appropriate spindle size. This might cause the measurements to be inaccurate and cause the comparison between the viscosities of different emulsions to be inaccurate and differ from the theory. In addition, after the emulsion had been taken out from the refrigerator, it has to been left for some time until room temperature is reached. This is to ensure that the emulsion that is to measured for its viscosity melts completely and is not in its solid state(ice). However, we just wait for a while and the emulsion have not reach to its room temperature. There may be some residues of solids(ice) that makes the viscosity measurement to be inaccurate since the emulsion has not completely melt.

(ii) Graph of the difference of viscosity (%) against the different oil contents.

The graph above shows the differences in viscosity versus different amount of mineral oil. The graph shows that an increase in the amount of mineral oil will increase the difference in viscosity except for emulsion IV (35mL mineral oil). Emulsion that contains 35mL of mineral oil decreases dramatically in viscosity difference. Theoretically, increasing amount of mineral oil will increase the viscosity difference. The emulsion type is oil-in-water emulsion. Hence the dispersed phase of oil droplets is believed to increase the viscosity. More oil composition in emulsion, more viscosity it will be.

The inaccurate results might be due to some errors that occurred during experiment. For example, we use different volume for the different types of oil. Hence, it causes the existence of two manipulated variables in one experiment. This causes the result to be inaccurate and difficult to be compared. The correct way is that we should fix the type of oil, which is mineral oil while varying the volume of the mineral oil in order for experiment result to be valid. Moreover, the exact amount of ingredients used to prepare the emulsion might not be accurate due to the error while weighing the ingredients or may be due to the unsuitable spindle used in the viscometer. On top of that, inaccurate result might be obtained if the same spindle is used without washing every time the measurement of the viscosity of the emulsion is made.

4. Plot graph ratio of phase separation against volume of mineral oil and give your comment.

Mineral Oil (mL)	Ratio of Phase Separation (x ± SD)
Emulsion I (20mL)	0.61± 0.05
Emulsion II (25mL)	0.61± 0.11
Emulsion III (30mL)	0.49± 0.27
Emulsion IV(35mL)	0.27± 0.04

From the experiment, the higher the volume of mineral oil, the lower the ratio of phase separation, due to the separated phase is lower than the initial phase. The phase separation ratio is not directly proportional to the volume of mineral oil. When the volume of mineral oil is increased, the volume of distilled water is decreased to make up 50 ml sufficiently.

Theoretically, the smaller the phase separation ratio, the higher stability of emulsions. If an emulsion is stable, it is difficult for the oil and aqueous phase to separate. When the volume of mineral oil used increases, the separation phase ratio is also increased. Thus, when increasing amount of oil is added, the tendency for the emulsion to be separated into oil and water also increase, this result in the emulsion produced is more unstable due to the presence of excess oil.

To prepare mineral oil emulsion using pestle and mortar, the thick (primary) emulsion must be prepared first. For wet gum method, the quantities for primary emulsion of oil, water and gum are four, two and one part respectively. 1 part of gum is triturated with 2 parts water to form mucilage, then the 4 parts oil is added slowly in portions while triturating. The mixture is triturated for several minutes after all of the oil is added to form the primary emulsion. Sometimes, the problems arose during prepared the primary emulsion, this may be caused by phase inversion, the product which has become a w/o emulsion cannot be diluted with water. The factors that may cause phase inversion are insufficient shear between the mortar base and the pestle head, inaccurate measurement of water or oil, cross contamination of oil and water, use of wet mortar, excessive mixing of gum and oil, too early or too rapid dilution of the primary emulsion or use of poor quality acacia.

5. What is the function of substance used in the preparation of this emulsion? How will the use of these different substances affect the physical properties and stability of emulsion?

Acacia is mainly used in oral and topical pharmaceutical formulations as a suspending and emulsifying agent. These materials form hydrophilic colloids which when added to water and generally produce o/w emulsions. Acacia is frequently used in the preparation of extemporaneous emulsions. Next is a high–molecular-weight alcohol, such as stearyl alcohol, cetyl alcohol, and glyceryl monostearate. These are employed primarily as thickening agents and stabilizers for oil in water emulsions of certain lotions and ointments used externally.

Vanillin is a vanilla extract was used as a flavouring agent in this product to masking an unpleasant taste. This is to ensure patient compliance as patients now expect and demand liquid medications that are pleasantly, or at least tolerably, flavored. This is especially true with children and older adults for whom solutions are most often prescribed. Syrup is used as diluents and sweetening agent also to mask unpleasant taste.

An emulsion is considered to be physically unstable if the internal or dispersed phase upon standing tends to form aggregates of globules, large globules or aggregates of globules rise to the top or fall to the bottom of the emulsion to form a concentrated layer of the internal phase, and if all or part of the liquid of the internal phase separates and forms a distinct layer on the top or bottom of the emulsion as a result of the coalescing of the globules of the internal phase. The emulsifying agent used will prevent this from happening by decrease the surface tension. In addition, an emulsion may be adversely affected by microbial contamination and growth and by other chemical and physical alterations.

CONCLUSION

The HLB value of the surfactant used will affect the stability of the emulsion formed. Stability of an emulsion depends both on the volume of the oil phase and aqueous phase, together with the amount and capacity of the emulsifying agent to function in the system. Based on the experiment, 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.

Based on the constituents and the intended application, liquid emulsions may be employed orally, topically, or parenterally; semisolid emulsions, topically. Many pharmaceutical preparations that are actually emulsions are not classified as such because they fit some other pharmaceutical category more appropriately.

Lastly, Homogenization causes the reduction in size of the globules in the emulsion and the emulsion formed is more homogenous and smooth.

Appendix