Characterisation of Emulsion Formulations

Introduction

Emulsion is a fine dispersion of minute droplets of one liquid in another in which it is not soluble or miscible. It is also a two-phase system that is not stable thermodynamically. It contains at least two immiscible liquids where one of them is dispersed homogenously in another liquid. In general, emulsion can be categorized into 2 types, oil-in-water (O/W) and water-in-oil (W/O). Emulsion is stabilized by adding emulsifying agent. 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, from 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:

HLB value =   (quantity surfactant 1)(HLB surfactant 1)+(quantity surfactant 2)(HLB surfactant 2)

                                                     Quantity surfactant 1 + Quantity surfactant 2

Objective

To determine:

1.      The effects of HLB surfactant on the stability of the emulsion

2.      The effects of different oil phase used in the formulation on the physical characteristics and stability of the emulsion

Procedures

1.      Each test tube is labelled and 1cm is marked from the base of the test tube.

2.      4ml of oil (according to table 1) and 4ml of distilled water is mixed into the test tube.

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 (refer Table 2). The test tube is closed and its content is mixed with vortex mixer for 45 seconds. The time needed for the interface to reach 1cm is recorded. The HLB value for each sample is determined. Step 1-3 are repeated in order 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 (1g) emulsion formed in a weighing boat and mixed homogeneously. The spread of the colour in the sample is compared. Some of the sample is spreaded on a microscope slide and observed under light microscope. The appearance and globule size formed is drew and described.

5.      A Mineral Oil Emulsion (50g) 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.      40g of emulsion is placed into a 50ml beaker and homogenized for 2 minutes using a vortex mixer.

7.      2g of emulsion (before and after homogenization) are took 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 are stated and compared.

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

Table 4

Readings			Viscosity(cP)				Average
	1	2	3	4	5	6
Before Temperature cycle
After Temperature cycle
Difference (%)

9.      5g of homogenized emulsion is placed into a centrifugation tube and centrifuged (4500 rpm, 10 minutes, 25 ºC). The height of the separation formed is measured and the ratio of the height separation is determined.

Table 5

Mineral Oil (ml)	Ratio of	separation	phase		Average	Ratio of separation phase
20
25
30
35

Apparatus

1.      Test tubes

2.      Measuring cylinder

3.      Pasture pipettes and droppers

4.      Vortex mixer

5.      Weighing boat

6.      Mortar and pestle

7.      Light microscope

8.      Microscope slides

9.      Pipette and bulb

10.  Beaker

11.  Centrifugation tube

12.  Viscometer

13.  Water bath

14.  Refrigerator

15.  Centrifugation apparatus

Materials

1.      Palm oil

2.      Arachis oil

3.      Olive oil

4.      Mineral oil

5.      Distilled water

6.      Span 20

7.      Tween 80

8.      Sudan solution

 Result ( Part 1 )

HYDROPHILIC-LIPOPHILIC BALANCE (HLB) VALUE:

Span 20: 8.6,

Tween 80: 15.0

HLB value =   (quantity surfactant 1)(HLB surfactant 1)+(quantity surfactant 2)(HLB surfactant 2)

                                                     Quantity surfactant 1 + Quantity surfactant 2

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 for phase separation (minutes)	41:50	37:20	33:20	27:06	26:01	25:24	3:55	1:17
HLB value	9.67	10.70	11.34	12.44	13.17	14.05	15.00	-
Spread of   the colour	Spread very slowly	Spread very slowly	Spread slowly	Spread slowly	Spread slowly	Spread moderately	Spread slowly	Spread immediately

Tube No.	Observation under microscope
1	-          Various sizes of A globules were unevenly distributed. -          Irregular shape formed
2	-          Various sizes of globules were formed. -          The distance between the globule sizes was further. -          Various shape formed
3	-Not uniform size distribution but smaller globule sizes can be observed. - Most of the globules are in circular shape.
4	- Globules are not well distributed but more small globule sizes can be observed. - Most of the globule sizes are in circular size.
5	-          The globules are further separated. -          All the globules are in circular shape but in various sizes formed.
6	- The globule sizes were in more uniform sizes and the distribution was more uniform. - All the globules are in circular shape.
7	- Large globules were observed but most are in uniform sizes. - The globules are not well distibuted
8	-          Only a few globules were formed. -          Irregular shapes are formed. -          Dispersion of 2 mediums did not occurs. -          Due to the absent of emulsifying agent, there was less formation of globules.                                                                      -

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 for phase separation( minutes)	-	-	57.54	11.40	32.24	14.43	37.49	16.5seconds
HLB value	9.67	10.70	11.34	12.44	13.17	14.09	15.00	-
Spread of the colour	Spread very slowly	Spread very slowly	Spread slowly	Spread slowly	Spread moderately	Spread moderately	Spread slowly	Spread immediately

Magnification (4 x 10)	Obseervation under light microscope
	The red spherical droplets packed very closely and there were some small and large size of globule. The emulsion is well dispersed.
	The red spherical droplets packed closely as well and almost same amount of small and large globule was formed. The emulsion is well dispersed.
	The red spherical droplets were distributed widely and the globule size was large, small and irregular.
	The red spherical droplets were closely packed, wide spread in the emulsion and the globule size was regular and small.
	The red spherical droplets are well distributed. There are small and large globules formed in the emulsion.
	The spherical globules were distributed. A few of large globules are formed in the emulsion but mostly are small globules.
	The spherical droplets were distributed evenly and there were low portion of small size globules and high portion of large size globules.
	The droplets were not well distributed in the emulsion. The size was quite large but also contained pretty small size globule.

  

Mineral oil:

Tube no.	Span 20 (drops)	Tween 80 (drops)	HLB value	Time for phase separation (min)
				T1	T2	Average
1	15	3	9.67	Interphase did not reach 1cm after 120 minutes	Interphase did not reach 1cm after 120 minutes	-
2	12	6	10.73	Interphase did not reach 1cm after 120 minutes	Interphase did not reach 1cm after 120 minutes	-
3	12	9	11.34	70	69	69.5
4	6	9	12.44	63	60	61.5
5	6	15	13.17	55	57	56
6	3	18	14.09	45	47	46
7	0	15	15.00	12	16	14
8	0	0	0.00	0.08	0.05	0.065

Tube No.	Observation under microscope
1	-          Various sizes of A globules were unevenly distributed.
2	-          Various sizes of globules were unevenly distributed. -          The distance between the globule size was further.
3	-  An uniform distribution and smaller globule sizes can be observed. - All the globules are in circular shape.
4	- A more uniform distribution and smaller globule sizes can be observed. - Most of the globule sizes are in circular size.
5	-          An uniform distribution was observed and the globules are in larger size. -          All the globules are in circular shape.
6	- The globule sizes were in various sizes and the distribution was less uniform. - All the globules are in circular shape.
7	- Large globules were observed.
8	-          Only a few globules were formed. -          Dispersion of 2 mediums did not occurs. -          Due to the absent of emulsifying agent, there was no formation of emulsion.                                                                      -

Arachis oil:

Tube no.	Span 20 (drops)	Tween 80 (drops)	HLB value	Time for phase separation (min)
				T1	T2	Average
1	15	3	9.67	95	-	95
2	12	6	10.73	94	-	94
3	12	9	11.34	93	-	93
4	6	9	12.44	42	-	42
5	6	15	13.17	31	-	31
6	3	18	14.09	61	-	61
7	0	15	15.00	8	-	8
8	0	0	0.00	1	-	1

Discussion

For the first part of the experiment, it clearly shows the phase separation of the emulsion.  The phase separation of the emulsion indicate the stability of the emulsion.  In a theoretical way it is determine by the HLB value of the emulsifier. HLB is stands for Hydrophilic-Lipophilic Balance. HLB value is an empirical expression for the relationship of the hydrophilic and lipophilic group of surfactant.  By calculating the HLB value, it enable the usage of combination surfactant to get the HLB value. The higher the HLB value, the more water soluble the surfactant.

The HLB value can be calculated by using this formula:

HLB value =	(quantity surfactant 1)(HLB surfactant 1) +( quantity surfactant 2)(HLB surfactant 2)
	Quantity surfactant 1 + quantity  surfactant  2

The properties and usage of HLB value is based on the purpose of the emulsion. Table below show the HLB range and its usage :

HLB range	Use
0 – 3	Antifoaming agent
3 – 6	Water-in-oil emulsifier
7 – 9	Wetting and spreading agent
8 – 18	Oil-in-water emulsifier
15 - 20	solubilizers

Therefore in order to make the stable emulsion the first thing that we not to know is the purpose of emulsifier to give what purpose to the emulsion. Then calculate the HLB value and start the formulation process.

 Table 1a shows the time taken for palm oil emulsion separation to occurs until it reaches 1cm. From the table, the interphase reach 1cm after 41 minutes  50 seconds in tube number 1. This show that it is the most stable emulsion with the HLB value 9.67. 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 minutes 17 seconds. The palm oil emulsion is not stable at all at this stage as it separates into distinct layer rapidly. In conclusion, the palm oil emulsion is most stable at HLB value 9.67 provided the emulsifiers are slightly hydrophilic, hence this is an oil-in-water emulsion.

From the table 1b, tube number 1, 2 and 3 shows the longest time taken for arachis oil emulsion separation to occurs until it reaches 1cm which is 95 minutes, 94 minutes and 93 minutes respectively. From tube number 4 until 7, the emulsions are not stable because the interphase are separated quickly. 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 1c, the olive oil emulsion at tube 1 and 2 are stable. The interphase 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 as the arachis emulsion is thermodynamically favourable at this stage. Since the emulsifiers also slightly hydrophilic, we assume this is an oil-in- water emulsion.

From the table 1d, mineral oil emulsion used in tube 1 and 2 showed the same results as the interphase did not reach 1cm after 120 minutes meaning that this emulsion is assumed to be stable. The emulsions are thermodynamically favourable due to the emulsifying agents form around the globules to reduce energy of the system. From tube 3 until tube 7, the emulsions are not stable because the interphase separated quickly and from the results taken, the fastest is 14 minutes. This happened due to the emulsifiers failed to reduce the interface tension and hence creaming, coalescence and breaking may occur. Appearance, odour and colour change are the physicochemical changes in poor emulsion. 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 0.065minutes. The mineral oil emulsion is not stable at all at this stage as it separates into distinct layer .Theoretically, the HLB values required for Mineral Oil are 10 and from the results, the range of HLB value is 9.67- 10.73 and it is acceptable.

Result ( Part 2 )

	Before homogenization	After homogenization
Picture under microscope
Texture	Coarse	Smooth
Consistency	Not homogenously dispersed	Homogenously dispersed
Degree of oily appearance	High	Low
Spreading of colour	Evenly spread	Evenly spread

Table 4

Mineral oil 20ml

Readings	Viscosity (cP)						Average
	1	2	3	4	5	6
Before Temperature cycle	16.80	13.20	16.00	13.80	15.20	14.50	14.90
After temperature cycle	31.60	39.00	35.00	36.40	37.60	33.80	35.60
Difference (%)	14.90/35.60 x 100% =41.85 %

Mineral oil 25 ml

Readings	Viscosity (cP)						Average
	1	2	3	4	5	6
Before Temperature cycle	2.40	1.20	2.40	1.20	2.40	1.20	1.8
After temperature cycle	6.00	12.00	1.20	2.40	6.00	4.80	5.4
Difference (%)	1.8/5.4 x 100% =33.3 %

Mineral oil 30ml

Readings	Viscosity (cP)						Average
	1	2	3	4	5	6
Before Temperature cycle	12.00	18.00	12.00	18.00	12.00	18.00	15.00
After temperature cycle	54.00	42.00	42.00	42.00	42.00	36.00	43.00
Difference (%)	15.00/43.00 x 100%=34.9 %

Mineral oil 35ml

Readings	Viscosity (cP)						Average
	1	2	3	4	5	6
Before Temperature cycle	72.00	84.00	78.00	78.00	78.00	72.00	77.00
After temperature cycle	102.00	150.00	144.00	222.00	78.00	72.00	128.00
Difference (%)	77.00/128.00 x 100% = 66.23 %

Mineral Oil(ml)	Distance of separation phase				Average	Ratio of separation phase
20	0.6	0.6	0.4	0.6	0.55	0.55/5= 0.11
25	1.5	1.2	1.4	1.3	1.35	1.35/5=0.27
30	3.3	3.3	3.2	3.2	3.25	3.25/5=0.65
35	2.5	2.5	2.5	2.5	2.50	2.5/5=0.50

Discussion

 This experiment shows an obvious change of the emulsion before and after homogenization. Before homogenization, the emulsion is not broken down into smaller globules. Therefore, the emulsifier, acacia is not well distributed around the globules in the emulsion. Unstable and large globules are formed and tend to coalesce with each other. This emulsion is unstable and tendency for destabilization process to occur is high.     

Sudan solution is a red color solution and it is used in this experiment to show the shape and physical characteristic of the emulsion. Sudan can be used to test the presence of lipid in the sample. Sudan solution can help to identify whether the formulation is an oily-in-water emulsion or water-in-oil emulsion because it can only be dissolved in the oily phase of the emulsion. This can be identified through comparing the amount of the globules in red color and the colorless globules. If the dispersed phase shows red color, meaning that it is an o/w emulsion and if the continuous phase shows red color, therefore it is an w/o emulsion.

        In this experiment, there is a significant change on emulsion before and after homogenization. Before homogenization process occur, the size of oil globule are not consistent, there are small and large oil globule distributed. The color of the emulsion are not well distributed and it can be seen that some part of the emulsion appears darker than other parts. Acacia is also not well distributed around the globules in the emulsion. The different size globules will tends to closely contact with each other and causes coalescence to occur, causing destabilization of the emulsion.

After homogenization, the oily phase will be broken down into small globules, globule size is much smaller than before homogenization occur. The palm oil occurs have a smoother texture and occur in a homogenous state. The oil and water phase are dispersed evenly, and emulsifier are adsorbing on the interfacial surface of globules, producing a stable emulsion. Therefore, we can conclude that homogenization process helps to make the oil globules disperse evenly in the emulsion and therefore producing a much stable emulsion when the dispersed globules can dispersed evenly in the aqueous phase.

The function of the ingredients used in the emulsion preparation including:

Ingredient	Function
Mineral oil	The oily phase in the o/w emulsion.
Acacia	Emulsifying agent to reduce the interfacial tension and maintain the separation of the droplets in the dispersed phase.
Syrup	Sweetener.
Vanillin	Flavoring agent.
Alcohol	Antimicrobial agent.
Distilled water	The aqueous phase in the o/w emulsion.

           The amount of ingredients can influence the physical characteristics and the stability of emulsion greatly because the amount of the palm oil used for the oily phase and the distilled water used for the aqueous phase used is important to determine the type of emulsion formed,, whether it is oil-in-water or water-in-oil emulsion. Phase inversion might occur when the ratio of dispersed phase to continuous phase is not appropriate. For example, the volume of the dispersed phase should not more than continuous phase to avoid phase inversion occur. The stable range for disperse phase is 30-60%. If the disperse phase exceed 70%, the formulations will become unstable and therefore phase inversion will occur.

          Mineral oil acts as internal phase in the oil in water emulsion. .Acacia used as an emulsifying agent in an emulsion preparation should be used in appropriate amount according to the HLB value. They help to stabilize emulsions preparation by forming a hydrophilic barrier between the oil and water phases to avoid flocculation and coalescence from occurring. If amount of acacia used is less than required, phase separation might occur due to the large interfacial tension formed between the dispersed phase and continuous phase.

           Syrup acts as sweetener and it helps to mask the unpleasant taste of the oil to increase patients’ compliance. However, suitable amount of syrup must be used as non-appropriate amount of syrup will affect the viscosity of emulsion and therefore affect the stability of the emulsion formed

          

Alcohol which acts preservative to prevent the growth of microorganisms in the emulsion, however they should not be used in excess to avoid toxicity. In the other hand, vanillin as a flavouring agent to enhance the taste to increase the patient’s compliance.

The physical stability of emulsion such as creaming is the migration of the dispersed phase of an emulsion, under the influence of buoyancy. The particles float upwards or sink, depending on the density of dispersed phase, and also the viscosity of the continuous phase. However as long as the particles remains separated and not coagulating, the process is known as creaming. This process produce a product which is not homogenous and therefore lead to poor uniform content. There is attractive forces between the dispersed phase molecules, which attract the droplets to come into contact with each other. When the droplets contact with each other and forming a larger drop which decreased the droplets surface area, coalescence occurs and this is thermodynamically unfavorable to the emulsion. The composition of palm oil used affects the physical properties and stability of the emulsion.

According to the theory, the separation phase ratio should be increasing with the increasing of the mineral oil contained in the formulation. The emulsion with highest value of separation phase ratio will produce the most unstable emulsion. Therefore, the separation ratio should be minimize as small as possible to prepare an emulsion with maximum stability and in homogenous condition.

The data obtained should be increases from 20 ml oil to 35 ml oil and the separation ratio for 35ml should be greater than 30ml oil, therefore we can conclude that error has occurred in some part of this experiment. The error occurred might be caused by the homogenous process that was not done properly. Besides, the quality of acacia is different for each group, so the emulsion produced might be unstable and causes some error in the data. Furthermore, during centrifugation process, the emulsion volume of each test tube might be not uniform and therefore causing inaccuracy in the result.

Conclusion

The effects of HLB surfactant on the stability of emulsion is determined. The effects of different oil phase used in the formulation of emulsion on the physical characteristics and stability of emulsion is determined.