Characterisation of Emulsion Formulation

Prepared by Group B2:

1) Muhammad Syakir Bin Mohd Shahrizan (A162715)
2) Akmal Hadawiah Binti Ismail Yatim (A162694)
3) Koo Bao Yi (A162843)

TITLE

Characterisation of Emulsion Formulations

OBJECTIVE

 To determine:

1.         The effects of HLB of 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

Emulsion is a two-phase system that is not stable thermodynamically. It contains at least two immiscible liquids where one of them (internal/dispersed phase) is dispersed homogenously in another liquid (external/continuous phase).

In general, emulsion can be categorised into 2 types, oil-in-water emulsion (o/w) and water-in-oil emulsion (w/o). Emulsion is stabilised by adding emulsifying agent. The HLB method (hydrophilic-lipophilic balance) is widely 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 (most lipophilic) to 20 (most hydrophilic). Usually a combination of 2 emulsifying agents 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

APPARATUS AND MATERIALS

Apparatus

8 Test tubes                                                              1 set of 5ml pipette and bulb

A 50ml measuring cylinder                                      1 50ml beaker

2 sets of pasture pipettes and droppers                     1 15ml centrifugation tube

Vortex mixer                                                             Centrifugation apparatus

Weighing boat                                                          Viscometer

1 set of mortar and pestle                                         Water bath (45°C)

Light microscope                                                      Refrigerator (4°C)

Microscope slides     

                                                  

Materials

Palm oil                                                                      Span 20

Arachis oil                                                                  Tween 80

Olive oil                                                                      Sudan III solution (0.5%)

Mineral oil                                                                   

Distilled water

PROCEDURES

Part A

1.   Each test tube was labelled and marked 1cm from the base of the test tube. Total of 8 test tubes were marked.

2.   4 ml of oil (the types differ following groups, according to table 1) was mixed with 4 ml of distilled water 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 were added into the mixture of oil and water (refer Table 2). The test tubes were closed with stoppers and the content were mixed with vortex mixer for 45 seconds. The time needed for the interface of the resulting mixture in each test tube to reach 1cm was recorded. The HLB value of the combined emulsifying agents for each sample was then determined.

Steps 1-3 were 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 were added to (1g) emulsion formed in a weighing boat and mixed homogenously. The spread of the colour in the sample was examined and recorded.

Some of the sample was spreaded on a microscope slide and observed under light microscope. The appearance and globule size formed were described and drawn.

Part B

5.         A Mineral Oil Emulsion (50g) was prepared from the formulation below by using wet gum method according to Table 3a & 3b below:

     Table 3 a

Mineral Oil	(refer Table 3b)
Acacia	6.25 g
Syrup	5 ml
Vanillin	2 g
Alcohol	3 ml
Distilled water qs	50 ml

  Table 3 b

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 was placed into a 50ml beaker and homogenized for 2 minutes using a vortex mixer.

7.   2 g of emulsion (before and after homogenization respectively) was taken, placed into a weighing boat and then labelled. A few drops of Sudan III solution were added and the emulsion was mixed homogenously. The texture, consistency, degree of oily appearance and the spreading of colour in the sample under the light microscope were stated and compared.

8. The viscosity of the emulsion formed after homogenization (15g in 50ml beaker) was measured using a viscometer that is calibrated with “Spindle” type LV-4.

    After that, the sample was first exposed to 45°C (water bath) for 15 minutes and then to 4°C (refrigerator) for another 15 minutes. After the exposure to the temperature cycle was finished and the emulsion had reached room temperature (10-15 minutes), the viscosity of the emulsion was again determined and recorded in the table below (Table 4).

    Step 8 was repeated again and an average value was 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 was placed into a centrifugation tube and centrifuged (4500 rpm, 10 minutes, 25°C). The height of the separation formed was measured and the ratio of the height separation was determined and recorded in the table below (Table 5).

 Table 5

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

RESULTS

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	5	0
HLB Value	9.67	10.73	11.34	12.44	13.17	14.09	15.00	0

 (HLB span 20 = 8.6, HLB Tween 80 = 15.0)

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

1) Time needed for interface to reach 1 cm

Palm Oil (Group 1)
Tube no.	1	2	3	4	5	6	7	8
Time taken for the interface to reach 1 cm (min)	Does not form interface after 90 min	Does not form interface after 90 min	80	60	46	23	9	2

Arachis Oil (Group 2)
Tube no.	1	2	3	4	5	6	7	8
Time taken for the interface to reach 1 cm (min)	Does not form interface  after 90 minutes	Does not form interface  after 90 minutes	Does not form interface  after 90 minutes	11	20	17	3	0.5

Olive Oil (Group 3)
Tube no.	1	2	3	4	5	6	7	8
Time taken for the interface to reach 1 cm (min)	3	Does not form interface after 90 mins	3	11	19	14	7	49

Mineral Oil (Group 4)
Tube no.	1	2	3	4	5	6	7	8
Time taken for the interface to reach 1 cm (min)	Does not form interface  after 90 minutes	Does not form interface  after 90 minutes	Does not form interface  after 90 minutes	56	11	9	7	0.1

Palm Oil (Group 5)
Tube no.	1	2	3	4	5	6	7	8
Time taken for the interface to reach 1 cm (min)	Does not form interface  after 90 minutes	Does not form interface  after 90 minutes	Interface did not reach 1 cm after 90 minutes	72 min 18 sec	Interface did not reach 1 cm after 90 minutes	43 min 9 sec	13 min 23 sec	3 min 45 sec

Arachis Oil (Group 6)
Tube no.	1	2	3	4	5	6	7	8
Time taken for the interface to reach 1 cm (min)	Does not form after 90 mins	40	74	27	33	20	5	1

Olive Oil (Group 7)
Tube no.	1	2	3	4	5	6	7	8
Time taken for the interface to reach 1 cm (min)	2	56	50	46	33	43	20	26

Mineral Oil (Group 8)
Tube no.	1	2	3	4	5	6	7	8
Time taken for the interface to reach 1 cm (min)	Does not form interface after 90 minutes	Does not form interface after 90 minutes	Does not form interface after 90 minutes	60	12	8	3	0.5

Palm Oil (Group 9)
Tube no.	1	2	3	4	5	6	7	8
Time taken for the interface to reach 1 cm (min)	34	29	66	55	40	51	32	3

Arachis Oil (Group 10)
Tube no.	1	2	3	4	5	6	7	8
Time taken for the interface to reach 1 cm (min)	150	135	120	110	49	43	39	1

Olive Oil (Group 11)
Tube no.	1	2	3	4	5	6	7	8
Time taken for the interface to reach 1 cm (min)	91	87	82	58	48	60	3	0.7

Mineral Oil (Group 12)
Tube no.	1	2	3	4	5	6	7	8
Time taken for the interface to reach 1 cm (min)	Does not form interface after 90 minutes	Does not form interface after 90 minutes	Does not form interface after 90 minutes	58	12	10	6	0.2

3) The appearance of the most stable emulsion formed.

Oil	Groups	Magnification (10x)	Describe the appearance	Colour distribution
Palm oil	1		The colour of the stained is light red. The size of the globules is small. The globules are distributed uniformly in the emulsion. Water droplets are seen dispersed in oil.	The red colour particles evenly dispersed.
	5		Homogenous, milky white and red particles mix together, the size of particles are about the same.	The red colour particles evenly dispersed.
	9		Milky white, the droplet sizes are not similar. Uneven distribution of droplets.	Red particles are hard to differentiate from the white particles.
Arachis oil	2		There are small droplets and a few bigger droplets. The droplets are closely packed to each other.	The colour is evenly dispersed.
	6		Milky white and a bit greasy. The droplets’ size is slightly not similar but packed closely to each other.	Evenly dispersed.
	10		The droplets of different sizes are packed closely.	Evenly dispersed.
Olive oil	3		Milky white, shiny	Evenly dispersed
	7		The globules’ sizes are varying and are closely packed.	Evenly dispersed
	11		Oil droplets dispersed in water. Thus, it is oil in water emulsion. The globule size is relatively uniform and packed. The HLB value is in the optimum range.	Sudan III evenly dispersed
Mineral oil	4		The globules are different in size and closely packed together.	The red colour is evenly dispersed
	8		The oil droplets are irregular in size and closely packed.	The red colour is evenly dispersed
	12		The droplets are different size and closely packed to each other.	The red colour particles evenly dispersed.

2) The texture, consistency, degree of oily appearance and the spreading of colour of mineral oil under the light microscope (Magnification x10).

Mineral Oil (ml)	Group	Picture Magnification 10x	Texture	Consistency	Degree Of Oily Appearance	Spreading Of Colour
20	1		Smooth and milky	Homogenous mixture	A bit  greasy	Evenly spread small particles
	5		Smooth and milky	Homogenous mixture, high consistency	The oil does not appear very clear due to lack of dye	Evenly spread small particles
	9		Smooth and milky	Homogenous mixture	Large and small globules appear, leaving a very oily appearance	Clear white and hard to see spreading of colour due to lack of dye added
25	2		Oily texture	Consistent size of globules	Small globules appear, Greasy	The colour is evenly spread
	6		Has a greasy texture	Has quite stable consistency or viscosity due to its oily texture	A bit greasy	Evenly spread
	10		Has an oily texture.	Has a high degree of consistency	Shiny and has a greasy texture	Shiny and evenly spread
30	3		Homo-geneous, smooth, milky, shiny surface	Very consistent	Little greasy	Shiny, evenly dispersed
	7		Homo-genous, smooth	Very consistent	Little greasy	Shiny, evenly dispersed
	11		Homo-geneous, milky	Very consistent	Little greasy	Shiny, dispersed evenly
35	4		Homo-genous, smooth, more milky	Consistent, more viscous	More greasy, less globules	Sudan III solution evenly distributed
	8		Homo-genous mixture	Consistent and viscous	Greasy	Evenly dispersed
	12		Smooth and homo-genous	Consistent, stable and more viscous	More greasy	Evenly distribution

3) The viscosity of the emulsion before and after the temperature cycle

Mineral Oil (ml)	Readings/ Groups	Viscosity (cP)
		Before Temperature Cycle			After Temperature Cycle			Difference (%)
		1	2	Average	1	2	Average
20	1	14.4	14.4	14.4	12.8	12.9	12.85	10.76
	5	3.50	3.31	3.41	4.69	4.44	4.57	34.01
	9	12.6	12.0	12.45	16.7	19.3	18.0	44.05
25	2	5.5	6.0	5.75	6.13	8.0	7.065	20.52
	6	12.7	10.5	11.6	14.6	12.6	13.6	17.24
	10	36.9	36.4	36.65	56.8	65.5	61.2	66.98
30	3	23.4	21.8	22.6	61.9	41.3	51.6	56.2
	7	24.1	26.8	25.5	58.1	59.0	58.6	33.1
	11	36.8	33.2	35.0	56.3	47.9	52.1	48.9
35	4	-	-	-	-	-	-	-
	8	-	-	-	-	-	-	-
	12	-	-	-	-	-	-	-

4) The ratio of the separation phase

Mineral Oil(ml)	Groups	Ratio of separation phase			Average
20	1	0.58	0.60	0.58	0.59
	5	0.73	0.71	0.73	0.72
	9	0.74	0.76	0.69	0.73
25	2	0.21	0.22	-	0.22
	6	0.70	0.65	0.67	0.67
	10	0.60	0.68	0.63	0.64
30	3	0.58	0.57	0.58	0.58
	7	0.54	0.58	0.60	0.57
	11	0.60	0.63	0.70	0.64
35	4	-	-	-	-
	8	-	-	-	-
	12	-	-	-	-

DISCUSSION

            Emulsion, a two phase substance is formulated to be stable thermodynamically. In the part one of the experiment, different emulsions were formulated using four different oil which are palm oi, arachis oil, olive oil and mineral oil and it’s formulated to be an oil in water emulsion. In order for an emulsion to be stable, an emulsifier need to be added. Two types of emulsifier were used which are Span 20 and Tween 80 and both have different HLB which are 8.6 and 15.0 respectively.  The emulsifiers were added in difference amount for a few times to investigate the most suitable HLB for the emulsion. Based on the result, the most stable palm oil emulsion can be achieved by using the first mixture of emulsifiers (with HLB of 9.67) and the second mixture of emulsifiers (with HLB of 10.73). This shows that both mixtures of emulsifiers have the amount of HLB which closest to the HLB of palm oil emulsion. For arachis oil, most group found that the first mixture of emulsifiers (with HLB of 9.67) provided the best emulsion. This proves that the HLB of arachis oil emulsion is closest to 9.67. Next, for the olive oil emulsion, two of the three group found that the second mixture of emulsifier (with HLB of 10.73) gave the most stable emulsion which shows that the olive oil emulsion’s HLB is closest 10.73. Lastly, the mineral oil emulsion were most stable when added with the first, second and third mixtures of emulsifiers with HLB of 9.67, 10.73 and 11.34. This shows that mineral oil emulsion has HLB closest to the first, second and third mixtures of emulsifiers. The method used to study the stability of the emulsion is by looking at the physical appearance of the emulsions. The time was recorded to see how long it take for the emulsion to form interface and reach 1 cm. The interface shows the instability of the emulsion thus it couldn’t stay in its one phase form. By looking at the tube no 8, the importance of emulsifier can be observed. Tube no 8 didn’t have any mixture of emulsifier thus the rate sedimentation is high. Therefore, tube no 8 had the shortest amount of time taken for the interface to reach 1cm. That is why emulsifier is a really crucial substance in formulating an emulsion. We also need to choose the best emulsifier which has the most suitable HLB to provide the most stable emulsion.

            The most stable emulsion of palm oil, arachis oil, olive oil and mineral oil were observed using microscope under magnification X10. Sudan III solution, an organic dye was added to aid in the observation of the emulsions. Under the microscope, we can see how emulsion is created. Most of the emulsion had the Sudan III solution’s red colour evenly dispersed. The globules or the droplet can be seen closely packed to each other with circle shape. It shows the typical appearance of emulsion under microscope.

            Emulsifier is an agent that will help to emulsify the two phases to become one. However, emulsifier is not the only thing matter when formulating an emulsion. Correct amount or formula of the emulsion is also an important element that is need to be looked upon. Different amount of substances used will provide difference in the stability of the emulsion. In the experiment, different amount of mineral oil were used in mineral oil emulsions. One of the way used to test the stability of an emulsion is by exposing the emulsion into extreme condition. In this experiment, the extreme conditions introduced involved two elements which are temperature and force. In the first part, the emulsions with different amount of mineral oil were involved in a temperature cycle. In this part, the stability of the emulsion was measured by looking at the viscosity of the emulsions. If the viscosity of the emulsions differ too much before and after it involved in the temperature cycle, then we can say that the emulsion wasn’t stable enough. The instability of the emulsion was triggered and initiated by the kinetic energy supplied by the temperature. The difference temperature exposed provided different range of kinetic energy thus playing a role in disturbing the stability of the emulsion. By looking at the result, the average difference percentage is high when a mineral oil emulsion was made with 30mL of mineral oil. The average percentage difference is 46% thus we can conclude that the emulsion was not stable enough to be exposed to the temperature cycle.

            At the second part of stability testing, the mineral oil emulsions were tested with a high amount of force provided by the centrifugation. Centrifugation accelerates destabilization of the product and thus simulates aging. Phase separation ratio is used to indicate the stability of an emulsion. After centrifugation, a creamy layer can be observed atop of another brown layer, and the creamy layer is actually the oil droplets that coalesce, causing the breaking of the originally stable emulsion. An unstable emulsion will have a high ratio of separation where two separated phase can be observed. The uniformity of drug in an unstable emulsion will be greatly altered and the accuracy of dose being administered into patient might be affected too. The instability of the emulsion can be observed by carefully measuring the difference in phase ratio. However, the result obtained did not really tally with the theory as the result obtained by the different groups did not really differ much and there were also some groups that differ totally from any other group. The average phase ratio of emulsion with 25mL of mineral oil is the lowest while the average phase ratio of emulsion with 20mL of mineral oil is the highest.

            There might be certain errors that were made which contribute to the inaccuracy of the results of the experiment. One of the mistakes that might happen was human error in measuring the substances needed and also in handling the substances. Different amount of substances used will provide a different result. Spillage or any wastage while handling the substance could also give inaccurate result. Other than that, the usage of machine such as viscometer might also produce inaccurate results when it’s not used properly. The machine should be handle with care and the spindle should be washed and cleaned thoroughly to not let any other fluid influence the viscosity of the emulsion measured. In order to avoid the errors and inaccurate result, we should be more careful and more focus when doing the experiment. Make sure that the experiment was carried out according to the procedure.

        

CONCLUSION

For emulsion using palm oil, it can be concluded that the test tube 1, 2 and 3 are more stable compared to others as they require longer time to achieve phase separation. Thus, the optimum HLB value to produce stable palm oil emulsion are in the range of 9 -13. Different oils required different HLB value to produce stable emulsion. Emulsifying agents should be use in an appropriate amount as insufficient of it will produce an undesirable emulsion which are not appropriate for medical use.

The stable emulsion formed should have considerably high viscosity to impede and oil droplets (internal phase) from approaching each other and coalescing. It should also show lower ratio of separated phase after centrifugation. However, we cannot effectively deduce the most stable emulsion in this experiment based on the two parameter due to huge gap in the collected data and inconsistency of the result. It is most likely due to error during the experiment.

REFERENCE

1. Mariana A. Montenegro, María L. Boiero, Lorena Valle and Claudio D. Borsarelli (2012). Gum Arabic: More
   Than an Edible Emulsifier, Products and Applications of Biopolymers, Dr. Johan Verbeek (Ed.), ISBN: 978-953-
   51-0226-7, InTech, http://cdn.intechopen.com/pdfs/31731.pdf
2. Klaus Tauer. Emulsions – Part 2: a little (theory) : emulsion stability, http://www.mpikg.mpg.de/886743/Emulsions_-2.pdf
3. British Pharmaceutical Codex 1973
4.  Aulton, Taylor. 2013. Aulton’s Pharmaceutics The design and manufacture of medicines (international edition). 4^th ed. Edinburgh, London, New York, Oxford, Philadelphia, St. Louis, Sydney, Toronto: Churchill Livingstone Elsevier.