PRACTICAL 2

UNIVERSITI KEBANGSAAN MALAYSIA

                  LABORATORY REPORT

DEVELOPMENT OF PHARMACEUTICAL

PRODUCTS 1

                           (NFNF 2213)

TITLE :	CHARACTERISATION OF EMULSION FORMULATION

GROUP	:	B
NAME	:	SITI NURFATIN ATHIRAH BINTI ALIAS CHRISTINE SHEK AI JIA	A148978 A148822
	:	NUR AMALINA BINTI AZMI LEE NING HUAN NUR LIYANA BINTI HASSAN NUR WAHIDAH BINTI HASSAN	A148533 A149304 A149008 A149020
LECTURER’S NAME	:	DR. NG SHIOW FERN

        SEMESTER 1, SESSION 2015-2016

              FACULTY OF PHARMACY

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

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 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

HLB (Hydrophile-Lipophile Balance) is an empirical expression for the relationship of the hydrophilic ("water-loving") and hydrophobic ("water-hating") groups of a surfactant. The table below lists HLB values along with typical performance properties. The higher the HLB value, the more water-soluble the surfactant.

The HLB system is particularly useful to identify surfactants for oil and water emulsification. There are two basic emulsion types:

Water-in-oil (w/o): water is dispersed in oil
Oil-in-water (o/w): oil is dispersed in aqueous phase, most common emulsion type.

Water-in-oil emulsions (w/o) require low HLB surfactants. Oil-in-water (o/w) emulsions often require higher HLB surfactants.

Surfactant selection for an o/w emulsion can be simplified if the HLB system is applied. Oils have required HLB numbers that identify the HLB necessary to give good o/w emulsification. Often the oil supplier can provide the required HLB value. Alternatively, there are a number of compiled lists in the literature on the required HLB for common waxes and oils. Since overall chemical structure (e.g., branched, linear, aromatic) is also a variable, a number of different surfactants with the required HLB should be examined. Not all surfactants having the same HLB value may be acceptable for an o/w emulsion. HLB values for surfactants can be calculated for simple alcohol ethoxylates. If a surfactant is not a simple alcohol ethoxylate, the HLB value must be determined experimentally. HLB values are additive; therefore, if two different surfactants or oils are present, the HLB will be the weighted average of the HLB values for each component. Example: An oil (HLB = 10.5) is a component in an aqueous cleaning solution.

Apparatus and Material

a. 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                   A 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                                                       

b. Materials

Palm oil                                                                      Span 20

Arachis oil                                                                  Tween 80

Olive oil                                                                      Sudan III solution (0.5%)

Mineral oil                                                                   

Distilled water

Procedures

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

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

                                                Table 1 

No.	Oil
1	Palm oil
2	Arachis oil
3	Olive oil
4	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 and their content were 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 determined. (Noted that the HLB value for Span 20 is 8.6 and Tween 80 is 15)

4.         Step 1-3 was 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

5.         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 was observedin the sample. Some of the sample was spread on a microscope slide and observed under light microscope. The appearance of globule size was recorded and described.

6.         Mineral oil emulsiom (50ml) from the from the formulation is prepared by using wet gum method according to table 3a & 3b :

7.                                                                                Table 3a

Mineral oil	Refer table 3b
Acacia	6.25g
Syrup	5ml
vanillin	2 g
alcohol	3ml
Distilled water qs	50 ml

8.                                                                               Table 3b

Emulsion	Group	Mineral oil (ml)
I	1,5	20
ii	2,6	25
iii	3,7	30
iv	4,8	35

9.         6.40g of emulsion is placed into a 50ml beaker and homogenized for 2 minutes using a vortex mixer

10.     7.2g of emulsion is taken (before and after homogenization)and is placed into a weighing boat and lable.Afew drops of Sudan III solution is added and mixed.the texture,consistency,degree of oily aappearance and the spreading of colour in the sample is observed and compared under light microscope.

11.     8.the viscosity of the emulsion formed after homogenization is determined.Using a viscometer that is calibrated with spindle type LV-4 is,15g in 50 ml is take using a beaker.The sample is exposed to 45 degree celcius(water bath) for 15 minutes and then to 4 degree celcius(refrigerator) for another 15 minutes.Determined the viscosity of emulsio after the exposure to temperature cycle is finished and emulsion had reached room temperature (10-15 minutes)Step 8 is repeated again to obtain an average value.

12.     9.5g of homogenised emulsion is placed into a centrifygation tube and centrifuged(4500 rpm,10 minutes,25 degree celcius).The height of the separation formed is measured and the ratio of the height separation is determined.

RESULTS:

MINERAL OIL

Test tube No.	Time needed for the interface to reach 1cm	HLB value	Spread of the colour in the sample	Observation under microscope
1	30 mins	9.67	-          The emulsion is milky but less viscous. -          Sudan III dye disperses unevenly. -          - Small bubbles can be seen on top.	-   Various sizes of Aglobules were unevenly distributed.
2	9 mins	10.73	-          The emulsion is very viscous and milky. -          Sudan III dye did not disperse through emulsion.	Various sizes of globules were unevenly distributed. -          The distance between the globule size was further.
3	4 mins 20 s	11.34	-          White milky emulsion was formed. -          Sudan III dye did not disperse rapidly when dropped into the emulsion. -          Very small bubbles were observed on the top.	-          An uniform distribution and smaller globule sizes can be observed. -          All the globules are in circular shape.
4	2 mins 45 s	12.44	-          Sudan III dye dispersed slowly but evenly in the emulsion.	-          A more uniform distribution and smaller globule sizes can be observed. -          Most of the globule sizes are in circular size.
5	1 min	13.17	-          The emulsion was milky and viscous. -          Sudan III dye dispersed evenly.	-          An uniform distribution was observed and the globules are in larger size. -          All the globules are in circular shape.
6	2 mins	14.09	-          White coloured emulsion was formed. -          Sudan III stains small bubbles after dropped into the emulsion.	-          The globule sizes were in various sizes and the distribution was less uniform. -          All the globules are in circular shape.
7	50 s	15	-          The emulsion was almost transparent when Sudan III dye was dropped. -          Sudan III dye dispersed throughout the emulsion.	-          Large globules were observed. -          All the globules are in circular shape.
8	5 s	0	-          Transparent and clear emulsion was formed when Sudan III dye was added. -          Sudan III dye dispersed rapidly on the top of the sample.	-          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.

RESULTS FROM OTHER GROUPS

1. PALM OIL

Tube no.	Time for phase separation
1	41 mins 50 s
2	37 mins 20s
3	33 mins 20 s
4	27 mins 06 s
5	26 mins 01 s
6	25 mins 24 s
7	3 mins 55 s
8	1 min 17 s

2. ARACHIS OIL

Tube no.	Time for phase separation
1	95
2	94
3	93
4	42
5	31
6	61
7	8
8	1

3. OLIVE OIL

Tube no.	Time for phase separation
1	-
2	-
3	57 mins 54 s
4	11 mins 40 s
5	32 mins 24 s
6	14 mins 43 s
7	37 mins 49 s
8	16.5 s

DISCUSSION

            Simple emulsions are either oil suspended in an aqueous phase (o/w), or water suspended in oil (w/o). in this practical,  the type of emulsions  are identified by using Dye Solubility Test. In this test an emulsion is mixed with a oil souble dye which is Sudan III and observed under the microscope. If the continuous phase appears red, it means that the emulsion is w/o type as oil is in the external phase and the dye will dissolve in it to give color. If the scattered globules appear red and continuous phase colourless, then it is o/w type. Similarly if a water soluble dye ( amaranth) is added to an emulsion and the continuous phase appears red, then it is o/w  emulsion.

An emulsion is an unstable system because there is a natural tendency for a liquid-liquid system to separate and reduce its interfacial area and, hence, its interfacial energy. Thus emulsifier is needed to stabilize the liquid-liquid system of the emulsion.  Emulsifiers work by forming physical barriers that surround the droplet and prevent the coalescence of droplet will finally resulting in breaking of emulsion into 2 separated phase. Tween 80 and Span 20 are actually surfactant which are one of the type of emulsifiers contain both a hydrophilic (water-loving, or polar) head group and a hydrophobic (oil-loving, or non-polar) tail. Therefore, emulsifiers are attracted to both polar and non-polar compounds. When added to an o/w emulsion, emulsifiers surround the oil droplet with their non-polar tails extending into the oil, and their polar head groups facing the water (Fig. 1). For a w/o emulsion, the emulsifier’s orientation is reversed: non-polar tails extend outward into the oil phase, while polar head groups point into the water droplet. In this way, emulsifiers lower the interfacial tension between the oil and water phases, stabilizing the droplets and preventing them from coalescing.

 

The stability of the emulsion is affected by the HLB value of the emulsifiers. Surfactants with a low HLB are more liphophilic and thus tend to make and stabilize a water in oil emulsion whereas those with a high HLB are more hydrophilic and tend to make an oil in water emulsion.  The HLB value of each surfactant is determined by an analysis of the characteristics of the surfactant. The HLB value for the combination use of more than 2 surfactant can be calculated by using the below formula :

        

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

  

Surfactant selection for an o/w emulsion can be simplified if the HLB system is applied. The selection of different surfactants in the preparation of either O/W or W/O emulsions is often still made on an empirical basis. A semiempirical scale for selecting surfactants is the HLB number developed by Griffin [18]. This scale is based on the relative percentage of hydrophilic to lipophilic (hydrophobic) groups in the surfactant molecule(s). For an O/W emulsion droplet, the hydrophobic chain resides in the oil phase, whereas the hydrophilic head group resides in the aqueous phase. For a W/O emulsion droplet, the hydrophilic group(s) reside in the water. Each range of HLB will determine the uses of surfactant in the formulation. For o/w emulsion, the prefer HLB range for the surfactant is 8-18. ( Application of surfactant in respective HLB range is shown in the table below  )

               

After the calculation , it is shown that test tube 1 contain combination of emulsifiers with HLB value 9.67 will form most stable emulsion as the time taken for the phase separation is the longest among all the test tubes.

Emulsion droplet sizes can range from less than a micron to more than 50 microns. Droplet-size distribution is normally represented by a histogram or by a distribution function.Emulsions that have smaller size droplets will generally be more stable. For water separation, drops must coalesce—and the smaller the drops, the greater the time to separate. The droplet-size distribution affects emulsion viscosity because it is higher when droplets are smaller. Emulsion viscosity is also higher when the droplet-size distribution is narrow (i.e., droplet size is fairly constant). Existing emulsions can be given increased stability by decreasing the size of the droplets either by impact or shearing the emulsion still further; the process is called homogenization. Homogenizing results in smaller and more uniform droplet sizes and a practical example is milk.  Milk is an emulsion of fat in water, which is not stable indefinitely as it separates on standing, into skim milk and cream. This is caused by the density differences between the fat and the water, the fat globules rising as predicted by Stokes' Law and coalescing at the surface to form a layer of cream. After homogenizing, this separation does not occur as the globules are much reduced in size. The globule size is also more uniform than the emulsion before homogenization. The oil globules are distributed more evenly. The size and shape of the oil globules are more consistent.

Function of each ingredient in the compounding of emulsion

Acacia is a natural macromolecular polymer that functions as an emulsifier. It will adsorb on the surface of droplets, decreasing the interfacial tension which prevents the flocculation and coalesces from occurring by ensuring there is a space between droplets. It will also increase the viscosity of among the interphase of oily and aqueous phase.

Syrup is a concentrated solution of monosaccharaides. It is usually used a sweeting agent and to increase the viscosity of the emulsion. It will be replaced with other alternatives when compounding for a diabetic patient because they cannot consume medication which contain high concentration of sugar.

Vanillin is an extract from the vanilla beans which is a phenolic aldehyde crystal. It acts a flavouring agent to the emulsion making it more palatable to the patients. It is a compulsory ingredient for compounding emulsion.

Alcohol is an organic compound which has a hydroxyl functional group is bound to a saturated carbon. In the compounding process, it usually refers to ethanol. It is natural synthesized by the fermentation of sugar, cereal and wheat by using yeast. Alcohol functions as a preservative which prevents the microbial growth and contamination of emulsion. This will allow a longer stability in terms of physiochemical properties of emulsion. It is not used in large amounts because it can cause toxicity so the quantity used must not exceed the one stated in the Pharmacopeia.

Distilled water is used as a diluent or vehicle that increases the volume of emulsion. It is the continuous phase of the emulsion. It is also to allow an easier acceptance and measurement of dose for the consumption of patients by volumes such as 5ml and 10ml. This allows a higher chance of the correct dose being delivered and compliance of patients. It is however should not be too high in quantity such as above 75% of the emulsion because it might cause a higher chance of creaming to occur. It is still able to be done but with a precise combination of surfactants to allow a stable emulsion to form.

Mineral oil is the liquid in the disperse phase of the oil in water emulsion. The ratio of the water and the oil must be specified when preparing the emulsion because it will affect the stability of the emulsion. The stability of emulsion formed will decrease if the volume of dispersed phase exceeds 50% of the emulsion. It will also be affected when there is less than 25% of the dispersed phase in the emulsion because there is a higher probability of creaming to occur. Phase inversion tends to occur for emulsions containing more than about 70% dispersed phase.

Sudan III test

Sudan III is an oil soluble dye which is red in colour. It will dissolve into the oil phase of the emulsion and stain it red in colour. That is why it will be used to identify the position, movement and the morphology of the oil globules in the emulsion. This is because the Sudan III will not stain the aqueous phase with the red colour and it will remain colourless. Not only that it will help in the identification of the type of emulsion being observed under the microscope which is either oil-in-water (o/w) emulsion or water-in-oil (w/o) emulsion. Oil-in-water emulsion will have red globules (dispersed phase) with a colourless background (continuous phase) while the water-in-oil emulsion will have colourless globules (dispersed phase) with a red background (continuous phase).    

The colour dispersion in the emulsions produced

The dispersion of colour in emulsion Ι, ΙΙ, ΙΙΙ and ΙV were uneven before homogenization due to the difference in distribution of oil phase and aqueous phase. 

Emulsion IV due to the globules (dispersed phase) being colourless and the background (continuous phase) is red in colour when observed under the microscope. It is concluded that the emulsion formed before homogenization is water-in-oil emulsion. After homogenization, the colour dispersion is even because the distribution of aqueous phase and oil phase is equal and well dispersed. It can be observed that the colourless globules are dispersed uniformly on a red colour background. The size of the globules is smaller than the size before homogenization. The emulsion still remains as a water-in-oil emulsion but with better distribution and size of oil globules in the aqueous phase.  

The physical appearance of the mineral oil emulsions produced

There were four types of mineral oils used to form an emulsion in this experiment. In each emulsion had is a different quantity of mineral oil depending on the type of mineral oil used.  Emulsion I contains 20ml of palm oil, emulsion II contain 25ml of arachis oil, emulsion III contains 30ml of olive oil and emulsion IV contains 35ml of mineral oil. Yet, each of the emulsion contains the same amount of ingredients excluding the amount of mineral oil in preparing the emulsion.

The physical appearance of emulsion IV before the homogenization there is an emulsion but it is coarse in texture and not homogenous when observed under the microscope. This is due to the difference in the distribution aqueous phase in the oil phase of the emulsion and also the sizes of the water globules are not uniform. There is a size variation of water globules from large to small .It is also because the other components in the emulsion have not fully been incorporated into the emulsion. However, the emulsion produced after homogenization is smooth and homogenous. This is because the homogenizer has increase the stability of emulsion, decrease the size of water globules and make it more uniform in size. The distribution of aqueous phase in oil phase is more even in the emulsion after homogenization. This physical appearance change is similar in all the other emulsion before and after the homogenization. There is a tendency for phase inversion to occur in emulsion. However, the emulsions produced after homogenization is more consistent and stable. Besides that, Emulsion Ι, ΙΙ, ΙΙΙ and ΙV are more greasy before homogenization compared to after homogenization.

Graph of sample viscosity before and after the temperature cycle vs. the content of mineral oil.

This graphs showed that after the temperature cycle there in an increase in the viscosity but for the case of emulsion IV there is a decrease in the viscosity after the temperature cycle. There is however no trend of obvious relation between the amount of oil. This might be due to the errors in the experiment which resulted in error on the results of experiments. The main reason is due to the different types of oil used in this experiment. Different types of oil needs different amount of acacia to obtain a good emulsion. Besides, this may be due to the homogenous process was not done properly. Inaccurate measurement of the highly viscous surfactant that is to be added into the formulation is also one of the reasons.

Theoretically, using the same type of oil, the viscosity of emulsion sample will increase when put in the water bath at 45 degree Celsius for 30 minutes in the temperature cycle. An increased temperature will cause a fall in apparent viscosity of the continuous phase and increased kinetic motion of the disperse droplets and the emulsifying agent at o/w interface. Subsequently, it is put into freezer at 40 degree Celsius for 30 minutes. At low temperature (40 degree Celsius), kinetic energy of the system is reduced and this will increase the viscosity of the continuous phase. This will decrease the rate of migration of the globules in the disperse phase. Thus, the viscosity of the emulsion will increase after the temperature cycle.

Graph of difference in viscosity (%) vs volume of mineral oil (ml)

From the graph it can be seen that there is an increase in difference in viscosity against the volume of oil used. However, at the volume 25ml mineral oil there is a slight drop in the difference in viscosity comparing at the volume of 20ml mineral oil. There might be an error at this point of the experiment resulting in this slight decline in difference viscosity instead of increase in viscosity as similar in theory. It could also be due to the difference in the type of oil used to prepare the emulsion Based on the theory that the higher the concentration of the oil phase (continuous phase) in the emulsion, the more viscous it will be. The graph is directly proportional showing that when there increase in volume of mineral oil used it will cause a directly affect to the difference in viscosity of emulsion.

Graph of separated phase ratio formed from the centrifugation process versus the different volume of mineral oil.

This graph shows that there is a decrease in ratio of separation phase when there is an increase in the volume of mineral oil. This makes the ratio of separation phase directly proportional to the volume of mineral oil. This also indicates that the stability of emulsion has increase, with the increase in volume of mineral oil due to a decrease in ratio of separation phase. In order to prepare a good emulsion, it needs to be stable, not easily going through coalesce and breaking which results in the formation of two layers. It cannot be denied there is no such thing as an ideal emulsion. However, unstable emulsions will easily go through separation resulting in a high ratio of phase separation. In this experiment it can be seen that the lowest volume of mineral oil (emulsion I) has the highest ratio of phase separation. This proves that it has the lowest stability while for the highest volume of mineral oil has the lowest ratio of phase separation. This makes the highest volume of mineral oil (emulsion IV) to be the most stable between the three emulsions.

This stability of emulsion is important because it is to ensure an appropriate dose is delivered to the patient. That is important to ensure that the therapeutic effect can be observed by the patient. Theoretically, the separation phase ratio should decrease with the increasing of the mineral oil contain in the formulation. This has been proven by the experiment done.

The inaccuracy of some of the data obtained may be due to some error in the process which the experiment is carried out. The main reason is due to the different types of oil used in this experiment. Different types of oil needs different amount of acacia to obtain a good emulsion. Besides, this may be due to the homogenous process was not done properly. Inaccurate measurement of the highly viscous surfactant that is to be added into the formulation is also one of the reasons. In addition, the height of the separated phase might not be measure accurately too.

Characteristic of emulsion

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

CONCLUSION

The stability of an emulsion is greatly affected by the amount of the emulsifier used. In this practical , combination of  15 drops Span 20 and 3 drops Tween 80 which has HLB value of 9.67 produce the most stable emulsion as the time taken for the phase separation is the longest among all the emulsion prepared.

REFERENCE

1. Al-Ghamdi, A. M., Noik, C., Dalmazzone, C. S. H., & Kokal, S. L. (2007, January 1). Experimental Investigation of Emulsions Stability in GOSPs. Society of Petroleum Engineers

2. Kokal, S., & Al-Juraid, J. (1999, January 1). Quantification of Various Factors Affecting Emulsion Stability: Watercut, Temperature, Shear, Asphaltene Content, Demulsifier Dosage and Mixing Different Crudes. Society of Petroleum Engineers.

3. Yang, M., Stewart, A. C., & Davies, G. A. (1996, January 1). Interactions Between Chemical Additives and Their Effects on Emulsion Separation. Society of Petroleum Engineers.