Particle size and shape analysis using microscope

Objective

• To analyse the particle shape and size under the microscope.
• To describe the distribution particle size and shape.

Date of Experiment

19 November 2015

Introduction

The dimension of the particulate solids are very important in order to achieve optimum production of efficacious medicines .The particle size of drugs is determined when the drug is synthesized and formulated .In the case ,particle size will influence the subsequent physical performance of medicines and pharmacological of the drugs There are many methods for the analysis of particle size and shape .In this case , analysis using microscope is one of the effective ways to determine the particle size and shape .It is an excellent technique because we can look at the particle directly and is relatively cheap .One of the disadvantages of using microscope for analysis the shape and size of particle is the elaborate sample preparation is slow . Moreover, it is not suitable for quality control.

Apparatus 

Microscope, weighing boat

Materials

sand with particle size of (150 mic, 355 mic, 500 mic, 850mic), lactose, sand with various sizes and MCC.

Procedure

1.     Sands with sizes of (150 mic, 355 mic, 500 mic, 850mic), sand with various sizes, lactose and MCC are placed in the different weighing boat by using spatula. The weighing boat were labeled according to the content.

2.     The microscope was set up and ready to be use.

3.     150 mic sand scattered on the glass slide and covered with the cover slip.

4.     The sand was observed under the microscope using 4x10 magnification.

5.     The particles were observed microscopically and the shape was determined.

6.     Steps 3 to 5 were repeated by using (355 mic, 500 mic, 850mic), lactose ,sand with various sizes and MCC.

Results

Question

1.Explain in brief the various statistical method that u can use to measure the diameter of a particle.

The first method is the Feret’s diameter.The Feret diameter or Feret's diameter is a measure of an object size along a specified direction.  It can be defined as the distance between the two parallel planes restricting the object perpendicular to that direction generally. Therefore ,it is also called the caliper diameter, referring to the measurement of the object size with a caliper. This measure is used in the analysis of particle sizes, for example in microscopy, where it is applied to projections of a three-dimensional (3D) object on a 2D plane. In such cases, the Feret diameter is defined as the distance between two parallel tangential lines rather than planes.

Moreover ,Martin diameter can be one of the method to determine the diameter of a particle .It is a specific method for measuring the diameter of irregular shaped particles, Martin’s diameter is the measured distance between opposite sides of a particle, and is measured transverse to the particle on a line that bisects the projected area. In essence, Martin’s diameter measures the chord of a particle and is useful for estimating the surface area of an irregular non-spherical particle.

Besides ,projected area can be one of the methods to measure the diameter of a particle .The projected area diameter is the diameter of a sphere having the same projected area as the particle. Projected area is two-dimensional area measurement of a three-dimensional object by projecting its shape on to an arbitrary plane. Besides, another useful method is the projected perimeter diameter which is based on the circle having the same perimeter as the particle. Both of these methods are independent upon particle orientation. They only take into account of 2 dimensions of the particle, thus inaccurate for unsymmetrical particle.

Lastly ,we can use Fourier analysis to determine the diameter of a particle. It provides an accurate quantification of particle morphology and texture. They describe the overall shape of soil particles which is known as morphology . A summary of higher order descriptors provides textural information which is related to local roughness features which is defined as texture.

2.State the best statistical method for each of the samples that you have analysed.

Martin's diameter and Feret's diameter of a particle depend on the particle orientation under which the measurement is made. Thus, obtaining a statistically significant measurement for these diameters requires a large number of randomly sampled particles which are measured in an arbitrarily fixed orientation. Since Martin's diameter, Feret's diameter, and projected area diameter are based on the two-dimensional image of the particles, they are generally used in optical and electron microscopy. 

 Discussion

Nowadays, pharmaceutical manufacture of products quality is more dependent on the particle size control as this is becoming increasingly apparent in the pharmaceutical industry. Precise particle size control technologies have also assisted in the development of drug delivery platforms for the delivery of a medicament to various part of the body.

In order to determine the particle size, several methods can be used such as microscopy, sieving, sedimentation techniques and etc. Besides, we can further analyze the particle size by using Feret’s diameter or Martin’s diameter. Martin’s diameter refers to the length of the line which bisects the particle image, while Feret’s diameter refers to the distance between two tangents on opposite sides of the particle, parallel to some fixed direction. This is because the Feret’s and Martin’s diameter is the best statistical method in which both of them use statistical diameter which are the average over many different orientations to produce a mean value for each particle diameter.because both of them use statistical diameter which are the average over many different orientations to produce a mean value for each particle diameter.These two methods consider the orientation of particles, hence this increase the accuracy of the results obtained.

Microscope is used in this experiment to examine the various particle size and shapes of sands and powder because the microscope magnification are sufficient enough to allow adequate characterization of small particles. Error can be minimised if magnification and the light is sufficient that the image of particles produced is sharp and clear. Whereby, we can examine each particle individually by observing the 2D shape, colour, etc. However, we have to know that we unable to obtain a 3-Dimensional orientation of particle size and shape through a microscope.

In this experiment, we had analyze 5 different types of sands and powders (MCC, lactose, 150 mc, 355 mc, 500 mc, 850 mc, various size ) by using a microscope. The particles which are observed microscopically were sketched and the general shape for each particle were determined. We found that the samples that we observed are varies in term of their shape and size. They can be characterized by the range of from low sphericity to high sphericity, from very angular, angular, sub-angular, sub-rounded, rounded and well-rounded. For instance, the size of 850mc sand is larger than 150mc sand as we can compare of the image of sample we got in which both are irregular shape and asymmetry.

The magnification which we used during this experiment set to be constant for all 6 sample which is 4x10 magnification. This is important so that we can compare the size and shape of the particle clearly and easily. Besides, we have to make sure that the sample particles is well-spread throughout the slide and dispersed evenly until it is a thin layer before observing it under a microscope. It is to avoid the agglomeration formed and ensure the image of specimen we obtain is accurate and clear in term of their shape and size.

There were several precaution steps taken in this experiment such as make sure that the fan is switched off and the sand granules and powder were carefully handled so that it will not spread all over the table and to ensure cleanliness of work place. Moreover, we are advised to wear goggles and mask to prevent the sand and powder gets into our eyes and to protect ourselves safety.

Conclusion

As from the observation of the image obtained from microscope by using a fix magnification, we can conclude that the shape and size of the sands and powder are different. All particles are varies in their size and have an asymmetrical and irregular shape.  

References

1. Physicochemical Principals of Pharmacy (2nd Edition) AT Florence and D.Attwood, The Macmillan Press Ltd.

2. Michael E.Aulton, 2007, Aulton's Pharmaceutics The Design And Manufacture Of    Medicines, Third Edition, Churchill Livingstone Elsevier (page 122-134)

3. Pharmaceutics, The science of dosage form design (2nd Edition) Michael E.Alton Edinburgh London New York Philadophia St Louis Sydney Toronto 2002.

Sieving

Objectives

• To determine particle size distribution of powder and the size of solid particle of  lactose and microcrystalline cellulose (MCC) by sieve nest.
• To determine the size of particles.  

Date of Experiment

19 November 2015

Introduction

Sieves are commonly used to break down agglomerates, and determine the size and size distribution of a particular powder. A sieve nest as diagram below is used to determine the particle size and the size distribution of the powders.

Apparatus And Materials

Lactose, microcrystalline cellulose (MCC), weighing machine, stack of sieves, mechanical sieve shaker

Procedure

1. 100g of lactose is weighed by using weighing machine.
2. The sieve nest in descending order (largest diameter to the smallest, from top to bottom) is prepared.
3. Lactose is poured onto the top of sieving nest.
4. The sieving machine is run for 10 minutes.
5. The weights of different sizes of lactose are weighed after the sieving process finished and a histogram is plotted for the distribution of size particle of lactose.
   
   
6. Step 1-5 are repeated for MCC.

Results

Lactose

Sieve diameter ( µm)	particle size range (µm)	Mass of lactose retained in each sieve (g)	Percentage of lactose retained (%)	Cumulative percentage retained (%)
<53	0 < x ≤ 53	8.139	8.139	8.139
53	53 < x ≤ 150	76.4572	76.457	84.596
150	150 < x ≤ 212	0.331	0.331	84.927
212	212 < x ≤ 300	12.7149	12.715	97.642
300	300 < x ≤ 500	0.0227	0.023	97.665
500	x > 500	1.412	1.412	99.077

MCC

Sieve diameter (µm)	Particle size range (µm)	Mass of microcrystalline cellulose (MCC)retained ineach sieve(g)	Percentage of microcrystalline cellulose (MCC)retained (%)	Cumulative percentage retained (%)
<50	0 < x ≤ 50	0.8598	0.8706	0.8706
50	50 < x ≤ 150	93.4919	94.6696	95.5406
150	150 < x ≤300	4.3167	4.3711	99.9113
300	300 < x ≤ 425	0.078	0.079	99.9903
425	x > 425	0.0096	0.0097	100

Questions

1. What are the average particle size for both the lactose and MCC?

          For Lactose,the total mass is 99.0768 g.

          Average particle size: total mass/6          

                                            : 99.0768/6

                                            : 16.5128 g

          For MCC, the total mass is 98.7560 g

          Average particle size: total mass/5

                                            : 98.7560/5

                                            : 19.7512 g

    2. What other method can you use to determine the size of particle ?

    i.            Laser Diffraction

Laser diffraction is the one of the most widely used particle sizing techniques and has become the standard method in many industries for characterisation and control. This type of particle size analyser relies on the fact that particles passing through a laser beam will scatter light at an angle that is directly related to their size. When particle size decreases, the observed scattering angle increases logarithmically. Scattering intensity is also subject to particle size, diminishing with particle volume. What this means is that large particles scatter light at narrow angles with high intensity while small particles scatter at wider angles with low intensity.

Laser diffraction has a wide dynamic range, from 0.2 to 2000 microns and is very fast and reliable. It is also very flexible as it can be applied to dry powders, aerosols and emulsions. In addition, laser diffraction does not require calibration but can be easily verified.

  ii.            Dynamic Light Scattering

Sometimes referred to as Photon Correlation Spectroscopy or Quasi-Elastic Light Scattering, this method is a non-invasive and sensitive technique used for measuring the size of molecules and particles in the submicron region. The results are expressed as particle hydrodynamic diameter. Dynamic light scattering is an accurate, reliable and repeatable technique.

iii.            Sedimentation

This is a traditional method widely used in the paint and ceramics industries. Equipment as simple as the Andreason pipette or as complex as centrifuges and X-rays can be used in this method. The main advantage of this technique is that it determines particle size as a function of settling viscosity. However, as the density of the material is needed, this method is no good for emulsions where the material does not settle or for dense material that settles too quickly. It is also based on spherical particles, so can give large errors for particles large aspect ratio.

 iv.            Image Analysis

This technology generates data by capturing direct images of each particle, providing users with the ultimate sensitivity and resolution. Image analysis systems are capable of high-resolution sizing ranging from 0.5µm – 1000µm. Subtle differences in particle size and shape can be accurately characterised using this method.

   v.            Acoustic Spectroscopy

Instead of using light, this technique employs ultrasound for collecting information on the particles that are dispersed in fluid. This can be done because dispersed particles absorb and scatter sound waves similarly to light. Acoustic spectroscopy can be used to measure particle size distribution for any particle in a fluid system and can measure at very high particle concentrations.

3. What are the importance of particle size in a pharmaceutical formulation?

The particle size distribution of active ingredients and excipients is an important physical characteristic of the materials used to create pharmaceutical products. The size, distribution and shape of the particles can affect bulk properties, product performance, accessibility, stability and appearance of the end product.

The link between particle size and product performance is well documented with regards to dissolution, absorption rates and content uniformity. Reducing particle size can aid the formulation of  normal curve equivalent’s with poor water solubility. Proper matching of active ingredient and excipient particle size is important for several process steps. Particle size analysis is an integral component of the effort to formulate and manufacture many pharmaceutical dosage forms.

Discussion

A sieve analysis is a practice or procedure used to assess the particle size distribution of a granular material. The size distribution is often of critical importance to the way the material performs in use. A sieve analysis can be performed on any type of non-organic or organic granular materials including sands, crushed rock, clays, granite, feldspars, coal, soil a wide range of manufactured powders, grain and seeds, down to a minimum size depending on the exact method. Being such a simple technique of particle sizing, it is probably the most common.

Sieve nest was prepared in descending order, from the largest diameter to smallest. In this experiment, 100 g of lactose or microcrystalline cellulose (MCC)was placed on uppermost sieve and sieving process was started. After 10 minutes, the sieving nest was removed and powder from each sieve was measured. The particle size of lactose and MCC is measured based on the principle that the particles cannot pass through certain sieve sizes due to the particle size larger than the sieve diameter.

From the result above, the particle size of lactose is estimated to be between 150µm to 300µm as the highest amount of lactose powder is retained at the sieve with diameter of 150µm. The particle size of MCC is estimated between 50µm to 150µm as the highest amount of MCC powder is retained at the sieve with diameter 50µm. Thus, we can draw a conclusion that lactose has bigger and more uneven particle size compared to MCC.

There are a few errors made in the experiment. There is loss of weight of lactose and MCC powder after sieving. For example, the initial weight of lactose before sieving is 100g, has reduced to 99.7475g after sieving and the initial weight of MCC before sieving is 100g, has reduced to 98.7560g. This may be due to some powders are blown out during the sieving process as the powders are light, some powders may stick to the sieve after the sieving process, small amount of powders may spilled out from the container when we moved it from one place to another place and the powders may not be completely removed from the sieves. Besides, the sieve net may be contaminated by other powders. Some powders are exposed to the air then absorb moistures and clump together. This affects the particle size distribution of the powders.

In order to reduce errors, a few precautions should be taken. First, when the sieve nest machine is operating, make sure the sieves are closed tightly so that the powders do not fly away or spill because of the high vibration of the machine. Besides, before conducting the experiment, the sieve net should be cleaned and dried to prevent contamination that will affect the result. We have to ensure we completely remove all the powder remained in each of the sieves. Last, the powders should be quickly poured into the sieve to minimize exposure to the air.

Conclusion

In this experiment,particle size distribution of powder and the size of solid particle of  lactose and microcrystalline cellulose (MCC) by sieve nest successfully determined.The size of particle is also determined.

References

• Jillavenkatesa A, Dapkunas S J, Lin-Sien Lum. 2001. Particle Size Characterization, NIST Special Publication
• Martin,A.N. 2006. Physical Pharmacy: Physical Chemistry Principles in Pharmaceutical Sciences. 5th Edition. Philadelphia: Lea & Febiger

Practical 3: Phase Diagrams (Part B) - Mutual solubility curve for phenol and water

Objective

·         To determine the mutual solubility for phenol and water

·         To determine the relationship between the temperature and solubility of the liquids.

·         To determine the critical solution temperature for phenol and water

 Date of Experiment

3 November 2015

Introduction

There are certain liquid pairs which are partially miscible. For example, if a small quantity of phenol is added into the water at ordinary temperature, it will dissolve in water completely. As the amount of phenol is increased, a stage is reached when no more phenol dissolves and two liquid layers are formed. The upper layer is a saturated solution of phenol in water while the lower layer is a saturated solution of water in phenol. These two solutions in equilibrium with each other are called conjugate solutions.

           At constant temperature, composition of the layers although different from each other, remains constant as long as two phases are present. Addition of small amount of phenol or water merely changes the relative volumes of the two layers and not their composition. As the temperature is raised, mutual solubility of the two liquid increases, until at a certain temperature the two liquids become completely miscible. This temperature is known as the mutual solubility temperature (MST).

            Generally, both liquids become more soluble with rising temperature until the critical solution temperature or consolute point is attained, and above this point the liquids become completely miscible. There is a big possibility that any  pair of liquids can form a closed system, whereby both upper and lower critical solution temperature exist, however it is not easy to determine both the temperature except for nicotine and water. At any temperature below the critical solution temperature, the composition for two layers of liquids in equilibrium state is constant and does not depend on the relative amount of these two phases.

Apparatus And Materials

Measuring cylinder, thermometer, beaker, boiling tube, distilled water, phenol, water bath

Procedure

1. In a tube, certain amounts of phenol and water were prepared.

Volume of Water (mL)	18.4	15.0	12.0	7.0	4.0
Volume of Phenol (mL)	1.6	5.0	8.0	13.0	16.0

     2. Each of the tubes was heated in a water bath. Remember, to always stir the tubes.

     3. The temperature for each of the tube at which the turbid liquid becomes clear was observed and          recorded

4. Then, it was removed from the hot water and allowed the temperature to reduce gradually. The temperature at which the liquid becomes turbid and two layers are separated was recorded. The cold water is used when it is needed.

     5. The average temperature for each tube at which two phases are no longer seen or at which two             phases exist was determined.

Results:

Test Tube	Phenol composition (%)	Volume of phenol (ml)	Volume of water (ml)	Temperature of solution when 1 layer is formed (°C)	Temperature of solution when 2 layers are formed (°C)	Average Temperature (°C)
A	8	1.6	18.4	50.0	46.0	48.0
B	25	5.0	15.0	62.0	60.0	61.0
C	40	8.0	12.0	68.0	65.0	66.5
D	65	13.0	7.0	65.0	64.0	64.5
E	80	16.0	4.0	54.0	54.0	54.0

Questions:

1. Plot the graph of phenol composition (horizontal axis) in the different mixtures against temperature at complete miscibility. Determine the critical solution temperature.

Phenol Composition, %

Critical solution temperature of water-phenol system is 66.5 ^oC.

2. Discuss the diagrams with reference to the phase rule.

The graph above in the results shows the graph of temperature at complete miscibility of solution against percentage of phenol composition in the solution. The region outside the curve shows that the solution is in complete miscibility and has only one phase, whereas the region inside the curve indicate the two phase system of the solutions. According to the phase rule,
F=C-P+2

F is degree of freedom

C is numbers of component

P is number of phase exist

F=2-1+2, thus the degree of freedom for this system is 3. This show that 3 intensive variable must be fixed in order to describe the system completely. As the pressure is fixed, F reduces to 2, and it is necessary to fix both temperature and concentration of phenol in the solution to define the system.

3. Explain the effect of adding foreign substances and show the importance of this effect in pharmacy.

The addition of foreign substances to binary system will results in ternary system. If the foreign substance is soluble only in one component, the mutual solubility will decrease. Thus, temperature at which the system becomes homogeneous is increased due to the salting out of water. However, if the foreign substance is soluble in both liquids, the solution will become soluble. The mutual solubility will increase. The critical solution temperature is lowered due to negative salting out effect. The effect of adding foreign substances is important to the industrial production of highly concentrated solutions of tar acids (phenols and cresols) used as disinfectants. Besides, the solubility of the substance is used to determine the purity of the substance.

Discussion

Phase rule is a useful device for relating the effect of the least number of independent variables like temperature, pressure and concentration upon the various phases (solid, liquid and gaseous) that can exist in an equilibrium system containing a given number of components. Phase rule can be expressed as F=C-P+2 where F is the number of degrees of freedom in the system, C is the number of components and P is the number of phases present. We may define a phase as a homogenous, physically distinct portion of a system that is separated from other portions of the system by bounding surfaces. The number of degrees of freedom is the least number of intensive variables (temperature, pressure, concentration, refractive index, density, viscosity, etc). When a two-component condensed system having one liquid phase, F=3 because F=2-1+2. However, the pressure is fixed so F is reduced to 2, hence we have to fix both temperature and concentration to define the system. When two liquid phases are present, F=2 because 2-1+2=2, but F is reduced to 1 as pressure is fixed. Hence, only temperature is needed to define the system.

           

Phenol-water system exhibit partial miscibility. The curve shows limits of temperature and concentration within two phases. The region outside the curve contain systems having one liquid phase whereas region inside the curve contain systems having two liquid phases. At point a, the system contains 100% water. Increasing percentage by weight of phenol in water at 50 ͦC will result in forming two liquid phases until the total concentration of phenol exceeds 63 ͦC at that temperature, and a single phenol-rich liquid phase is formed. The maximum temperature at which two phases region exists is termed as critical solution temperature. From the curve, the critical solution temperature is 66.8 ͦC, whereby any combinations of phenol and water above this temperature are completely miscible and yield only a single liquid phase.   

In order to improve the accuracy of the results, some precaution should be taken in this experiment. When we sealed the tubes, we have to ensure that all the tubes are tightly sealed to prevent evaporation of phenol once the phenol is mixed with water. Evaporation of phenol will affect the result of this experiment. Since phenol is a carcinogenic compound, extra caution and care need to be taken. The results show a deviation of critical solution temperature. This may be due to the evaporation of some of the phenol.  The result is not accurate is also caused by the slightly changes in time taken to observe the temperature. The temperature may not be taken at the exact time when two phases exist or two phases are no longer seen.

Conclusion
The critical solution temperature is 66.5ºC. The plotting of mutual solubility curve of water-phenol system is achieved.

References
1.         Martin's Physical Pharmacy and Pharmaceutics 6th Edition

2.          A. S. Negi, S. C. Anand. 2004. A Textbook of Physical Chemistry.