Lab Report : Suppository

EFFECTS OF DIFFERENT COMPOSITIONS OF MATERIALS ON THE CHARACTERISTICS OF SUPPOSITORY FORMULATION.

Date of Experiment: 6th May 2013

Lecturer’s Name: Dr. Ng Shiow Fern

Aims:

1. To determine the effects of different composition of base on the physical characteristics of suppositories.

2. To determine the effects of different composition of base on the rate of drug release.

Introduction:

Suppository is a medicated formulation which consists of different sizes and shapes. Making the drug more suitable to be administered to patient through rectum, vagina or urethra. It is generally conical or bullet shape. The ideal suppository bases must be able to melt at body temperature, 37⁰C, non irritating, physically and chemically stable, inert, high viscosity when melted and then to release the drug locally or systemically. Bases that is more widely used in a formulation is cocoa butter which are innocuous, bland , non reactive and melts at body temperature. The amount of dose of drug incorporated into a suppository depends on the release properties of the base.

Suppository is taken when a person unable to take medicine orally and is mainly used to treat constipation. The advantage of suppository is that it can prevent first pass effect ,and to avoid the digestive enzyme and thus increase bioavailability. The suppository can be targeted delivery system and get to site of action with lower dose. This will reducing systemic toxicity. If there is severe nausea or vomiting it is advisable to use suppository as well. But, suppository have some disadvantages. It can cause mucosal irritation, patience compliance, erratic and undesired absorption. If patient with diarrhea and disease state will affect absorption.

The action level of suppository depends on the nature of drug, types of bases, concentration of drugs and absorption rate of targeted site. The pH of the mucosal is important for drug absorption. Weak acids and bases can be absorbed more effective than strong acids and bases. This is due to highly ionized drugs are poorly absorbed. Drug has to be incorporated into a suitable suppository base in order to deliver the drug efficiently to the targeted site. A different composition of suppository base will influence the drug release rate. If a drug is more soluble in base, this will slow down the release rate of drug from suppository. On the contrary, a drug which is less soluble in base will be more readily released. A good base must not cause toxicity, does not cause irritation, does not react with the drug and easy to form suppositories. In this experiment, water-soluble base, polyethylene glycol is used.

Apparatus:

Weighing balance                                            Water-bath(37⁰C)

Weighing boat                                                   Dialysis bag (10cm)

Spatula                                                                 2 strands of thread

50ml beaker                                                      Glass rod

100ml beaker                                                   5ml pipette set and pipette bulb

Hotplate                                                              1 plastic cuvette

5ml measuring cylinder                               UV spectrophotometer

1 set of suppository mould

Materials:

Polyethylene glycol (PEG) 1000

Polyethylene glycol (PEG) 6000

Paracetamol

Procedure:

1. Saturated stock solution of paracetamol is prepared. (1g of paracetamol is added to mixture of PEG 1000 and PEG 6000).

2. 10g of paracetamol suppository is prepared using the following formula:

Suppository	Group	Ingredient (g)			Total (g)
		PEG 1000	PEG 6000	Paracetamol stock solution
I	1,5	9	0	1	10
II	2,6	6	3	1	10
III	3,7	3	6	1	10
IV	4,8	0	9	1	10

3. Suppositories are shaped by using suppository-mould. The shape, texture, and colour of the suppositories are being examined.

4. 1 suppository is placed into the beaker containing 10ml of saline water at 37⁰C and time required to melt the suppository is recorded.

5.1 suppository is filled into dialysis bag. Two terminal end of the bag is assured to be tied up neatly. The dialysis bag is placed into the 100ml beaker which containing 50ml saline water being heated up to 37⁰C.

6. An aliquot sample with 3-4ml is pipette at 5 minutes interval. The rate of release of paracetamol from the suppository is determined using UV-visible spectrometer. Make sure saline water is stirred by glass rod before sample is taken out.

Results:

1. Compare the physical appearance of the suppository formed.

Characteristics	I	II	III	IV
Shape/Appearance	Bullet-shape, Half transparent solids
Colour	White
Texture	White Paracetamol drug is seen dispersed in the half transparent PEG suppository base
Hardness	+	++	+++	++++
Greasiness	++++	+++	++	+
Stickiness	++++	+++	++	+
Hygroscopicity	++++	+++	++	+

According to the results obtained, the shape and the appearance of the suppositories are the same which are bullet-shaped, half-transparent solids, because all the suppositories were made from the same type of suppository mould. The different between four of suppository are: different contents of PEG 6000 is used.

Besides that, the colures of all suppositories is white. The intensity of this suppositories with different contents of PEG 6000 is undistinguishable. We can say that the differences in formulation have no effect on the colour intensity of the suppository as there is no difference in the colour of the raw PEG 1000 and PEG 6000.

The hardness of the suppositories increases when the amount of PEG 6000 increases  but the stickiness decreases. This is because PEG 6000 contains higher content of hydroxyl groups within the structure. More of intra-molecular and intermolecular hydrogen bonds are formed between PEG molecules, thus rendering the overall structure strength, and the suppositories formed are less sticky in nature. Higher energy or forces is needed to break up these interactions, so the suppositories are harder and harder.

Moreover, the greasiness of the suppositories decreases from suppository I to suppository IV. However, PEG is a water-soluble suppository base. Thus, there will not be any feeling of greasiness on the suppositories produced.

Hygroscopicity means the ability of absorbing moisture. The hygroscopicity of the suppositories decreases when the amount of PEG 6000 increases. This may be due to the increasing percentage of hydrogen bonding within structures. The abundant hydrogen bonds between PEG are stronger than the bonding between the PEG and water molecules in the air. As a result, it is becoming more and more difficult for the suppositories to absorb water readily, with increase the amount of PEG 6000. The higher amount of PEG 1000 will absorbed more of water from air.

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

Suppository	Group	Time needed to melt the suppository
I	1	55 minutes 14 seconds
II	2	30 minutes
III	3	36 minutes 46 seconds
IV	4	55 minutes 47 seconds
I	5	37 minutes 37 seconds
II	6	56 minutes 18 seconds
III	7	30 minutes 14 second
IV	8	55 minutes 52 seconds

Amount of PEG 6000 (g)	0	3	6	9
Mean time (min) (x ± SD)	46.425  ± 8.805	43.15  ± 13.15	33.5 ± 3.27	55.825 ± 0.045

Graph 1

Polyethylene glycol (PEG) is a polyether compound which being applied in many application from industrial manufacturing to medicine. In this experiment, PEG is used as the suppository base. Two types of PEG which are PEG 1000 and PEG 6000 are being used in this experiment.

A graph of mean time versus amount of PEG 6000 has been plotted. The suppository is placed in a beaker containing 10mL distilled water with temperature of 37⁰C and the time needed to melt the whole suppository is recorded.

There are comparisons of mean time needed to melt the suppository with a different amount of PEG 6000. When the amount of PEG 6000 is none, the mean time needed is 46.425 minutes, while the mean time needed shows 43.15 minutes when amount of PEG 6000 is 3g. The mean time needed is 33.5 minutes when the amount of PEG 6000 is 6g and when amount of PEG 6000 is 9g, the mean time is 55.825 minutes. The mean time needed is decreasing when the amount of PEG 6000 is increasing. However, there is an inaccuracy of result occur when the mean time increase when using 9g of PEG 6000 compare to 6g of PEG 6000. This may be due to some errors when experiment is carried out. For example, the water temperature has not reach 37ᴼC when the suppository is put in the water bath.

Melting point of PEG is generally above body temperature. The high melting point also means that the bases do not melt in the body but dissolve and disperse the medication slowly, providing a sustained effect.

3. For each 5 minutes, an aliquot sample (3-4mL) is pipette and determines the release of Paracetamol from the suppository by the spectrometer UV-visible. Make sure the distilled water is stir with the glass rod before the sample is taken.

Time (min)	UV-visible absorption
	0	5	10	15	20	25	30	35	40	45	50	55	60
UV absorption at 520nm	0.0	0.083	0.023	0.032	0.023	0.060	0.058	0.073	0.091	0.083	0.102	0.089	0.118

Graph 2

Based on the graph plotted, at the first 5 minutes, the UV absorption was very high, decreasing after 5 minutes and generally, as the time increases, the UV absorption also increases. Unfortunately, looking closer into it, there’s slightly decrease after each elevated UV absorption. This may be due to instrumental or operational error during the experiment as the ideal UV-visible absorption should increase gradually over time.

Our group had prepared suppository II which contain 6g of PEG 1000, 3g of PEG 6000 and 1g of Paracetamol. Polyethylene glycols are polymers of ethylene oxide and water, prepared to various chain lengths, molecular weights, and physical states. Polyethylene glycols (PEGs) having average molecular weights of 300, 400, and 600 are clear, colorless liquids, while those with molecular weights of 600-1000 are semisolids. Those having average molecular weights of greater than 1000 are wax-like, white solids with the hardness increasing with an increase in the molecular weight. These polyethylene glycols can be blended together to produce suppository bases with varying: melting points, dissolution rates and physical characteristics. Drug release depends on the base dissolving rather than melting.

Polyethylene glycols are water soluble polymers that often melt at temperatures higher than the rectal or physiological temperature of 37°C. Consequently, when used in rectal formulations, the drug is released gradually, as a result of the progressive dissolution of the PEG excipients in an aqueous dissolution medium. High molecular weight PEGs have high melting points, and consequently have slower dissolution rates, compared to PEGs of intermediate molecular weight and this was the basis of formulating the PEG 1000/6000 blend. PEG 6000 released paracetamol at a slower rate than PEG 1000.

Therefore, a few steps of precaution need to be taken in order to achieve a better and accurate result. Before the sample is taken to the UV spectrometer, the distilled water inside the beaker must be stirred first. Besides, the cuvette must be wiped before place it inside the spectrometer. Other than that, the UV spectrometer must be under a good condition in order to have a good result.

4. Plot graph of UV absorption against time for the suppository formulation with different compositions. Discuss and compare the results. 

Time		UV absorption average at 520nm (x ± SD)
		0	5	10	15	20	25	30	35	40	45	50	55	60
suppository	I	0	0.022	0.023	0.024	0.0255	0.0265	0.0295	0.0385	0.040	0.0435	0.0465	0.056	0.06
		±	±	±	±	±	±	±	±	±	±	±	±	±
		0	0.0127	0.0141	0.0156	0.0148	0.0148	0.0177	0.0219	0.0212	0.0205	0.0233	0.0226	0.0184
	II	0	0.0575	0.0295	0.0365	0.0350	0.0535	0.0530	0.0625	0.072	0.07	0.0815	0.079	0.107
		±	±	±	±	±	±	±	±	±	±	±	±	±
		0	0.03606	0.00919	0.00636	0.01697	0.00919	0.00707	0.01485	0.02687	0.01838	0.02899	0.01414	0.01556
	III	0	0.0305	0.034	0.036	0.038	0.0445	0.0525	0.06	0.066	0.067	0.0745	0.0815	0.0935
		±	±	±	±	±	±	±	±	±	±	±	±	±
		0	0.01202	0.01273	0.01414	0.01556	0.01485	0.01202	0.024	0.0269	0.024	0.0332	0.0361	0.0431
	IV	0	0.0175	0.0205	0.0255	0.03	0.034	0.0365	0.0375	0.044	0.051	0.059	0.0625	0.0655
		±	±	±	±	±	±	±	±	±	±	±	±	±
		0	0.00636	0.00778	0.00212	0.00141	0.00424	0.00778	0.0064	0.0085	0.0085	0.0099	0.0092	0.0050

Graph 3

Graph 4

Ultraviolet spectrometer or ultraviolet-visible spectrophotometry refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region. It allows particle size distributions to be measured in concentrated systems without dilution. By using this technique, a light is passed through the sample that we are going to measure. When light travels through the sample, it loses energy, also known as attenuated. Therefore, if the sample contains the suspended particles, the attenuation of light will change due to a variety of scattering and absorption patterns. The changes of energy of the light are then be measured to indicate the size distributions of the particles in the sample.

Dialysis tubing is actually a semi-permeable membrane when it is used in water. It is made up from regenerated cellulose. Dialysis tube has been used in the experiment for illustrating osmosis and pressure gradients across a membrane. As it is semi-permeable, therefore it only allows certain molecules to pass through by diffusion and it will block some molecules such as polar compound to pass through the membrane. Diffusion is the movement of particle from the region of high concentration to the region of low concentration until equilibrium is reached.

In the experiment, dialysis tube had been use to determine the ability of the paracetamol of suppository to pass through the membrane and enter into the water. The amount of the sample that passed through the dialysis tube is measured by using the ultraviolet spectrophotometry. Dialysis tube indicates the human’s biological barrier. Based on the results, we can see that UV absorption at 524 nm is increasing with time for each of the suppositories. Increasing in UV absorption indicates that there is increasing in the numbers of particle of the suppositories diffuse through the dialysis tube membrane.

From the graph above, the value that has been obtained is not accurate because in this experiment, the suppository I has lowest drug release (given as the UV absorption). However, it should be the highest among the four suppositories since it has the highest amount of the PEG 1000. In theory, the hardness of the polyethylene glycol will increase with increasing molecular weight. So, as the suppository becomes harder, it should required longer time to dissolve the drug and passing through the dialysis tube membrane. Polyethylene glycol with molecular weight from 600 to 1000 is presence in the semisolid form and molecular weight higher than 1000 is wax-like form. Therefore, suppository I should has the highest UV absorption.

High proportion of high polyethylene glycol produce suppository which release drug slowly and also brittle. Suppository IV has the highest proportion of PEG 6000 which will produce the hard suppository that difficult to dissolve. The result of the experiment can be considered same as the theory. In order to prepare less brittle suppository with release drug more readily, high molecular weight should be mixing with medium or low molecular weight of polyethylene glycol.

Since PEG is nonionic substances, they are quite soluble in hard water or in other aqueous solution of various salts. Suppositories can be formulated with much higher melting point to be melting at body temperature and dissolve in body fluids. Graph become not accurate may be because of the error that has been done during the experiment. May be error occur in taking sample by pipette to measure dispersion of paracetamol using spectrometer UV-visible.

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:

Different percentage of combination of PEG 1000 and PEG 6000 affects the physical characteristics of the suppository and the rate of release of the active ingredient.

References:

1. Rita Bhatta and Mohammad Salim Hossain, 2011. Evaluation of Kollidon SR based Ketorolac Tromethamine Loaded Transdermal Film. Journal of Applied Pharmaceutical Science 01 (08); 2011: 123-127. http://japsonline.com/vol-1_issue-8/23.pdf

2. onlinelibrary.wiley.com/doi/10.1002/jps.2600640706/pdf

Lab Report: Ointment

Effect of Different Ingredients on the Characteristics of an Ointment

Date of Experiment: 22nd April 2013

Lecturer’s Name: Dr. Haliza Katas

Aim: To study the effects of different ointment composition on the physical characteristics of ointment formed and the rate of drug released from it.

Introduction:

Ointment formulation is a semisolid dosage form which is suitable for external application on skin. It is oily preparations that contain one or more active ingredients that is soluble or spread homogenously. A good ointment must have an appealing texture, easy to use on skin characteristic as well as releasing it active ingredient from it.

Generally, ointment composed of active ingredient either powder or liquid that is incorporated into the oily semisolid continuous phase. In Pharmaceutics, ointment preparation is used to act as local treatment at application site, increasing the moisture of the skin (emollient effect).

Apparatus:

Weighing balance                                                        2 Threads

Weighing boat                                                               Glass rod

100ml beaker                                                               Water Bath

Heater                                                                              Pipette

Glass slab & spatula                                                    Plastic Kuvette

Mortar & pestle                                                            UV spectrophotometer

Dialysis bag (10cm)  

Materials: 

Emulsifying wax                                                         Acetylsalicylic acid

White soft paraffin                                                     Distilled water

Liquid paraffin

Procedure:

1. Emulsifying ointment (50g) is prepared using the formula below:

Emulsifying Ointment	Group	Materials (g)			Total (g)
		Emulsifying wax	White Soft Paraffin	Liquid Paraffin
I	1,5	21	25	4	50
II	2,6	17	25	8	50
III	3,7	13	25	12	50
IV	4,8	9	25	16	50

2. 5g of ointment formed is taken out and put into the weighing boat and it is then labelled. The texture, clarity, colour of ointment, spreadibility, greasiness, and hardness of the ointment is explained and compared.

3. 1.5g Acetylsalicylic acid powder is incorporated into the 15g ointment prepared by using levigation technique. If necessary, the acetylsalicylic acid is first mashed into fine powders by using mortar and pestle.

4. The acetylsalicylic acid ointment is put into dialysis beg and both end of the bag were tied up neatly.

5. The bag consisting acetylsalicylic acid ointment is put into 250ml beaker containing 100ml distilled water that has been heated to 37ºC.

6. At time interval of 5 minutes, 3-4ml aliquot sample is pipette and the release of acetylsalicylic acid from the ointment base is determined by using UV spectrophotometer. Ensure that the distilled water is mixed by using glass rod before taking out the sample.

Results and Discussion:

1. Comparison of the physical appearance of the each ointment.

Group	Texture	Clarity	Colour	Greasiness	Spreadability
Emulsifying Ointment V	Hardest	Higly Opaque	White	Least greasy	Very difficult
Emulsifying Ointment VI	Hard	Opaque	White	Less greasy	Difficult
Emulsifying Ointment VII	Soft	Clear	White	Greasy	Less Difficult
Emulsifying Ointment VIII	Softest	Very Clear	White	Very greasy	Easily

 

2. Plot UV absorption graph against time. Give explanation.

From this experiment, distilled water in the beaker represents the blood circulation in the body. Since distilled water is hypotonic so acetylsalicylic acid inside the dialysis bag can diffuse out from the dialysis bag. Hence, acetylsalicylic acid will diffuse out to the hypotonic solution against concentration gradient.  As a result, the content of acetylsalicylic acid in the distilled water increases. Dialysis bag acted as phospholipid bilayer and this experiment is conducted in 37ºC because the temperature mimics human body temperature. The UV absorption value obtained from the spectrophotometer represents the amount of acetylsalicylic acid released from the ointment into the distilled water through the dialysis beg.

Based on the graph above, it shows that the UV absorption value increases with time. This means that amount of acetylsalicylic acid in the distilled water increases with time. The gradient of the graph represents the rate of drug (acetylsalicylic acid) release to the blood circulation (distilled water). The gradient or rate of acetylsalicylic acid release is not consistent, some is steeper but some is less steep. When the gradient is low or at the less steep part, the distilled water that pipetted out for the test in spectrophotometer is saturated. This is because it is not stir evenly before taking out the sample. When the UV absorption value increases, the acetylsalicylic acid release from the dialysis bag into the surrounding solution also increases. If the saturation occurred, the graph become constant and the rate of release becomes nearly zero because the concentration of acetylsalicylic acid in the distilled water is isotonic to the concentration of ointment.

3. Plot a graph of UV absorption against time for the ointment preparation that contain different composition. Compare and discuss the result.

Time (min)		Average of UV absorption at 300nm (x+ SD)
		0	5	10	15	20	25	30	35	40	45	50	55	60
Emulsifying Ointment  I	Group 1	0	0.423	0.443	0.464	0.521	0.579	0.586	0.591	0.636	0.648	0.655	0.730	0.738
	Group 5	0	0.098	0.105	0.120	0.127	0.153	0.175	0.183	0.192	0.214	0.255	0.271	0.296
	Average(x)	0	0.261	0.274	0.292	0.324	0.366	0.381	0.387	0.414	0.431	0.455	0.500	0.517
	SD	0	0.163	0.169	0.172	0.197	0.213	0.206	0.204	0.222	0.343	0.200	0.229	0.221
Emulsifying Ointment II	Group 2	0	0.104	0.168	0.183	0.219	0.278	0.321	0.237	0.278	0.300	0.362	0.383	0.428
	Group 6	0	0.145	0.186	0.193	0.226	0.305	0.318	0.338	0.352	0.353	0.407	0.412	0.435
	Average(x)	0	0.125	0.177	0.188	0.223	0.292	0.320	0.284	0.315	0.327	0.385	0.398	0.432
	SD	0	0.021	0.009	0.005	0.004	0.014	0.002	0.051	0.037	0.027	0.023	0.015	0.004
Emulsifying Ointment III	Group 3	0	0.047	0.049	0.146	0.158	0.183	0.220	0.236	0.253	0.277	0.289	0.319	0.387
	Group 7	0.052	0.114	0.122	0.141	0.150	0.154	0.200	0.220	0.274	0.293	0.341	0.360	0.417
	Average(x)	0.026	0.081	0.086	0.144	0.154	0.169	0.210	0.228	0.264	0.285	0.315	0.340	0.402
	SD	0.026	0.034	0.037	0.003	0.004	0.015	0.010	0.008	0.011	0.008	0.026	0.021	0.015
Emulsifying Ointment IV	Group 4	0.028	0.051	0.068	0.086	0.140	0.175	0.204	0.183	0.205	0.214	0.215	0.206	0.230
	Group 8	0	0.036	0.040	0.065	0.082	0.109	0.122	0.141	0.177	0.180	0.225	0.246	0.250
	Average(x)	0.014	0.044	0.054	0.076	0.111	0.142	0.163	0.162	0.191	0.197	0.220	0.226	0.240
	SD	0.014	0.008	0.014	0.011	0.029	0.033	0.041	0.021	0.014	0.017	0.005	0.020	0.010

Ultraviolet spectrometer or ultraviolet-visible spectrophotometry refers to absorption spectroscopy or reflectance spectroscopy in ultraviolet-visible spectral region. It allows particle size distributions to be measured in concentrated systems without dilution. By using this technique, a light is passed through the sample that we are going to measure. When light travels through the sample, it loses energy, also known as attenuated. Therefore, if the sample contains the suspended particles, the attenuation of light will change due to a variety of scattering and absorption patterns. The changes of energy of the light are then be measured to indicate the size distributions of the particles in the sample.

Dialysis tubing is actually a semi-permeable membrane when it is used in water. It is made up from regenerated cellulose. Dialysis tube has been used in the experiment for illustrating osmosis and pressure gradients across a membrane. As it is semi-permeable, therefore it only allows certain molecules to pass through by diffusion and it will block some molecules such as polar compound to pass through the membrane. Diffusion is the movement of particle from the region of high concentration to the region of low concentration until equilibrium is reached.

In the experiment, dialysis tube had been use to determine the ability of the acetylsalicylic acid of ointment to pass through the membrane and enter into the water. The amount of the sample that passed through the dialysis tube is measured by using the ultraviolet spectrophotometry. Dialysis tube indicates the human’s skin. Based on the results, we can see that UV absorption at 300 nm is increasing with time for each of the ointments. Increasing in UV absorption indicates that there is increasing in the numbers of particle of the ointment diffuse through the dialysis tube membrane.

From figure 1, it was observed that the highest UV absorption is emulsifying ointment I and it is followed with the emulsifying ointment II and emulsifying ointment III. While emulsifying ointment IV has the lowest average UV absorbance. The higher the average UV absorbance, the higher the penetration rate of the drug from the dialysis tube and vice versa.  Therefore, emulsifying ointment I that has the highest average UV absorption can indicate that it has the highest diffusion of the acetylsalicylic acid through the dialysis tube.

The variation in the penetration rate of the drug through the dialysis tube may be the amount of emulsifying wax and liquid paraffin (oil phase). Emulsifying wax influence the capability of ointment to hold water. Emulsifying wax will prolonged the the contact of ointment with the dialysis tube membrane which will lead to the formation of occlusive layer. The higher the amounts of emulsifying wax in the formulation, the greater the capability of the ointment to hold water. However, liquid paraffin that increasing amount of liquid paraffin led to a retardation of drug release from formulation. This is because increasing liquid paraffin may decrease the thermodynamic activity of the drug which can be expressed in term of relative solubility of the drug. This leads to the retardation of drug release from the ointment base.

Four emulsifying ointments that prepared in the experiment are made up of different proportion of the emulsifying wax and liquid paraffin. Ointment I has the most amount of the emulsifying wax and the least amount of liquid paraffin, therefore the acetylsalicylic acid is more efficient in penetration of the dialysis tube and has the highest average UV absorption. However, ointment IV has the least amount of the emulsifying wax and the highest amount of the liquid paraffin. Therefore, the salicylic is more difficult to penetrate the dialysis tube membrane and resulted with the lowest average UV absorption.

4. What is the function of each ingredient that is used in the ointment? How the different composition of the emulsifying wax and liquid paraffin affects the physical properties of the ointment and the release rate of a drug from it?

Emulsifying wax acts as an emulsifying agent which decreases the interfacial surface tension so that the drug particles can be distributed uniformly in the ointment and to prevent any sedimentation from occurring.

White soft paraffin is an ointment base which are used as an emollient and moisturizer. To increases the greasiness of the ointment so that it can penetrate through the skin hydrophobic lipid bilayer more easily.

Liquid paraffin acts as the base too. It softens the ointment formed. It also decreases the ointment viscosity and acts as emollient.

When the proportion of the emulsifying wax decreases and liquid paraffin increases, the spreadibility and greasiness of the ointment increase while the hardness decreases and thus the drugs can be released from the formulation and penetrate through the skin lipid bilayer more readily. Therefore, the rate of release of drug increases. However, this is different from results of formulation IV which may be due to the experiment errors such as the distilled water is not stirred before sampling. So, ideally, the formulation IV should have the highest rate of release.

Conclusion:

Different composition of the ointment can affect the physical properties of the ointment formed and also the rate of release of the drug from the formulation. Therefore, it is important to ensure that the components of the formulation are in suitable proportion to produce a drug formulation which has desired therapeutic effects.

References:

1. Aulton, M.E. 1998. Pharmaceutics: The science of dosage form design. Edinburgh: Churchill Livingstone.

Lab Report: Suspension

Experiment 2: Effects of different ingredients on the characteristics of suspension formulation

Date of Experiment: 18th March 2013

Lecturer’s Name: Dr. Haliza Katas

Aim:

To determine the effects of different amount of Tragacanth used to the formulation of suspension.

Introduction:

Suspension formulation is a type of dispersed system where the solid molecules do not dissolve homogenously in the liquid phase. A good suspension must stay in homogenous state after being shaken, be easy to pour out from the container and having a uniform solid particle size and also attractive taste and texture. Precipitation formed from the storage must be easily dispersed again after shaking and the dispersion formed must be homogenous.

Normally, suspension composed of active ingredient (solid phase) dispersed in the liquid carrier, wetting agent, flavoring agent and coloring agent. Wetting agent (eg: Tragacanth) is used to reduce the surface tension between solid particle and liquid. Suspension is classified as coarse suspension where the particle diameter is more than 1 µm, or colloidal suspension where the particle size is less than 1 µm. In pharmaceutics, suspension is used to improve the stability, the taste and the bioavailability of the active ingredient.

Materials and Apparatus:

Apparatus:                                                         Ingredients:

Weighing instrument                                     Chalk

Weighing boat                                                   Tragacanth

Mortar and pestle                                           Concentrated Peppermint Water

150 ml plastic bottle                                      Syrup BP

50 ml measurable cylinder                         Double-strength chloroform water

200 ml measurable cylinder                      Distilled water

1 set of 1 ml pipette and pipette bulb

1 centrifugator tube 15 ml

100 ml beaker

Coulter counter instrument

Centrifugator

Viscometer

Procedures:

1 formulation of Pediatric Chalk Mixture (150 ml) was prepared by the following formulation.

Chalk	3 g
Tragacanth	(referred to the Table 1)
Concentrated Cinnamon Water	0.6 ml
Syrup BP	15 ml
Double Strength Chloroform Water	75 ml
Distilled Water, q.s.	150 ml

Table 1

Pediatric Chalk Mixture	Group	Tragacanth (g)
I	1,5	0.0
II	2,6	0.1
III	3,7	0.3
IV	4,8	0.5

5 ml of suspension was poured into the weighing boat and labeled. Texture, clarity and color of the suspension formed was described and compared.

50 ml of suspension was poured into 50 ml measurable cylinder. Height of the solid phase precipitated in the cylinder was measured at the interval of 0, 5, 10, 15, 20, 25, 30, 40, 50 and 60 minutes.

The rest of the suspension (95 ml) was poured into 100 ml beaker and the viscosity of the suspension was measured by a viscometer.

10 ml of suspension was poured into centrifugator tube and the height of the solid phase was measured after centrifugation (1000rpm, 5 minutes, and 25°C).

Results and Discussion:

1. Compare the physical appearance of formed suspension and gives comments.

Suspension	Texture	Clearity	Colour
I	Very dilute	Clear when still, cloudy when shaken	Cloudy
II	Dilute	Cloudy when still, becomes more cloudy when shaken	Light milky white
III	Concentrated	Opaque	Milky White
IV	Very Concentrated	Very opaque	Milky white

In this experiment, the physical appearances of suspension in those formulations are compared by varying the amount of tragacanth. Tragacanth is a suspending agent where it helps prevent caking at the bottom besides to facilitate redistribution of a suspension on shaking. One of the properties of a well-formulated suspension is that it can be easily resuspended by the use of moderate agitation.

Based on the observation done, the absent and present of tragacanth affect the texture, clearity, and colour of suspension formulated. Texture for Formulation I is very dilute due to lack of tragacanth as the suspending agent. As the amount of tragacanth increase, the texture of suspension produce becomes more viscous and concentrated. This is good for the appearances of a suspension. In terms of clearity, the present of tragacanth increase the cloudiness of suspension formulated even when they are not shaken. In the absent of tragacanth in Formulation I, the colour of the suspension is clear white and it turns to milky white when tragacanth is added. Besides that, tragacanth is partly soluble in water, thus forming viscous solution with water. Due to this factor, higher amount of tragacanth will form more viscous and concentrated solution which is like Suspension IV.

2. Plot a graph of height of sediment vs. time. Give explanation.

Time (min)	0	5	10	15	20	25	30	35	40	45	50	55	60
Height (mm)	0	3	3	4	5	5	5	5	5	5	5	5	5

The graph above has shown the height of sedimentation of a suspension, Pediatric Chalk Mixture types II which formulated with 0.1g Tragacanth powder over one hours. According to the graph, the height of the sediment formed at the 5^th minutes is low which is 3mm only and it is remained until 10 minutes. After 10minutes, the height of sedimentation had increased to 4 mm at 15 minutes and it was continued to increase to 5mm at 20 minutes. Then, the height of the sedimentation reached the maximum sedimentation level with the height of 5 mm for the following 40 minutes till the end of experiment.

According to this graph, the suspension does not contain any sediment at the beginning of the experiment. However, as the time passes, sedimentation will start to form. The height of sedimentation increases as the time increases. This is because the forces of interaction between the inter–particular attractive forces are stronger than the inter-particular repulsive forces of the Tragacanth powder on suspension. Hence, the height of sedimentation will increases with time until a maximum sedimentation level is achieved. Tragacanth that included in the formulation acts as the suspending agent that allows the insoluble solute particles to suspend in the dispersed medium. The amount of tragacanth that added into the suspension can determine the duration for the dispersed phase to remain suspend in the dispersed medium. 0.1g of tragacanth that added is able to allow the insoluble solute to disperse for 5 minutes and it started to settle down.

3. Plot a graph of height of sedimentation against time for the formulations of suspension with different contents of Tragacanth. Discuss.

Time (min)		Average height of sedimentation (mm) (mean+SD)
		0	5	10	15	20	25	30	35	40	45	50	55	60
Amount of Tragacanth (g)	0.0	0	18	15	12	11	10	10	9	9	9	9	9	9
	0.1	0	3	3	4	5	5	5	5	5	5	5	5	5
	0.3	0	2	4	6	7	8	8	9	9	10	10	10	10
	0.5	0	1	3	7	11	15	18	20	20	21	21	21	21

For suspension 1 which is the one that without tragacanth, there is a sharp increase in the height of sedimentation from 0 to 18 mm from 0 to 5 minutes. After 5^th minutes, the height of sedimentation had been decreased gradually until it reached the height where equilibrium is reached. The sharp increase in the height of sedimentation is due to the absence of tragacanth in the formulation. Particles tend to regroup and flocculate. Therefore, the dispersed phase settles to the bottom quickly. At the beginning of sedimentation, there is a lot of space that present in between the particles.  As time passing, the height of sediment decreases gradually due to the particles filled in the spaces between the porous structures to make the sediment become more compact. The sediment forms a cake at the bottom of the measuring cylinder. In compacted cake, stronger forces are involved. At the end of the sedimentation process, the spaces between the particles already filled with the particles and no more space available. Thus the sediment do not reduce I height at the end of experiment.

For suspension 2 (with 0.1g of tragacanth), there is an increase from 0 to 20 minutes which is then remained unchanged at height 5mm. The sedimentation of suspension 2 is increased in height as time passed. However in suspension 1, the sedimentation increased initially and started to decrease gradually. This is because of the presence of tragacanth which is acts as the suspending agent that allows the active ingredients suspended in the dispersed medium by increasing the viscosity of suspensions. As time passing, the height of the sediment remain unchanged like in the suspension 1. This is also due to the filling of spaces between the porous structures which in turn makes the sediment to become more compact and forms cake at the bottom of the measuring cylinder as some crystals knit together at point of contact at the bottom of the container and all the space already filled with the particles.

For suspension 3 that with 0.3g of tragacanth, the height of the sediment increases slowly and remains constant from 45 to 60 minutes. From 0 to 5 minutes, the height of sedimentation only increased with 2 mm and the increament is less than the increament in suspension 2. This is due to the presence of greater amount of tragacanth in the suspension which makes suspension 3 become more stable than suspension 2. The greater the amount of suspending agent used, the longer the time taken for the sediment to form since the suspension is more stable. The height of sediment remains unchanged from 45 to 60 minutes also due to the same reason that already mentioned above.

For suspension 4 (with 0.5g tragacanth), the sediment that formed from 0 to 5 minutes only has 1mm and it is the smallest increased in the height among four different suspension since it has the highest amount of the tragacanth. However, the height of the sediment increases dramatically from 10 to 35 minutes. The height of the sediment is then remains constant from 45 to 60 minutes. With a high amount of tragacanth, the particles remain suspend in the suspension which counteract the gravity force, thus there is no caking of suspension. It is readily to be redispersed and it can be considered as a stable suspension.

4. Explain briefly the mechanism of viscometer analysis. Plot a graph of viscosity of the suspension versus the amount of Tragacanth (g). Give explanation.

Tragacanth(g)	Viscometer reading
	1	2	3	4	5	6
I	0.0	0.0	2.0	2.0	2.0	3.5
II	2.0	2.0	2.0	3.0	2.0	3.0
III	2.5	1.5	2.0	2.5	3.0	3.5
IV	0.0	10.0	5.5	3.0	3.0	3.0

Tragacanth content (g)	0.0	0.1	0.3	0.5
Viscosity (cP) (x ± SD)	1.58 ± 1.24	2.33 ± 0.49	2.5 ± 0.65	4.08 ± 3.09

The graph of viscosity of the suspension versus the amount of tragacanth (g)

By theory, the viscosity of the suspension should be directly proportional to the amount of the tragacanth powder in the suspension. The tragacanth powder acts as a suspending agent which suspends the chalk in the liquid phase and form a suspension. Most suspending agents perform two functions. Besides acting as a suspending agent they also imparts viscosity to the solution. Suspending agents form film around particle and decrease interparticle attraction. Suspending agents also act as thickening agents. They increase in viscosity of the solution, which is necessary to prevent sedimentation of the suspended particles as per Stoke’s’law.

From this experiment, chalk is suspended by tragacanth (suspending agent) in the liquid phase and form suspension. From the graph obtained, the viscosity of suspension is increased when the weight of tragacanth added is increased. Increasing the weight of tragacanth, the gel-like structure formed will be stronger and thus the suspension formed will be more viscous.

5. Plot a graph of the height ratio vs the amount of Tragacanth used in the suspension. Give comments.

Amount of Tragacanth (g)	0.0	0.1	0.3	0.5
Height Ratio (mm)	7:1	11:1	17:1	20:1

Based on the graph, it is shown that the height ratio upon centrifuging is increasing with the increased of the amount of Tragacanth. Tragacanth is used as a suspending agent to slow down the sedimentation rate of the powder in the suspension by increasing the viscosity of the vehicle. It forms a less sticky suspension and is a better thickening agent than acacia. Therefore, the more the amount of the Tragacanth used, the height ratio should be decreased as the suspension becomes more viscous and harder to sediment. This is because of the ability of the Tragacanth to hold the heavy and insoluble solids of the suspension, thus increase the difficulty of the separation. However, during the experiment, due to some error, the result is not as expected.

6. What is the function of each ingredients used in this suspension formulation? How does different amount of Tragacanth used can influence the physical characteristics and the stability of certain suspension?

Chalk	Active ingredient which acts as absorbent which is to treat diarrhea.
Tragacanth	Suspending agent to form a less sticky suspension
Concentrated Peppermint Water*	Flavouring agent
Syrup BP*	Sweetening agent
Double Strength Chloroform Water*	Preservative
Distilled Water	Diluents in suspension

* To make the preparation more palatable

As the amount of Tragacanth used increased, the suspension will become more viscous and stable against coagulation. Tragacanth may increase the viscosity of the aqueous vehicle and thus slow the rate of sedimentation of the particles but they will also form adsorbed layers around the particle so that the approach of their surfaces and aggregation to the coagulated state is hindered. Therefore, there will be less flocculation and once the sedimentation occurs; the suspension becomes more difficult to redisperse.

Conclusion:

The amount of different Tragacanth powder will affect the rate of sedimentation and the height of sediment formed in the suspension. As amount of tragacanth powder increase, sedimentation rate will be occurring more slowly and thus sediment height will be lower.

References:

1. Pharmaceutics: the science of dosage form design, Aulton, M.E.2002

2. British Pharmaceutical Codex 1973

3. http://www.tokisangyo.com/pdf/R85E.pdf

Lab Report: Emulsion

Evaluation of the Effects of Certain Content on the Characteristics of an Emulsion Formulation 

Date of Experiment: 11th March 2013

Lecturer’s Name: Dr.Ng Shiow Fern

Aims:

1. To identify the effects of HLB surfactant on the stability of the emulsion.
2. To determine the physical and stability effects on the emulsion formulation due to the different of emulsifying agents used.

Introduction:

Emulsion is a thermodynamically unstable two phase system. It contain at least two immiscible liquids which one of them is dispersed homogenously (dispersed phase) in another liquid (continuous phase). Emulsion can be classified as two types: water in oil emulsion and oil in water emulsion. Emulsion can be stabilised by adding the emulsifying agent. Emulsifying agent can be divided into four type namely hydrophilic colloid, finely divided particles, surface active agent or surfactant.

HLB method is being used to identify the quantity and types of surfactant that is needed in order to produce a stable emulsion. Each surfactants, is numbered based on the HLB scale which is 1 (lipophilic) to 20 (hydrophilic). Usually, the combination of two emulsifying agents is used to produce a more stable preparation of emulsion.

Apparatus:

8 test tubes, 50mL measuring cylinder, 2 sets of pasture pipette and droppers, Vortex mixer, weighing boat, mortar and pestle, light microscope, microscope’s slides, 5mL pipette and a bulb, 50mL beaker, 15mL centrifuge tube, Coulter Counter, centrifuge machine, viscometer, 45°C water bath, 4°C fridge.

Materials:

Palm oil, Arachis oil, Olive oil, Mineral oil, distilled water, Span 20, Tween 80, Sudan III solution (0.5%) and ISOTON III solution.

Procedures:

1. Each test tube was labelled and a straight line was drawn 1cm from the bottom of the test tube.

2. 4mL of the given oil for each group and 4mL of distilled water were mixed in the test tube.

Group	Test Oil
1, 5	Palm Oil
2, 6	Arachis Oil
3, 7	Olive Oil
4, 8	Mineral Oil

3.Span 20 and Tween 80 were dropped into the mixture. The test tube was sealed and left to the Vortex mixture for 45 seconds. The time taken needed to reach the interface of 1cm was recorded. The HLB value of each samples were determined.

No. of tubes	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
HLB value
Time of interface (min.)
Stability

4. A few drops of Sudan III solution were added to 1g of emulsion that was formed on a weighing boat and flattened. The colour dispersion in the sample was determined and compared under the light microscope. The shape and size of the globules formed were drawn, explained and compared.

5. 50g (of the given oil) Emulsion was prepared by using the wet gum method and the following formula.

Arachis Oil	25mL
Acacia	6.25g
Syrup	5mL
Vanillin	2g
Alcohol	3mL
Distilled water (qs)	50mL

6. 40g of the formed emulsion was added into a 50mL beaker and homogenization process was formed for 2 minutes by using a homogenizer.

7. 2g of the formed emulsion (before and after the homogenization) was put on the weighing boat and labelled. A few drops of Sudan III solution was added and flattened. The texture, consistency, degree of the oily shape and colour dispersion of the sample was determined and compared under the light microscope.

8. The formed emulsion viscosity (15g in the 50mL beaker) after the homogenization was determined by using the viscometer that has been calibrated with spindle type LV-4. The sample then was exposed to the temperature of 45°C (water bath) for 30 minutes and then to the temperature of 4°C (fridge) for 30 minutes. The viscosity of the emulsion was determined after the exposure and reached the room temperature for about 10-15 minutes.

Readings	Viscosity (cp)			Average + SD
	1	2	3
Before the exposure	419.9	419.9	389.9	409.90±199.51
After the exposure	857.9	842.9	731.9	775.37±66.63
Difference (%)	89.16%

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

Interface	Heights (mm)
Separate phase	20
Original emulsion	39
Height ratio	39/20

Results and Discussion:

1. What are the HLB values that form a stable emulsion? Discuss.

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
HLB value		9.67	10.73	11.34	12.44	13.17	14.09	15.00	0.00
Time needed for interphase to reach 1cm (min)	Group 1	Interphase did not reach 1cm after 120 minutes.			58.00	61.00	45.00	25.00	0.50
	Group 5	Interphase did not reach 1cm after 120 minutes.			16.00	30.00	39.00	16.00	7.00
	Average	–			37.00	45.50	42.00	20.50	3.75
Stability		Yes	Yes	Yes	No	No	No	No	No

Arachis 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
HLB value		9.67	10.73		11.34	12.44	13.17	14.09	15.00	0.00
Time needed for interphase to reach 1cm (min)	Group 2	Interphase did not reach 1cm after 120 minutes.				27.00	40.00	55.00	19.00	9.00
	Group 6	Interphase did not reach 1cm after 120 minutes.				38.00	49.00	61.00	19.00	25.00
	Average	–		–	–	32.50	44.50	58.00	19.00	17.00
Stability		Yes	Yes		Yes	No	No	No	No	No

 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
HLB value		9.67	10.73	11.34	12.44	13.17	14.09	15.00	0.00
Time needed for interphase to reach 1cm (min)	Group 3	Interphase did not reach 1cm after 120 minutes.		8.19	14.48	87.35	58.35	19.49	0.33
	Group 7	Interphase did not reach 1cm after 120 minutes.				63.00	Interphase did not reach 1cm after 120 minutes.	45.00	2.50
	Average	–				75.18	–	32.25	1.42
Stability		Yes	Yes	No	No	No	No	No	No

Mineral 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
HLB value		9.67		10.73		11.34	12.44	13.17	14.09	15.00	0.00
Time needed for interphase to reach 1cm (min)	Group 4	119.00			114.00	108.00	94.00	80.00	34.00	8.00	0.50
	Group 8	Interphase did not reach 1cm after 120 minutes.			82.00 50.00	24.00	28.00	29.00	15.00	18.00	0.50
	Average	–	o			66.00	61.00	54.5	24.50	13.00	0.50
Stability		Yes		No		No	No	No	No	No	No

An emulsion can be considered as stable if it takes longer duration for the phase separation to occur. Span 20 and tween 80 is the surface active agent that added into the emulsion to improve the stability of the emulsion. The amount of span20 and tween80 that needed to add depends on the stability that required for the emulsion. HLB system gives the guidelines for the selection of emulsifier and the amount that need to add to achieve the satisfactory stability. The HLB system assigns a numerical value for each of the emulsifier and known as HLB. HLB of the emulsifier is the balance of the size and strength of the hydrophilic (water-loving or polar) and the lipophilic (oil loving or non-polar) groups of the emulsifier.

An emulsifier that is lipophilic in character is assigned a low HLB number (below 9.0), and one that is hydrophilic is assigned a high HLB number (above 11.0). Those in the range of 9-11 are intermediate. Appropriate HLB value is important in determining the stability of the emulsion. Span is hydrophobic and it is used to make the w/o emulsion while tween is hydrophilic and is used to form o/w emulsion. In the stabilization of oil globules, it is essential that there is a degree of hydrophilicity to confer an enthalpic stabilizing force and a degree of hydrophobicity to secure adsorption at the interface. So, a combination of both offers a suitable HLB value which matches with the system and produces a stable emulsion.

For palm oil, arachis oil and olive oil, the HLB value that brings to stable emulsion is 11.34. This means that the stable emulsion of palm oil can be prepared by adding 12 drops of Span 20 and 9 drops of Tween 80. On the other hands, the HLB value of the mineral oil that can give stable emulsion is 10.73 which mean 12 drops of Span 20 and 6 drops of Tween 80 are required for the formation of stable emulsion. These can show that different types of oils as the dispersed phase required different HLB values of emulsifier. Thus it requires different combination of emulsifiers in order to obtain a stable emulsion.

From the experiment for arachis oil which is done by our group, it was found that the emulsion in test tube 1, 2 and 3 with the HLB values as 9.67, 10.73 and 11.34 respectively which can give the most stable emulsions. The phase separation for the emulsion in test tube 1, 2 and 3 do not occur after 120 minutes (2 hours) which means they require longer time for phase separation to occur. Thus we can conclude that they are the most stable emulsion with the HLB value that ranges from 9-13. Meanwhile, emulsions from tube 7 and 8 give the lowest stability where the phase separation time is the shortest. This is because the absence of surfactant as an emulsifying agent in tube 8 while in tube 7, only Tween 80 present and with a high HLB value that more than 11. HLB value that more than 11 means a hydrophilic emulsifier has been used and it unable to stabilize the emulsion.

From the experiment for palm oil, it was found that the emulsion from test tube 1, 2 and 3 with the HLB values as 9.67, 10.73 and 11.34 respectively give the most stable emulsions. The phase separation for the emulsion in test tube 1, 2 and 3 do not occur even after 120 minutes (2 hours), which means they require longer time for phase separation to occur. Thus we can conclude that they are the most stable emulsion with the HLB value that ranges from 9-13. However, the emulsions from tube 7 and 8 give the lowest stability where the phase separation time is the shortest. The reason is just the same as the above which is due to the absence of surfactant as an emulsifying agent in tube 8 while in tube 7, only Tween 80 present and with a high HLB value that more than 11. HLB value that more than 11 means a hydrophilic emulsifier has been used and it unable to stabilize the emulsion.

For the emulsion of olive oil in the experiment, emulsions from test tube 1 and 2 with HLB value 9.67 and 10.73 respectively have shown the most stable emulsion. Their interphase do not reach 1cm after 120 minutes (2 hours) which mean that they require a longer duration for the phase separation to occur. The emulsion in test tube 8 is the least stable emulsion since there is no emulsifier added into the emulsion to stabilize it. Thus the emulsion just dispersed less than 3 minutes and separate into 2 phases.

For the emulsion of mineral oil, the most stable emulsion is from the test tube 1 with the HLB value 9.67. The interphase of emulsion in test tube 1 do not reach 1cm after 2 hours which means that the emulsion is stable and allow the even dispersion of the oil globules in the water. As the same with the others emulsion of different oil, the emulsion from test tube 8 is the least stable due to the absence of the emulsifier. Phase separation of emulsion in test tube 8 is occurred as soon as the removal of swirling force.

2. Compare the physical appearance of arachis oil emulsion and give comment about it. What is Sudan III test? Compare the colour distribution in emulsions formed and give comments about it.

Sudan III test is a test using Sudan III which is oil soluble to show amount and location of lipids. Sudan III is also a fat-soluble dye used for staining of triglycerides in frozen sections, some protein bound lipids and lipoproteins on paraffin. Sudan III has the appearance of reddish brown crystals. It stained red in oil. . It is not water soluble. When the emulsion is oil in water, the Sudan III does not disperse in the emulsion because they will not mix together. While in the water in oil emulsion, the Sudan III colouring will disperse in the emulsion.

Magnification (40×10)	Physical appearance	Colour distribution
Test tube 1	Water droplets dispersed in oil. This is water in oil emulsion. This emulsion is not dispersing very well due to error.	Sudan III colour dispersed in the emulsion. The emulsion stained light orange.
Test tube 2	Water droplets dispersed best in oil. This is water in oil emulsion. HLB value of the emulsion in this test tube is not in the optimum range.	Sudan III colour dispersed in the emulsion. The emulsion stained light orange.
Test tube 3	Water droplets dispersed better in oil compared to test tube 1. This is water in oil emulsion. However, the HLB value of the emulsion in this test tube is not in the optimum range.	Sudan III colour dispersed in the emulsion. The emulsion stained light orange.
Test tube 4	Water droplets are not properly dispersed in oil. HLB value of the emulsion in this test tube is not in the optimum range.	Sudan III colour dispersed in the emulsion. The emulsion stained light orange.
Test tube 5	Water droplets are not properly dispersed in oil. HLB value of the emulsion in this test tube is not in the optimum range.	Sudan III colour dispersed in the emulsion. The emulsion stained light orange.
Test tube 6	Water droplets are not properly dispersed in oil. HLB value of the emulsion in this test tube is not in the optimum range.	Sudan III colour dispersed in the emulsion. The emulsion stained light orange.
Test tube 7	Water droplets are not properly dispersed in oil. HLB value of the emulsion in this test tube is not in the optimum range.	Sudan III colour dispersed in the emulsion. The emulsion stained light orange.
Test tube 8	The emulsion is totally not formed without surfactant, phase separation occur very fast.	Sudan III does not disperse in the emulsion, globules of Sudan red form on surface of emulsion.

3. Plot and discuss

Group		Viscosity (cP)
		Before the temperature cycle			After the temperature cycle
20	1	80.0	90.0	100.0	90.0	100.0	100.0
	5	100.0	110.0	120.0	130.0	140.0	140.0
25	2	389.9	419.9	419.9	779.8	779.8	659.9
	6	419.9	419.9	389.9	857.9	842.9	731.9
30	3	239.9	210.0	210.0	539.9	449.9	389.9
	7	50.0	60.0	50.0	50.0	50.0	50.0
35	4	650.0	650.0	600.0	300.0	300.0	300.0
	8	740.0	820.0	920.0	900.0	920.0	960.0

i. Graph of sample viscosity before and after the temperature cycling vs the different content of oil.

Content of mineral oil (ml)	Average Viscosity (cP) (Average ± SD)		Difference Viscosity (%) (Average ± SD)
	Before the temperature cycle	After the temperature cycle
Palm oil (20)	100.00±12.91	116.67±20.55	16.67%
Arachis oil (25)	409.90±199.51	775.37±66.63	89.16%
Olive oil (30)	136.65±83.98	254.95±209.53	86.57%
Mineral oil (35)	730±111.05	613.33±313.83	-15.98%

The viscosity is the measure of internal friction in a liquid or the resistance to a flow. In the experiment, we use four different types of oil which are palm oil, arachis oil, olive oil, and mineral oil. Besides that, we also use different volume of each type of oil. In the experiment, the emulsion for each sample oil is heated at 45°C for 30 minutes then the emulsion is put into refrigerator for freezing at 4°C for 30 minutes. The exaggeration of the temperature fluctuations subjected to the emulsion is used to compare the physical instabilities of the emulsion under the normal stored condition. When the emulsion is heated and frozen, the small ice crystals will be formed continually. This will disrupts the adsorbed layer of the emulsifying agent at the oil-water interface. As a result, the weakness in the structure of the film will become more apparent quickly.

From the graph, the volume of oil is increased, the viscosity of emulsion will be increased. For example, the emulsion II has higher viscosity than emulsion I. Besides that, in which emulsion I (20 ml of turpentine oil) has the lowest viscosity before and after the temperature cycle. On the other hand, viscosity of the emulsion containing 35ml of mineral oil is the highest. For emulsion IV (35 ml of mineral oil), the viscosity of emulsion of  before heating is higher than after heating.

Theoretically, the formation of ice crystal leads to the decrease in the viscosity of the emulsion. As the consequence, the sample viscosity before temperature cycling is actually higher than that of after temperature cycling. Besides that, the drop in the sample viscosity of the mineral oil after temperature cycling may be due to the occurrence of phase inversion. This means that the initially o/w emulsion is now converted into w/o emulsion. However, the graph obtained does not follow the pattern theoretically. This is due to errors while conducting the experiments. Some precautions was occurred, like we did not waiting until room temperature then directly measure the viscosity of emulsion. The temperature of emulsion is too low after take out from refrigerator. This cause the viscosity of emulsion is  increasing. 

ii. Graph of the viscosity difference (%) versus various amount of mineral oil

In this experiment, emulsion containing 25ml of arachis oil shows the greatest difference in the viscosity, that is 241.18%. The smallest difference in viscosity, which is 16.03%, 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 arachis oil is the most unstable emulsion while emulsion with mineral oil is the most stable one. However, emulsion with palm oil actually should have the smallest viscosity difference value and is the most stable one as it contains the least amount of palm oil. This is because when there is a higher 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 mineral oil is not the most stable emulsion. These show that errors occur during the experiment.

4. Plotting graph of Seperated Phase Ratio Against Different Type and Volume of Oil.

Readings	Height (mm)
	Palm oil (20ml)			Arachis oil (25ml)
	Group 1	Group 5	Average	Group 2	Group 6	Average
Separation phase	30	35		36	20
Original emulsion	49	48		46	39
Height ratio	0.61	0.73	0.67	0.78	0.51	0.645

Readings	Height (mm)
	Olive oil (30ml)			Mineral oil (35ml)
	Group 3	Group 7	Average	Group 4	Group 8	Average
Separation phase	35	37		29	30
Original emulsion	50	50		50	45
Height ratio	0.70	0.74	0.72	0.58	0.67	0.625

Mineral Oil (ml)	Ratio of Phase separation (X± SD)
Palm Oil (20 ml)	0.67±0.06
Arachis Oil (25 ml)	0.645±0.135
Olive Oil (30 ml)	0.72±0.02
Mineral Oil (35 ml)	0.625±0.045

Phase separation ratio is used to indicate stability of an emulsion. A high ratio of phase separation will be resulted in unstable emulsion which it will have two separated, non-homogenised phase. 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 decrease from 20mL mineral oil emulsion to 25mL mineral oil emulsion, increase from 25mL mineral oil emulsion to 30mL mineral oil emulsion and decrease from 30mL mineral oil emulsion to 35mL mineral oil emulsion. Emulsion with 30mL mineral oil by using olive oil has highest separated phase ratio while 35mL mineral 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 is the function of each ingredient used in the emulsion preparation? How can the different amount of ingredients influence the physical characteristics and the stability of the emulsion?

	Function	Note
Palm oil, Arachis oil, Olive oil and mineral oil	The oily phase in the o/w emulsion.	Amount of the mineral oil (oily phase) and the distilled water (aqueous phase) used is important to determine the type of emulsion formed, whether o/w or w/o emulsion. The volume of the dispersed phase should not be more than the volume of the continuous phase. Or else, phase inversion will occur.
Acacia	It is a natural product and it can act as an emulsifying agent which can reduces the interfacial tension and maintains the separation of the droplets in the dispersed phase. Acacia which acts as the emulsifying agent should be used in appropriate amount according to the HLB value. If the inappropriate HLB value used, the large interfacial tension between the dispersed phase and the continuous phase formed and the separation of phase will occur.	Since acacia is a natural product, it can be a good medium for the growth of microorganisms.Thus, agent antimicrobial such as benzoic acid 0.1% is added to stabilize the emulsion from microbial growth. It is different from the surfactant which reduce the surface tension.
Syrup	Increase the viscosity of the emulsion and acts as sweetening agent that used to mask the unpleasant taste of the mineral oil in order to increase the patient compliance. Moreover, it can be used to increase the viscosity of the emulsion since it is a viscous liquid. Therefore the amount of syrup that added into the emulsion has to be controlled to ensure the suitable viscosity of emulsion.	Viscosity of the emulsion will affect on the physical stability and the rheological characteristic of the emulsion. We have to consider the ease of pouring out the emulsion from the container. Therefore, the viscosity of the emulsion has to be strictly monitored to avoid the rheological problem.
Vanillin	As flavoring agent which can improve the taste of emulsion thus can increase patient compliance.	Mostly emulsion has a bad taste that most of the patient unable to accept. Vanillin aids in the taste of emulsion.
Alcohol	Alcohol is an antimicrobial agent. In this formulation, alcohol acts as a preservative but the amount of alcohol used should not too high to prevent toxicity occur.	In this formulation, there is a high proportion of water present in the emulsion which making it more susceptible to microbial contamination. A suitable antimicrobial agent should be used to prevent instability of emulsion.
Distilled water	It is the continuous phase in the emulsion in which the oily phase is homogenously dispersed with the aid of the surfactants.	The distilled water which is the aqueous phase and the oils which is the oily phase in the emulsion, thus the amount of each phase or the volume ratio in certain emulsion is determined by the desired type of emulsion, either o/w or w/o emulsion.

Conclusion:

The combination of different surfactants will results in an accurate HLB value that is required to form a stable emulsion. Furthermore, different types of emulsifying agent will results in emulsion with different physical characteristics and stability. Emulsifying agent adsorb onto the oil and water interface thus lowering the surface interfacial tension which then in turn will lower the free energy of the system hence stabilizing the emulsion.

Reference:

1. Aulton, M.E. 1998. Pharmaceutics: The science of dosage form design. Edinburgh: Churchill Livingstone.

2. Shariza, Rudy, Ng Shiow Fern, Thomas. 2011. Pharmacy Practice: Guide to Compounding and Dispensing. Penerbit UKM: UKM Press.