EXPERIMENT 3: OINTMENT

Title: Impact assessment of different ingredients on the characteristics of an ointment formulation

Objective:Study the effects of different ointment composition on the physical characteristics of an ointment that is formed and the rate of drug release from it.

Introduction:Ointment formulation is a semi-solid dosage forms that is suitable for application on the skin, for external use only. An ointment is an oily base preparation and consists of one or more drugs distributed homogenously in base and can be absorbed through the skin for systemic action. Ointments are formulated using hydrophobic, hydrophilic, or water-emulsifying bases to provide preparations that are immiscible, miscible, or emulsifiable with skin secretion. Ointment bases are intended to soften but not melt when applied to the skin. The purpose of an ointment bases include as a vehicle from which the drug may be absorbed by the skin and as a protective layer or emollient for the skin. A good ointment must have an interesting texture, easy to spread on the skin and release the active ingredient.     A desired base should not interfere with the normal functioning of the skin by preventing radiation of heat from the skin or preventing the excretion of various secretion. It must not inactivate or interfere in any way with any incorporated medicament. Different composition of base will greatly affect the pharmacologic effect of a particular drug. This includes stability, penetrability, solvent property, approximately neutral with regard to pH, and ease of application and removal. Therefore in pharmaceutical aspects, choice of bases should be made carefully to obtain optimum drug distribution.

Apparatus:Weighing balance, 1 weighing boat, 1 100mL beaker, heater, I set of slap and spatula, 1 set of mortar and pestle, 1 10 cm dialysis bag, thread, glass rod, water-bath, pipette and pipette bulb, Spectrophotometer UV.

Materials:Emulsifying wax, White soft paraffin, Liquid paraffin, Acetylsalicylic acid, distilled water.

Procedure:

1.  50g of emulsifying ointment is prepared through formulation as following:

2.  5g of ointment is taken from the total of 50g and is placed in weighing boat and is labelled. The texture, clarity and colour of ointment is observed and compared.

3.  1.5g of Acetylsalicyclic acid is incorporated into 30g of made ointment by using levigation. (Acetylsalicyclic acid powder is crushed into finer particles using mortar and pestle if required.)

4.  The Acetylsalicyclic acid ointment is filled into dialysis bag. Both ends of the bag are being tied.

5.  The dialysis bag is placed into 100ml beaker which contains 50ml of saline water and is heated in water-bath to 370C.

6.  One aliquot sample (3-4ml) is pipetted at an interval of 5 minutes. Rate of release of Acetylsalicyclic acid from the ointment tube is determined by using UV-visible spectrometer. The saline water is stirred by glass rod before transferring of sample from beaker to pipette is carried out.

1.      Compare and describe the physical appearance of the ointment.

Physical appearance	Emulsifying ointment
	I	II	III	IV
Emulsifying wax	21	17	13	9
White soft paraffin	25	25	25	25
Liquid paraffin	4	8	12	16
1)Texture
-spreadibility	+	++	+++	++++
-greasiness	+	++	+++	++++
-hardness	++++	+++	++	+
2) Clarity	++++	+++	++	+
3) Color	white	White	white	white

From the table, we can see that the all the 4 types of emulsifying ointments have the same clarity and colour. However, the spread ability and greasiness of ointment slowly increases from ointment I to ointment IV while the hardness of the ointment decreases from ointment I to ointment IV.

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, it is not enough to produce “interlocking wax” structure, thus ointment IV is very soft while ointment I with 21g of emulsifying wax is very hard. 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.

In addition, increasing the proportion of liquid paraffin in the ointments will increase the greasiness of the ointments and decreases hardness of the ointments due to its function to soften the ointment. This will aid in the ointment penetration through the skin. However, increasing greasiness will make it difficult to be removed from the skin.

2.      Graph of UV absorption against time is plotted. Explain.

In this experiment, the dialysis bag indicates the skin membrane where the absorption of drug is entering the circulation by passing through it. The concentration of drug in the distilled water represents the amount of drug absorbed into the circulation that is the bioavailability of the drug.

UV absorption refers to the amount of drug that passes through the membrane. Based  the graph, we can see that UV absorption at 310mm which carried out at 37 °C is proportional to the time throughout the experiment. Theoretically, UV absorption is proportional to the time of acetylsalicylic acid release from the tube that is exposed to the dialysis environment. The UV absorption of the ointments increases when the time increases, which means that the longer the time, the higher the amount of drugs passing through the membrane or the higher the bioavailability is.

The gradient of the graph refers to the releasing rate of the acetylsalicylic acid from the membrane of the tube. From the graph, we can see that the gradient of the graph increases as the time increases until time 30^th. This is because in the beginning of the experiment, distilled water is much hypotonic to the ointment which consists acetylsalicylic acid  in the dialysis bag. Acetylsalicylic acid which is hypertonic than distilled water will move from higher concentration in the dialysis bag to lower concentration in the distilled water.  As the experiment proceeds, the solution in the dialysis bag becomes isotonic to the distilled water as some of the acetylsalicylic acid has been moved to the distilled water. This shows that the equilibrium for diffusion of acetylsalicylic acid into and out of the tube is achieved. Hence, the gradient of the graph decreases. This indirectly shows that the drug had achieved its bioavailability.

In this experiment, some errors have been made and resulted in the fluctuation of the graph. This may due to the insufficient stirring of the distilled water until uniform before sample is taken out for measurements. There are some precautions that we need to consider when carry out the experiments to avoid errors. We need to control the temperature in order to get the uniform temperature at 37oC. On the other hand, the dialysis bag also has to be tied tightly and immersed completely in the distilled water in order to get an accurate result. The smooth surface of the cuvette should not face the source of UV light and the smooth surface should be cleaned before it is placed into the device. Lastly, the technique of using spectrophotometry device also plays a vital role in getting accurate results.

3.   Plot graph UV absorption against time for the ointment formulation with different composition. Compare and discuss the results.

In this experiment, we are instructed to make four ointments with different formula. In the formulation, emulsifying wax and liquid paraffin are modified to investigate the permeation of acetylsalicylic acid from the ointment. The ratio of emulsifying wax and liquid paraffin respectively in all four formulae are different.

Generally, the UV absorption of all the formulations increases with time. This implies that the longer the time of ointment remains in the water bath, the more the amount of acetylsalicylic acid diffuses out of the dialysis bag. Hence, more UV is absorbed by acetylsalicylic acid. From the graph plotted above, the steepest graph of emulsifying ointment I show that it has the highest UV absorption that is the highest amount of permeation of acetylsalicylic acid in the sample. This is followed by emulsifying ointment 2, 3 and 4.

The amount of acetylsalicylic acid found in the experiment I is the highest which should be the lowest due to lowest amount of paraffin in the formulation. This is because the oil phase paraffin will allow the acetylsalicylic acid to diffuse out of the bag into the solution. The amount or proportion of the paraffin in the experiment I is the lowest. The bar chart shown indicates that formulation 2 has higher concentration of acetylsalicylic acid in distilled water than formulation       
Theoretically, as the amount of emulsifying wax decrease and amount of liquid paraffin increases, the acetylsalicylic acid can diffuse through the membrane faster. In formulation 2, the percentage of emulsifying wax, white soft paraffin and liquid paraffin should be 34%, 50%, 16% respectively. Meanwhile, in formulation 3, the percentage of emulsifying wax, white soft paraffin and liquid paraffin should be 26%, 50%, 24% respectively. Both formulations shows only 4% difference with the ideal formulation. Thus, these formulations encourage the diffusion of drugs into the distilled water through the membrane.   In formulation 4, the amount of paraffin is the highest but the amount of acetylsalicylic acid found is the lowest because the proportion of the emulsifying wax is too low for the acetylsalicylic acid to disperse well in the ointment.
 There are several factors contributing to the errors which we should take note of. First of all, the amount of emulsifying ointment filled into the dialysis bag in each group may differ due to difficulty to fill a small bag with greasy semisolids. This will affect the accuracy of readings as more drugs are able to diffuse out from the dialysis bag with more emulsifying ointment. Since the ointments are prepared by different groups using different techniques, the consistency of products is not guaranteed. Moreover, the outer membrane of dialysis bag may be contaminated with the emulsifying ointment during the filling of ointment into the bag.

4.  What is the function of each of the material used in preparing the ointment? How are the used of different composition of Emulsifying wax and liquid paraffin affect the physical characteristics of ointment and its releasing rate of the active ingredient?

Emulsifying Wax	Substance added to an emulsion to prevent the coalescence of the globules of the dispersed phase. They are also known as emulgents or emulsifiers. They act by reducing the interfacial tension between the two phases and forming a stable interfacial film. It can increase the water solubility of the ointment.
White Soft Paraffin	It is reserved for products with colourless, white or pale ingredients. It inhibits water loss from the skin by forming a greasy layer, and this in turns improves the hydration of the skin in dry scaly conditions.
Liquid Paraffin	Liquid paraffin increases the lipophilicity of ointment, so that it can penetrate the membrane. It also used to reduce the viscosity of the ointment.
Acetylesalicylic acid	An active ingredient used as an analgesic to relieve minor aches and pains, as an antipyretic to reduce fever, and as an anti-inflammatory medication.

Liquid paraffin increases the lipophilicity, greasiness and spreadability of ointments. Hence, greater proportion of the liquid paraffin will produce a watery, greasy and soft ointment and increases the penetration of drugs through the membrane.

On the other hand, emulsifying wax that acts as the emulsifier to stabilise the emulsion by preventing the coalescence of the globules of the dispersed phase and increases the viscosity of the ointment will increase drug release from the formulation and penetrates into the skin and also the water solubility of the ointments. Thus, greater proportion of emulsifying wax will produce harder and lower spreadability ointments due to its increased viscosity.

  Conclusions:

     Different compositions of ointment formulation will affect the rate of release of the active ingredient. High Emulsifying Wax proportion in an emulsifying ointment will release drug faster, but will produce a hard ointment. On the other hand,  high liquid paraffin proportion will increase the spreadability of the ointment which  make the ointment easy to be applied on skin but the ointment will become more greasy and have slower drug release. Thus, we need to balance between the amount of emulsifying wax and liquid paraffin in an  ointment formulation to achieve an ideal ointment without affecting the rate of drug release.

 References:

1.  http://en.wikipedia.org/wiki/Aspirin
2.  Paul Beringer, Remington: The Science And Practice Of Pharmacy, 21th Edition, 2005, Lippincott Williams & Wilkins.
3.  John F. Marriott, Pharmaceutical Compounding And Dispensing, 2006, Pharmaceutical Press.
4.  Aulton, M.E. 2002. Pharmaceutics: The science of dosage form design. Edinburgh: Churchill Livingstone.
5.  Florence,A.T. & Attwood, D. 1998. Physico-chemical principles of pharmacy. 3rd Ed. New York: Macmillan. 

Appendices

1.  Ointment being inserted into dialysis bag and heated in waterbath :

   2.    Aliquot sample is taken every 5 minutes :

EXPERIMENT 2: SUSPENSION

Title : Effects of different ingredients on the characteristics of  suspension formulation

Objective        : 

To know the effects of using different amount of Tragacanth to the formulation of suspension.

Introduction:

       Since some drugs will not dissolve in solvents suitable for medicinal use, they are made into suspensions. Suspensions consist of a finely divided solid dispersed in a water-based liquid. Suspension formulation is a type of dispersed system where the solid molecules do not dissolve homogenously in the liquid phase. It is a heterogeneous mixture containing solid particles that are sufficiently large for sedimentation. Usually they must be larger than 1 micrometer. The internal phase (solid) is dispersed throughout the external phase (fluid) through mechanical agitation, with the use of certain excipients or suspending agents.

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.

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

Procedure:

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

Table 1

2. 5 ml of suspension was poured into the weighing boat and was labeled. Texture, clarity and color of the suspension was described and compared.
3. 50 ml of suspension was poured into 50 ml measurable cylinder. Height of the solid phase precipitated in the cylinder was measured at the duration of 0, 5, 10, 15, 20, 25, 30, 40, 50 and 60 minutes.
4. The rest of the suspension (95 ml) was poured into 100 ml beaker and the viscosity of the suspension was measured by a viscometer.
5. 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 the suspension formed and give the analysis.

Physical appearance	Paediatric Chalk Mixture
	I	II	III	IV
Tragacanth (g)	0.0	0.1	0.3	0.5
1)Texture
-smoothness	+	++	+++	++++
-viscosity	+	++	+++	++++
-‘redispesibility’	++++	+++	++	+
-ease to precipitate	fast	slow	Slow/not precipitate	Not precipitate
2) Clarity	++++	+++	++	+
3) Color	white	white	white	white

       The color of the suspension I to IV is white. Tragacanth powder is the suspending agent. Suspension without Tragacanth powder will separate into two layers with clear boundary. One is the clear liquid phase and another one is the solid phase. The Chalk mixture I contains no Tragacanth powder and the amount of Tragacanth powder is increasing to Chalk mixture IV. By using Tragacanth powder, suspension is formed where the Chalk is suspended in the liquid and the suspension becomes not clear and more viscous. By using larger amount of Tragacanth powder, the suspension formed becomes more viscous. The time for particles to precipitate is longer and the ability of redispersibility becomes less. This is because as more Tragacanth powder is used, more Chalk will be suspended and the rate of precipitation will become slower.

2.  Plot the graph of height of sedimentation versus time. Give explanation.

       

For the Pediatric Chalk Mixture type IV, 0.5g Tragacanth powder is used as a suspending agent, there is no sediment formed at the beginning. This is because Tragacanth which used as suspending agent has stabilized the formulation from forming sedimentation and help in the formation of viscous suspension. It forms film around particle and decrease interparticle attraction.

      

               However, after the Periodic Chalk Mixture has been left to stand for a period of time, the sediment start to form and the height of sediment increase as the time increase. This is because particles in a suspension will settle out if the suspension is allowed to stand undisturbed. Particles in a suspension are larger than those in the solution, and therefore will settle out if left to stand. These particles can be redispersed when it is shaken and the sediment formed does not show decreased in  height. This is due to the present of suspending agent. The addition of the tragacanth, at some critical concentration, negates the surface charge on the suspended particles and allows the formation of floccules or clusters as particles are held loosely together by weak van der Waals forces. Since the particles are linked together only loosely, they will not cake and may be easily redispersed by shaking the suspension.

3.  Plot the graph of height of solid phase against time for different amount of tragacanth in the suspension. Give your comment.

    

 For the Pediatric Chalk Mixture type II, III, IV using the Tragacanth powder, sedimentation generally does not occur at the beginning due to the presence of Tragacanth powder as a suspending agent. However, the sedimentation starts and the height of sediment increases with time. This is due to the inter–particular attractive forces are stronger than the repulsive forces of the Tragacanth powder on the chalk particles. Therefore, the sedimentation formed increases with time until a maximum sedimentation level.

     The Pediatric Chalk Mixture type II, III, IV contains 0.1g, 0.3g, and 0.5g of Tragacanth powder respectively. Based on theory, the more the amount of Tragacanth powder present, the lower the sediment formed and the time needed for sedimentation to occur is longer. This is due to the sediment formed in the suspension containing higher amount of Tragacanth powder is more stable and less flocculation occur.   They need more time for sedimentation to occur compared to suspension containing less amount of Tragacanth powder. Therefore, the amount of Tragacanth powder in the formulation will influence the height of sediment formed and the time for sedimentation of suspension to occur.

But based on the result we observed, there is increase in volume of sedimentation when increase amount of tragacanth. This is maybe due to some errors occur during experiment. We having a problem during observed and measured the height of sediment. This is because it is quite difficult for us to observe the sedimentation height. So, maybe there is error during observation and reading the height of sediment.

4.      Explain briefly the mechanism of viscometer analysis. Plot a graph of

     viscosity of the suspension versus the amount of Tragacanth (g). Give explanation.

Groups	A1	A2	A3	A4
Amount of tragacanth (g)	0.0	0.1	0.3	0.5
Viscosity (cP)(average +SD)	1.83 + 0.55278	5+0	6+4.89898	17.67+1.24722

The motor of the viscometer spin with speed of 300rpm which is fixed by the control centre. There are discs which are connected with the spiral spring at angle more than 180° used to spin the stirrer. The change of the spin angle is directly proportional to the viscosity of the fluid and the rate of spin. The infrared radiation from LED passes through the discs before detected by the photo diod. The change of the angle of spinning disc will affect the infrared radiation and will be detected by the photo diod.

       The viscosity of the suspension is 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. As the amount of Tragacanth powder increases, the suspension formed will be more viscous.

A suspending agent will slow down the sedimentation rate of suspension by increasing the viscosity of the fluid medium. This agent follows the Stokes law which states that the settling velocities of small particles in a fluid medium are resisted by the viscosity of the liquid. Moreover, suspending agents will form film around the insoluble solid particles and decrease the surface tension. Based on the results of the experiment, when we increase the amount of tragacanth used, the viscosity of the suspension will increase. As our group used the highest amount of tragacanth, our suspension has the highest reading of the viscometer.

          Tragacanth solution is viscous in nature. It is a better thickening agent than acacia.It also  provides thixotrophy ability to the solution. Thixotrophy is one of the properties of a well-formulated suspension is that it can be easily resuspended by the use of average shear force. When agitation is applied the viscosity is reduced and provide good flow characteristic from the mouth of the container.

4.  Plot a graph of sediment height ratio after centrifugation versus the amount of Tragacanth (g). Give explanation.

Group	1	2	3	4
Height ratio (mm)	80 : 7	80 : 12	80 : 15	80 : 20

Suspension I

X = 7 / 80

    = 0.0875

Suspension  II

X = 12 / 80

    = 0.15

Suspension  III

X = 15 / 80

= 0.1875

Suspension  IV

X = 20 / 80

= 0.25

When tabulate the data:

Group	1	2	3	4
Height ratio (mm)	0.0875	0.1500	0.1875	0.2500

     

                

         By theory, the more the amount of Tragacanth powder present in the formulation, the lower is the sediment formed. Therefore the height ratio should be lower for those containing higher amount of Tragacanth powder. The Tragacanth powder acts as a suspending agent that suspends the solid phase (chalk) in the liquid phase to form a stable suspension. With the addition of the large amount of Tragacanth powder in the formulation, it gives a more stable suspension and less flocculation will formed compared to those containing less amount of Tragacanth powder.

       However, the experimental results show that the amount of sediment increases with the amount of Tragacanth powder present in the formulation except for the mixture IV. This may be due to the experimental errors that occur during preparing the suspension. The amount of chalk to be suspended maybe not accurately measured. This will affect the height of sediment. Also, the amount of the suspending agent, Tragacanth powder may affect the results if not accurately measured.

5.  What is the function of each ingredient used in the suspension preparation? How does the different amount of Tragacanth powder affect the physical characteristics and stability of the suspension formulation?

Chalk is an indiffussible solid that is suspended in the Pediatric Chalk Mixture. The Tragacanth powder acts as a suspending agent to suspend and stabilise the chalk in the suspension. The concentrated peppermint water is the carminative agent and flavouring agent. Syrup BP increases the viscosity of the suspension and acts as a sweetening agent. Double strength chloroform water acts as the flavouring agent, preservative and solvent. Distilled water is the solvent.

      The Tragacanth powder acts as a suspending agent that suspends the solid phase (chalk) in the liquid phase to form a stable and uniform suspension where no two different layers of ingredients present. By using the Tragacanth powder, the suspension will be looked cloudy, viscous and smooth. If higher amount of Tragacanth powder is used, less sedimentation will occur and it will take longer time to occur. The suspension containing large amount of Tragacanth powder is more stable and less flocculation will occur or occur after longer duration of time compared to those having smaller amount of Tragacanth powder.

Conclusion:

 The amount of different Tragacanth powder will affect the rate of sedimentation and the height of sediment formed in the suspension.

 Reference:

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

2.      http://global.britannica.com/EBchecked/topic/567002/Stokess-law

3.      http://formulation.vinensia.com/2011/12/list-and-characteristic-of-suspending.html

Appendices

Suspension from Group 1A to 4A