Wednesday, May 21, 2014

SUPPOSITORY

TITTLE : TEST ON THE EFFECT OF DIFFERENT CONTENTS ON THE 

CHARACTERISTICS OF SUPPOSITORY FORMULATION.

Introduction
Suppository is a solid formulation with various sizes and shape suitable for rectal administration. A good suppository should melt after administration into the rectum and release drugs for local or systemic effect.
The drugs should be distributed in a suitable suppository base. A good base should be non-toxic, no irritation, no reaction with the drug and easy to be form into a suppository. A different base composition will influence the rate and limit of drug release from the suppository.
In this experiment, the effect of different base composition to the physical characteristic of formed suppository and the effect of drug release will be studied.

Apparatus
Weighing scale
1 weighing boat
Spatula
1 beaker (50ml) and 1 beaker (100ml)
Hotplate
1 measuring cylinder (5ml)
1 set of suppository mould
Water bath (37°C)
1 dialysis bag (10cm)
2 threads
1 glass rod
1 pipette (5ml) and pipette bulb
1 plastic covet
Spectrophotometer UV/Vis

Materials
Polyethylene glycol (PEG) 1000
Polyethylene glycol (PEG) 6000

Paracetamol 

Procedure

     1.       A concentrated solution of paracetamol was prepared (10 g on 5 ml of distilled water).  
     2.       A paracetamol suppository (10g) was prepared using the formula.

     1.       The suppository was formed using the suppository-mould. The shape, texture and the colour of the suppository were compared.
     2.       One suppository was inserted into a beaker containing distilled water (10 ml, 37°C) and the time taken for the suppository to melt was recorded.
     3.       One suppository was placed into a dialysis bag and both ends of the bag were tied. The bag was placed into a beaker (100ml) containing distilled water (50ml) which has been heated to 37°C.
    4.       At each interval of 5 minutes, an aliquot sample (3-4ml) was pipette and the release of paracetamol from the suppository was determined using the spectrometer UV-visible. The distilled water was stirred before the sample is taken.

RESULTS AND DISCUSSIONS

      Compare the physical appearance of the suppositories and give explanation

            The different quantities of Polyethylene Glycol (PEG) which are PEG 1000 and PEG 6000 for each base formulation of suppositories will produce different physical characteristics of those suppositories. Based on the experiment, all the suppositories have the shape of a bullet since the mould that is being used is of this shape.
            For the formulation that has the highest amount of PEG 1000, which means that it has the lowest quantity of PEG 6000 shows greasy surface compared to the other suppositories. In other words, the higher the quantity of PEG 6000 in a formulation, the less greasy the suppository will be. Besides, all the formulation of suppositories has smooth texture. As for the hardness, all the suppositories produced are hard. Theoretically, the higher amount of PEG 1000 compared to PEG 6000 will give softer suppository and vice versa. The inaccurate result may due to some errors occurred throughout the experiment. The softer suppository provides ease to be administered to the patient compared to the hard one and it may lead to pain in the process of administering. Besides, it will easily melting within the body and also dissolves in body fluids.   
             For the colour of suppositories, since the active ingredient that we used is paracetamol which is white in colour, the colour of the suppositories produced also white but differ in the transparency degree. The colour changes from evenly white to uneven white with decreasing PEG 1000. This means that the formulation with the lower amount of PEG 1000 is more transparent compared to the others. 

2. Plot a graph of time needed to melt suppository against the content of PEG 6000 in the formulation. Compare and discuss the result.



Based on the graph, 9g PEG 6000 melts the longest followed by 6g, 0g and 3g. Theoretically, less PEG 6000 will melts first. In this experiment, 0g is not the fastest to melt instead, 3g is. Therefore, there may be some mistake occurred and as we are group 1, we will discuss about our experiment only. During experiment, the temperature of water bath is not constantly at 37°C because there are many other groups using the same water bath hence the temperature of water inside beaker may be lower than 37°C. The deviation between our group and group 5 is considered big that is 8.1 and their group melts at 54 minutes. This may be due to the cooling of suppository in freezer take too long hence the suppository freezes and take time to defrost before melts.

3.       Plot a graph of UV absorption against time. Give explanations.


From the graph obtained above, the UV absorption at 520nm is the highest during 10 minute. The UV absorption at 520nm is in an increasing order except there is slightly drop during the 25 minute and 60 minute. Theoritically the longer the period the dialysis bag stays in the solution, the more the amount of paracetamol diffuses out of it, and hence the higher the value of UV absorption because more UV light is absorbed. In this experiment, dialysis bag resembles the lipid bilayer membrane while the distilled water simulates the human plasma which represents the drug diffussion mechanism in our body. At 37 oC, the drug in the suppositories will diffuse into the systemic circulation due to the concentration gradient between the melted suppositories in the dialysis bag and dilutes solution outside the dialysis bag. This may due to some error in handling the sample and inapproriately use of machine. Other reason may be impurities in the sample container due to incomplete washing.

4.  Plot a graph of UV light absorption against time for the suppository formulation which contained different compositions. Give explanation.

Mean:
I=0.098
II=0.075
III=0.027
IV=0.020



From the graph above, the suppository I has the highest UV absorption at 520nm, which means it has the highest concentration of paracetamol in the distilled water, followed by formulation II, III and IV.  The results are tally to the theoretical value because different formulations of suppository give rise to different rate of drug release profile. The releasing rate of paracetomol is measured by spectrometry. Theoretically, the greater amount of PEG 6000, the slower the drug will be released from suppository. This is because the higher amount of hydrogen bonds formed between the base molecules and the paracetamol molecules hence the drug molecules (paracetamol), to be held stronger in the formulation and thus exert a lower releasing rate. Although the graph obtained from this experiment is very fluctuated, however generally there showed an increased concentration of paracetamol over time and the rate of drug releasing decreased with increased amount of PEG 6000 in each formulation. The graph obtained should be increasing initially and then become constant gradually as all the drug is released into the water..
          The fluctuation of the graph may be due to several errors occurred while handing the UV spectrometry or human errors, such as the incorrect way in the suppositories preparation or the presence of impurities in the formed suppositories. Besides, the uneven stirring process before the content of the mixture is pipetted also can contribute to the inaccuracy of the results which forming a few small fluctuations in the line graph. Some precaution steps should be carried out to obtain an accurate result. The surface of dialysis bag should be cleaned before immersing it into the distilled water and suppository should be filled into the bag carefully to prevent breakage of the bag.

5. What is the function of each ingredient that is used in the preparation of these suppositories? How does the usage of different content of PEG 1000 and PEG 6000 affect the physical properties of suppository formulation and rate of releasing of drug from it?
Paracetamol is active ingredient in the suppositories. Paracetamol is used as analgesic and antipyretic. It appears as white, odourless and light powder. PEG 1000 and PEG 6000 are the bases for the active ingredient, paracetamol of the suppository. They allow a smoother drug delivery of the suppository into the rectal. They also allow the absorption of paracetamol by the membrane to occur. A suitable combination of PEG allows an optimum drug releasing to occur, in which the drug will not be held strongly in the base and can be easily released. This is important to allow an optimum drug bioavailability to take place as the drug can be absorbed by mucosa membrane of the rectal.
The physical characteristic and the rate release of the suppository preparation can be interfered by the different combination of PEG1000 and PEG6000. As the proportion of PEG6000 increases, the drug becomes more difficult to be released from the suppository. Besides, the suppository also will become hard, crystal like and with a clear white colour. Proper combination of base should be determined to achieve a balance between hydrophilic and lipophilic characteristics.

CONCLUSION
From the experiment, we can conclude that the higher the amount of PEG 6000 in the formulation of suppository, the lower the rate of drug released. 




OINTMENT

Tittle: Effects of different component to the formulation of ointments

Objectives:
    1)      To determine whether the difference in ointment component will affect their physical characteristic.
    2)      To determine the rate of drug release from ointment.


Introduction:

        An ointment is a homogeneous, viscous, semi-solid preparation, most commonly a greasy, thick oil (oil 80% - water 20%) with a high viscosity, that is intended for external application to the skin or mucous membranes. They are used as emollients or for the application of active ingredients to the skin for protective, therapeutic, or prophylactic purposes and where a degree of occlusion is desired.
Ointments are used topically on a variety of body surfaces. These include the skin and the mucous membranes of the eye (an eye ointment), vulvaanus, and nose. An ointment may or may not be medicated.
Ointments are usually very moisturizing, and good for dry skin. They have a low risk of sensitization due to having few ingredients beyond the base oil or fat, and low irritation risk. There is typically little variability between brands of generics and name brand drugs. They are often disliked by patients due to greasiness. Therefore it need to be attractive and easy to apply on skin for patient compliance and also need to be in good rate of drug release.


Chemical and apparatus:   

     a)      Apparatus

Weighting scale, weighting boat, 500 ml beaker, heater, slap & spatula set, mortar & pestle set, dialysis bag, thread, glass rod, water-bath, 5ml pipette & pipette-bulb, Spectrometer UV-visible,

    b)      Chemicals

Emulsifying wax, White soft paraffin, liquid paraffin, Acetylsalicylis Acid, distilled water.

Procedure:

    1.      The Emulsifying Ointment (50 g) is prepared by using this formula



 2.      5g of ointments is put into the weighing boat and labeled. The ointment texture, clarity and colour had been compare and discuss.

       3.      1.5 g of Acetylic acid had been incorporated with the 30g of ointment with levigation technique.
       4.      The acetylic acid ointment is filled in the dialysis bag and tied at both edge.

      5.      The base was put in the 500ml beaker that contain distilled water that been heated to
       37 0C.
       6.      The aliquot sample is pipette out about 3-4 ml in every 5 minute intervals and the release of Acetylic              acid is determined from the ointment base by using Spectrometer UV-visible. The distiied water need to        be stir first with glass rod before take the sample.

RESULT


 DISCUSSION

    1.       Compare the physical texture of the ointment formed and give explanation.

The hardness of the ointment decreases from ointment I to ointment IV because the content of emulsifying wax is decreased from ointment I to IV. In ointment IV with emulsifying wax of only 9g, is not enough to produce “interlocking wax” structure, thus ointment IV is very soft and clear while ointment I with 21g of emulsifying wax is very hard and opaque. Emulsifying wax prevents the coalescence of globules of the dispersed phase and increases the viscosity of the ointments. Thus, high proportion of emulsifying wax in the ointment will decrease the spread ability of the ointments causes it difficult to be applied onto the skin.
The greasiness of the ointments increases as liquid paraffin amount is increases. Liquid paraffin’s function is to soften the ointment. This will aid in the ointment penetration through the skin but give greasy feeling on skin and difficult to remove by washing with water.

2. Plot graph of UV absorption against time



This graph is fluctuated but on the whole there shows an increase of UV absorption against time. The UV spectrometer measure the releasing of acetylsalicylic acid from the ointment in the dialysis bag immersed in the 100 ml 37˚C distilled water.                                  
            The great increase in the UV absorption value at the first 5 minutes is due to the great concentration difference between content in dialysis tube and the distilled water. The content of in the dialysis bag is hypertonic to the distilled water. So, large amount of ointment with acetylsalicylic acid diffuses to the distilled water with high concentration gradient different. The amount of ointment diffuse is proportional to the acetylsalicylic acid. The high value of UV absorption may due to the presence of residual at both end of the dialysis bag, left during the insertion of ointment into the dialysis bag. So when the dialysis bag is immersed in the distilled water, the residual will dissolve in the water.
This makes there are content of acetylsalicylic acid that are not from the diffusion through the dialysis bag. After the residual at both end of dialysis bag has dissolved, the acetylsalicylic acid diffused out only through the pore of dialysis bag, so the reading after 5 minutes shows regular increase. A sudden large increase in the UV absorption does not occur after 5 minutes as the concentration gradient across the dialysis bag decrease, the amount of acetylsalicylic acid in the distilled water increases with time. The gradual increase in the UV absorption indicates the gradual release of acetylsalicylic acid from the dialysis bag. The concentration of content in distilled water gradually increased until equal the concentration of the content in dialysis bag. This will result in the reduced increase in the UV absorption when times pass by.
            Due to time constriction, the results of this experiment are not complete. However, we can predict the pattern of the graph as it will increase gradually until a constant is achieved. If the experiment is continued beyond 60 minutes, it may result in a straight line graph as the concentration of acetylsalicylic acid in the distilled water equals to the concentration of acetylsalicylic acid at the dialysis bag. The content in dialysis bag is isotonic to the distilled water content concentration.
            Some of the precautions steps can be taken during conducting the experiment are to avoid over stirring the distilled water as this might break the dialysis bag and result a large amount release of the ointment from it. Besides, the residual that retained at both end of the dialysis bag should be removed before immersing the dialysis bag in the distilled water to ensure accuracy of the result. The rope should tie both end of the dialysis bag tightly to prevent leakage of the ointment. The cuette should be cleaned for the fingerprint around the surface of it so that the measurement is correct. The distilled water in the beaker should cover the whole dialysis bag to ensure consistent diffusion.
  


4. What are the functions of each material used in this preparation of ointment? How does the different emulsifying wax and liquid paraffin components affect the physical appearance of the ointment formed and the release rate of the drug from the ointment?
                Emulsifying wax is an emulsifying agent that is used to make the oil droplets in ointment becomes soluble and mixable with water in order to allow the formation of the ointment. It also allows the ointment to have self-emulsifying property. Liquid paraffin and soft paraffin are the hydrophobic bases of the ointment. Liquid paraffin softens the ointment formed and reduces the viscosity of the ointment in the presence of emulsifying wax. It increases the spreadibility and decreases the hardness of the ointment. White soft paraffin enhances the penetration of acetylsalicylic acid through skin surface as well as the absorption through the membrane into circulation.
                 From the experiment, different proportions of materials are used to produce 50g of emulsifying ointment. High concentration of emulsifying wax hardened the ointment while a high concentration of liquid paraffin produces a watery, greasy, and soft ointment due to the reduced viscosity of ointment. Therefore, higher concentration of emulsifying wax and lower concentration of liquid paraffin produce harder and less greasy ointment compared to lower concentration of emulsifying wax and higher concentration of liquid paraffin in an ointment formulation.In this formulation, the increased concentration of emulsifying wax reduces the rate of drug released from ointment. This is because higher amount of emulsifying wax will decrease the drug diffusion from ointment by holding the active ingredient (acetylsalicylic acid) tightly and thus retard the drug released. On the other hand, diffusion rate of acetylsalicylic acid from the dialysis tube is increased in a formulation of ointment with optimum concentration of liquid paraffin because acetylsalicylic acid which is hydrophilic is tend to diffuse into the surrounding distilled water. Hence, the release rate of drug is increased. However, the diffusion rate of acetylsalicylic acid will decrease when the concentration of liquid paraffin is beyond the optimum value. Thus, an optimum ratio of the phases is needed to produce an ointment with good physical property and also the drug releasing rate.

CONCLUSION
From the experiment, we can conclude that the rate of drug released from the ointment is reduced with increased concentration of emulsifying wax in the formulation of Ointment.

EMULSION

Title: Effects of different component to the formulation of emulsion

Objectives:
(1) To determine effects of HLB in stability of emulsion
(2) To identify effects of different surfactant to the surfactant in term of physical changes and stability.

Introduction:

       Emulsion is a thermodynamically instable two phase system. It consisted of at least two liquids which immiscible in one another being dispersed homogeneously in another liquid. Emulsion can be classified into two: oil in water emulsion and water in oil emulsion. In an oil in water emulsion, oil is the dispersed phase being dispersed in water (continuous phase) while water is being dispersed in oil phase for water in oil emulsion.  Multiple emulsions are available also, including a “water-in-oil-in-water” emulsion or an “oil-in-water-in-oil’ emulsion. An unstable emulsion tends to separate into oil and water phase when there are no energy inputs such as stirring, shaking or homogenizing. A stable emulsion can be achieved by adding emulsifier. Emulsifier can be classified into four: hydrocolloid, fine solid particles and surface active agent or surfactant. In this experiment, Tween 80 and Span 20 were used in combination at different ratio to determine the effects of different quantity of surfactant towards the stability of emulsion formulation. Tween and Span are both non-ionic surfactant which stabilise emulsion by positioning themselves on the interfaces between oil and water. They are stable in mild acids, alkalis and electrolytes and do not react with ionic ingredients.

         HLB (hydrophilic-lipophilic balance) has been used to determine the quantity and type of surfactant to prepare a stable emulsion. Each surfactant has a HLB value ranged from 1(most lipophilic) to 20(most hydrophilic). Normally, combinations of 2 surfactants produce a more stable emulsion. In this experiment, Tween 80 with HLB value 15 and Span20 with HLB value 8.5 were used. Combination of Tween 80 and Span 20in different ratio and their effects of the combination in different formulation of emulsion were investigated. HLB value of surfactant combination can be calculated using the formula below:

Chemical and apparatus:

(a)    Apparatus:

8 test tubes
1 50ml measuring cylinder
2 set pipette and droppers
Vortex
Weighing boat
Light microscope
Microscope slide
1 set 5ml pipette and bulb
1 50ml beaker
1 15ml centrifuge tube
Coulter Counter
Viscometer
Water bath (45˚C )
Refrigerator (4˚C)

(b)    Chemicals

Palm oil
Arachis oil
Olive oil
Mineral oil
Distilled water
Span 20
Tween 80
Sudan III (0.5%)
ISOTON III

Procedure:

      1.      Each test tube was labelled and a clear line was drew 1cm from the bottom of each test tube.                   
      2.      4ml oil and 4ml water were mixed in test tube according to table 1.

      3.      Span 20 and Tween 80 was added to the mixture according to the volume stated in Table 2. Test tubes         were closed and mixed well by using the Vortex for 45 seconds. Time taken for the interface to reach            1cm mark was recorded. HLB value for each samples were determined. 


     4.      Few drops of Sudan III were added to 1g of emulsion spread in weighing boat. Spreading pattern of the       emulsion was recorded and compared.  A little sample was spread on microscope slide and observed           under light microscope. The shape and size of globule formed was drew, recorded and compared.

     5.      By using wet gum method, mineral oil emulsion (50g) was prepared using the formula below:


       6.      40g emulsion was poured into 50ml beaker and homogenized using the homogenizer for 2 minutes. 
  
       7.      2g of emulsion (before and after homogenized) was put into weighing boat and labelled. A few drops of          Sudan III were added. The texture, consistency, oily degree and colour spread ability under light                   microscope.

       8.      Viscosity of the emulsion formed after homogenized (15g in 50ml beaker) was determined using                   Viscometer (calibrated with LV-4 Spindle). After that, sample was left in water bath (45˚C) for 30                 minutes and then refrigerated (4˚C) for 30 minutes as well. The viscosity of emulsion after fluctuation of          temperature and viscosity of emulsion achieved room temperature (10-15minutes) were determined.

       9.      5g homogenized emulsion was poured in centrifuging tube and centrifuged (4500rpm, 10minutes, 25˚C         The height of emulsion separation produced was measured and the height ratio were determined.

Result

 Comparison Of Phase Separation Time Between Different Test Tubes


Examination On Palm Oil Emulsion





Test tube 1: The spherical shaped globules are differing in size. The smaller globules are arranged in long chain and tend to stick around the edge of bigger globules. There are also some red-coloured globules disperse unevenly in the sample. 

Test tube 2: The shapes of globules are relatively irregular and loosely packednot in spherical shape. The smaller globules tend to stick around the bigger globules.  The difference in size of globules is relatively large also. Red-coloured globules dispersed in the continuous phase.

Test tube 3: The spherical globules formed are relatively uniform but smaller in size. They are most closely packed compared with other formulation and tend to stick together.

Test tube 4: The spherical globules are arranged loosely and similar to that in test tube 1. The sizes of the globules are not uniform and the smaller one tends to stick on the bigger globules. The globules are stained red and dispersed in continuous phase with flocculation.

Test tube 5: The globules formed are spherical in shape. The size of globules is different.  . Flocculation occurred between small globules as the small globules dispersed in the continuous phase with some stick with other globules

Test tube 6: There are globules in spherical and also irregular shape. They are varies in size and loosely packed. Some of the globules stained red while some do not but they sticked together.

Test tube 7: The spherical shape globules have a great different in size. The globules are not closely packed also. Only small and medium size globules are stained red.

Test tube 8: A few of globules formed in this formulation. The globules formed are irregular in shape and scattered apart in the continuous phase. The size of the globules form n test tube 8 is the smallest among these test tubes.  The continuous phase is in red colour with small globules dispersed in it. There is no flocculation because the globules do not stick to each other.

 Calculations:


Viscosity of Mineral Oil Emulsion (20ml of Mineral Oil)




Discussion

1.      What is the HLB value that produces stable emulsion? Discuss.
Hydrophilic-Lipophilic Balance (HLB) value is a ratio of polar and non-polar group in the surface active agent or surfactant. In other words, HLB value is the balance of oil soluble substance and water soluble substance in a surfactant. HLB has been used to determine the quantity and type of surfactant to prepare a stable emulsion. Generally, a surfactant is 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. The adsorption of the surfactant between the oil and aqueous phase will reduce the surface tension and this will stabilized the emulsion formed. A stable emulsion will remain in dispersion form longer as the phase separation occurs more slowly. Therefore, the slowest the phase separation time, the greater the stability of emulsion formed.
Normally, combinations of two surfactants produce a more stable emulsion. In the experiment, Tween 80 with HLB value 15 and Span20 with HLB value 8.5 were used. Different types of oil used to prepare an emulsion will need different optimum HLB values. Our result shows that the optimum HLB value for Palm Oil to produce the most stable emulsion is 11.29 where the time for phase separation to occur is the longest (80 minutes). The longer phase separation time, the more stable the emulsion. Besides, HLB values of 10.67 and 14.07 also produce stable emulsion as the time for separation phase to occur are 68 minutes and 67 minutes respectively.
From the experiment, tube 8 gives the lowest stability of emulsion produced with HLB value of 0. This is because, there is neither Span 20 nor Tween 80 is used as surfactant 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). The phase separation occurs in the shortest time that is 10 minutes only. Besides, use of only one type of surfactant will affect the stability of emulsion. In tube 7, only Tween 80 is used and the phase separation occurs in shorter time (24 minutes) which indicates not stable emulsion is produced.  
Although a very useful tool, the HLB system does have some limitations. For example additional water phase ingredients are not considered but still may impact the stability. The method also does not provide information as to how much surfactant is needed, but 2 to 4% surfactant is a good starting point to begin further optimization for stability. 

2.      Compare the physical appearance for the mineral oil emulsions formed and explain. What is the Sudan III Solution? Compare the colour dispersion in the emulsions formed and explain.
The physical appearance is compared in terms of globule shape and size, texture, greasiness and consistency; before and after homogenization.
Before homogenization, droplets are of a combination of intermediate and smaller sizes. After homogenization, smaller spherical droplets are formed. During the compounding of emulsion, vigorous mixing is incorporated. This low-energy mixing reduces extremely large droplets to a medium size range. Homogenizer, however, provides high energy which reduces the medium-size droplets to a small-size range. Since higher energy is needed to reduce a smaller droplet than that needed to reduce a larger droplet, premix is done before subjecting the sample to homogenizer so that the energy of homogenizer is not expended on reducing extremely large droplets to a medium size range, when this can be done with low energy mixing.
The emulsion before homogenization is greasy, most probably due to non-uniform sizes of intermediate and smaller oil droplets in water during premix stage. The emulsion is less greasy after homogenization as the droplets achieve uniform smaller sizes. The texture of the emulsion before homogenization is more slippery than that after homogenization, due to the presence of larger droplets of dispersed phase.
The droplets are more consistent in size and arrangement after homogenization. Before homogenization, low forces are applied to disrupt the emulsion. During homogenization, large forces are applied to the emulsion, disrupt and deform the droplets into smaller sizes. Rapid adsorption of surfactant leads to small stable droplets. (J. Weiss, 2008) Cavitation theory (APX, 2009) explains that due to large pressure drop in valve of homogenizer which produces high velocity of emulsion flow, the vapour pressure of the liquid exceeds the ambient pressure causing formation of vapour bubbles or cavities in the liquid. When the cavitation bubbles implode, shock waves are generated in the liquid. These shock waves break apart the dispersed droplets. Hence, smaller consistent droplets are formed after homogenization.
Sudan III solution is a lysochrome (fat-soluble dye) diazo dye. It is used to determine whether an emulsion formed is an oil-in-water emulsion or a water-in-oil emulsion. This can be done by its ability to dissolve in oil phase. When it dissolves in oil phase, it stains them red, which can be easily detected and distinguished from colourless phase. In this experiment, Sudan III solution stains the droplets red and the continuous phase remains colouless. This indicates that the droplets are oil phase, and this is an oil-in-water emulsion.
The colour dispersion is more consistent in the emulsion containing 20ml of mineral oil, as the ideal volume ratio is achieved. The colour dispersion becomes more inconsistent as the volume of mineral oil increases, due to the emulsifying agents are gradually less able to emulsify the emulsion. Besides, as the volume of mineral oil increases, the droplet size becomes bigger, the emulsion formed is greasier, more slippery texture, and there are more red droplets observed. A phase inversion may probably occur when the oil phase is in excess.

      3.      Plot and discuss on: 

     (a)   Viscosity before and after cycle of temperature against different volume of mineral oil graph


In the experiment, 4 different emulsions were prepared by varying the amount of mineral oil which is 20, 25, 30 and 35 ml. Theoretically, an emulsion produced is more viscous with increasing amount of mineral oil in its formulation. This is because oil has higher viscosity compared to water. From the graph above, the viscosity of the emulsion before the temperature cycle increases consistently as the proportion of the mineral oil increases. The trend of graph is compared with the plots for after temperature cycle. Also, the graph shows an increase in the viscosity of the emulsion as the amount of mineral oil increases. Therefore, it can be concluded that the emulsion with 20 ml of mineral oil has the lowest viscosity and emulsion with 35 ml of mineral oil has the highest viscosity for both before and after temperature cycle.   
Besides, after the temperature cycle, all emulsion should have a higher viscosity compared to those before temperature cycle. This is because heating of the oil in water emulsion will cause conversion to the water in oil emulsion. This is referred as reverse emulsion. Usually, emulsion of water in oil has a higher viscosity compared to the oil in water emulsion. This phase inversion shows instability of the emulsion. The experiment shows that instable emulsion can be influenced by temperature cycle thus affect its viscosity. The purpose of treating the emulsion with exaggeration of the temperature fluctuations (temperature cycling) is to compare the physical instabilities of the emulsion. The temperature cycle will disrupt the adsorbed layer of emulsifying agent at the oil/water interphase and hence affect the stability of the emulsion. As the result, the emulsion becomes unstable. Thus, its viscosity increases.        
From the result, emulsion with 30 ml and 35 ml of mineral oil shows a drastic increase in viscosity after temperature cycle compared to before temperature cycle. This is due to higher instability of the emulsion. In opposite, 20 ml and 25ml of mineral oil had shown a decrease in viscosity after the temperature cycle compared to before temperature cycle. This result differs from the theory in which it could be due to some possible errors encountered during the experiment. One of the errors could be from improper rinsing of the viscometer spindle that might affect the concentration of the emulsion components. Besides, the inaccurate result may also due to the different sizes of spindles that we used to measure the viscosity of the emulsions. The emulsion that might have not being exposed to the temperature according to the duration stated in the procedure also affects its viscosity.

   (b)   Difference of viscosity (%) against different amount of mineral oil (ml) graph


In this experiment, emulsion containing 20ml of mineral oil shows the greatest difference in the viscosity, that is 171.43%. The smallest difference in viscosity, which is 23.30%, is shown by the emulsion containing 35ml of mineral oil.

The higher the difference in viscosity, the less stable is the emulsion. From the results obtained in this experiment, emulsion with 20 ml mieneral oil  is the most unstable emulsion while emulsion with 35 ml mineral oil is the most stable one. This is because when there is a lower oily phase present in an emulsion, the emulsion is actually becoming more unstable. Therefore, when these different types of emulsion are subjected to temperature cycling, the amount of ice crystals formed is usually directly proportional to the extent of the instability of the emulsion, or the volume of the oil used. The more the ice crystals are formed, the greater is the reduction in sample viscosity, and thus the greater is the viscosity difference (%). Therefore, emulsion with 20 ml mineral oil is not the most stable emulsion.

 4.      Plot a graph of separated phase ratio due to centrifugation against different amount of turpentine oil. Give comments.

Average ratio of phase separation for different amount of oil



Phase separation ratio is used to determine the stability of an emulsion. A high ratio of phase separation will be resulted in unstable emulsion which it will have two separated phases. The non-homogenous emulsion is easily separated as compared with homogenous emulsion. The uniformity of drug in the emulsion will be altered and inaccurate dosage is being administered.
Based on the graph plotted, separated phase ratio increase from 20mL mineral oil emulsion to 25mL mineral oil emulsion, decrease from 25mL mineral oil emulsion to 30mL mineral oil emulsion and increase from 30mL mineral oil emulsion to 35mL mineral oil emulsion. Emulsion with 25mL mineral oil by using arachis oil oil has highest separated phase ratio while 30mL olive oil emulsion has lowest separation phase ratio.
According to theory, as the amount of oil increase, the separated phase ratio will increase. This is because the added amount of oily phase in emulsion has exceeded the oil amount at which stable emulsion is formed. Therefore, separation will occur in a faster rate.
However, the results obtained from graph do not follow this theory. This may be due to several errors that occur during experiment. For example, inaccuracy in measuring amount of oil before forming the emulsion, insufficient homogenisation that has been carried out on emulsion or the height of separated phase is not measured accurately. Besides, if the volume of each test tube is not equal during centrifuge, the result of centrifuge will be inaccurate. Using of wrong method of preparation of emulsion, that is, the wet gum method may affect the result too. If good emulsion is failed to be produced, it will affect stability of emulsion which will then affect the result of the experiment.

    5.      What are the functions of each ingredient used? How these different ingredients affect the               physical characteristics and stability of an emulsion formulation? 
   
                 Mineral oil- Function as oil phase (internal phase) in the oil in water emulsion (o/w emulsion),Acacia, Span 20 and Tween 80 are emulsifying agents used to emulsify two immiscible liquids into a miscible form called emulsion. The hydrophobic tails will be in contact with the oily phase while the hydrophilic head group will be in contact with the aqueous phase.  They can also increase the viscosity of the interphase between the oily and aqueous phase. However, it is a suitable medium for the growth of microorganisms; hence antimicrobial agents should be added to prevent the growth of the microorganisms.
Alcohol is used as a preservative in this oil-in-water emulsion, because the high proportion of water present in the emulsion is very susceptible to microbial contamination and also deterioration of emulsion by other external environmental factors.
Besides that, syrup which contain high amount of sugar act as sweetening agent to mask the unpleasant taste of the mineral oil to increase patient compliance. It can also be used to increase the viscosity of the emulsion. . However, the amount of syrup used should be controlled so that the emulsion produced is not too viscous and form a layer on the side of the container.
Vanillin acts as flavouring agent which can increase the taste of emulsion because an emulsion always has a taste not preferred by most of the patients. Thus by adding vanillin, patient compliance can be increased.
Distilled water can function as aqueous phase (continuous phase) in oil-in-water emulsion.
The type of emulsion, either o/w or w/o emulsion can be determined by the amount of each phase or the volume ratio. If there is too much oily phase in an o/w emulsion, the emulsion will become very unstable, and phase inversion will occur where it is converted into w/o emulsion. Hence, suitable emulsifying agents with suitable HLB value should be selected in order to produce a stable emulsion. Sometimes, a combination of the surfactant can be used to improve the stability of the emulsion. Unsuitable surfactants will produces emulsions with different physical properties such as globule size, texture, consistency, oily phase dispersion, etc. These may affect the therapeutic effects of the emulsion. The use of different type of mineral oil will affect the physical characteristics and chemical stability of emulsion. For example, palm oil has anti-oxidant properties which increase the chemical stability of the emulsion. This type of emulsion will be less prone to oxidation than using other types of oil. The quantity of syrup is limited to avoid rheological problem and physical properties of the emulsion. 

Conclusion:
The more mineral oil in the emulsion, the more viscous the emulsion formed.

References:
1.      APV (2009). Homogenizer Handbook: Processing of Emulsions and Dispersions. SPX http://www.apv.com/pdf/catalogs/Process_E_D_By_Homog_3005_01_06_2008_US.pdf
2.      J. Weiss (2008). Emulsion Processing: Homogenization http://people.umass.edu/mcclemen/FoodEmulsions2008/Presentations(PDF)/(5)Emulsion_Formation.pdf
3.      Micheal E. Aulton, Aulton’s Pharmaceutics – The Design & Manufacture of Medicines (3rd Ed), Elsevier Inc.