TF 1 Lab - Report on Polarized Microscope

Introduction

The polarizing microscope is also called as petrographic microscope. It is mainly used in geological studies but also in medicine and biology.  Polarized microscope used a polarized light that the light waves will vibrate in one direction. However for normal microscope, light vibrate in random directions. Polarized light microscope can analyse structures that are birefringent; structures that have two different refractive indices at right angles to one another. This microscope containing a polarizer and analyzer, a circular rotating stage, special plates placed between the object and light path and the Bertrand lens (if necessary).

It is the best choice to study materials like minerals, polymers, ceramics, wood, urea, substances of natural and synthetic fibers with those birefringent properties, cellophane, and also botanical and insect specimens and fish scales. But with polarizing microscopy, it is possible to determine the color absorption, structure, composition and refraction of light in isotropic (gases and liquids – one refractive index) and anisotropic substances.

To study the pathway of the light, actually The light passes through a polarizing filter called the polarizer (the polarizer is fixed in an east to west vibrational way, but it can be rotated if necessary. There is one more polarizing filter called the analyzer. It is usually situated above the objectives and can be moved in and out of the optical path). Passes through the birefringent specimen. The polarizer is usually fixed in an east to west vibrational direction, but it can be rotated as required. There is one more polarizing filter called the analyzer. It is usually situated above the objectives and can be moved in and out of the optical path.

Objective

1)      To study the used of polarized microscope.

2)      To measure the diameter of particle or droplet under the polarized microscope.

3)      To examined the pharmaceutical formulations using a polarized microscope.

Materials

Gaviscon suspension

Scott’s emulsion cod liver oil

Microscope slides and cover slips

Application sticks

Polarizer

Procedure

1)      A thin layer of Gaviscon suspension was spread on a clean and dry microscope slide by using an application stick.

2)      The sample was covered with a coverslip gently.

3)      The suspension was observed and gave a detail microscopic description under bright light at 10x.

4)      Snapshot was taken under a bright light and the average particle sizes was measured. An average of 10 readings was taken from different snapshots.

5)      The polarizer was placed on the top of the light source.

6)      The polarizer was turned until a crossed polar field was obtained. The suspension was observed under the polarized light at 10x and the microscopic description was given.

7)      Step 1 - 6 was repeated with Scott liver emulsion.

Results

1.      Images of suspension and emulsion under bright field at Mag x10.

1.      Images of suspension and emulsion under polarised field at Mag x10.

3.  Microscopic description of suspension and emulsion under bright and polarized light.

For suspension, we could see clearly the shape of the crystal. Each particle is not equal in size. Under polarized light, the crystals look like stars with variety of shape. In addition, the particle has uneven distribution. The characteristics of the crystals look like rocks with rough surface.

For emulsion, we could see clearly the oil in water emulsion. The bubble form is oil while the surrounding is water. There is no overlapping between the oil and water. The emulsion is evenly distributed and has variety of size. Basically the oil has round shape.                                                                                                                                                                                                                 4. Measure the particle/droplet size (diameter). Take an average of at least 10 readings from different snapshots.

Reading (µm)	Gaviscon Suspension	Scott’s Emulsion
1	24.1	45.7
2	45.8	60.0
3	55.2	116.4
4	39.0	51.6
5	49.2	49.9
6	32.8	44.8
7	21.1	27.4
8	56.9	73.2
9	43.2	40.8
10	26.7	56.1
Average	39.4	56.6
SD	12.17	22.98

DISCUSSION

 Plane polarised light is a term used to describe the polarisation state of the source light used in polarising microscopes. Polarised light is light that vibrates in a single direction due to its passage through a polariser. In a polarising microscope the lower polariser is usually orientated so that light vibrates parallel to the E-W direction. If the upper polariser (the analyser), is not inserted the view is said to be under plane polarised light, often abbreviated to PPL.

1.      Polarizing light microscopes employ two polarizers, set at 90 degrees to each other. Two polarizers in this configuration will allow virtually no light to pass through. If a sample is placed between these two polarizers, then certain properties will become apparent if the material changes the rotation of the light. Thus, polarized light leaving one polarizer strikes the object, and is rotated; only if it is rotated can it pass through the second polarizer. 

These techniques are useful for any compounds that rotate light; typical uses include examining crystals, including minerals.

A normal or binocular microscope is any microscope with two eyepieces; a polarizing light microscope may be a binocular scope (many are), but most binocular scopes do not have polarizing light microscopy ability. A good polarizing scope will run several thousand dollars. The easiest way to pick between the two is that the polarizing scope will almost always have a rotating stage so the sample may be rotated. Similarly, because of the polarizers, a polarizing scope will have no light visible through the eyepiece when both polarizers are in place. 

Note that when some people refer to a binocular scope, they actually mean stereo scope. Stereo scopes have two separate optical paths, so the object appears to be in three dimensions.

2.      While it is not essential that molecules pack in an orderly crystalline environment, it is usually observed that drug substances do so. The crystalline environment provides a thermodynamically more favorable arrangement than does a disorderly “amorphous” form and has a higher, more efficient, density packing of molecules. As a direct result, crystalline arrangements of molecules typically give rise to more chemically stable drug substances, are less hygroscopic and give products that have better flow properties allowing for a more readily processed and formulated product. The simplest and most cost effective analytical technique used in pharmaceutical development is polarized light microscopy (PLM). PLM can be used to determine many physical properties of pharmaceutical compounds and plays critical role in most laboratories due to its simplicity of use and the expediency with which information can be gained. Two important qualities that are instantly observed when one examines a solid are the presence of birefringence under cross polarization indicates that the substances is crystalline, while crystal habit provides insight as to how well a material might process.

3.      The advantages of a microscope in determining droplet and particle size in pharmaceutical formulations are it is easier to determine the size and we can get accurate result in correct manner. Besides, we can differentiate between the particles and the foreign substances with the presence of plane polarized light. Moreover we can see and determine the crystal shape of particles and also look at the distribution of particles in a solution or suspension. However, the disadvantages are sometimes human error can occur if we wrongly look or measure the particles. Besides, we must also measure a lot of particles to get the mean of distribution and the particles size. Hence, it is a little bit hard because we must find the average measurement of particle sizes.  

CONCLUSION

Based on the objectives, we are able to understand the used of the polarized microscope, measure the diameter of particle or droplet under the polarized microscope, and to examine the pharmaceutical formulations using a polarized microscope. From our results, it seems that the average diameter of Gaviscon suspension is smaller than the Scott’s emulsion. The readings for Gaviscon suspension is 39.4 µm while Scott’s emulsion is 56.6 µm.  Thus, the standard deviation for  Gaviscon suspension is 12.17 while Scott’s emulsion 22.98. The images of suspension and under polarised field at Mag x10 shows shining things. Those images are differs between under bright microscope and polarized microscope.

Lab Report - Particle Size Analysis (Metasizer and Zetasizer)

INTRODUCTION
Particle size analysis is a laboratory technique to determine the size range, average or mean size of
particle in a sample. Eg, in powder or liquid.

This can be perform with a variety of techniques or methods. Particle size analysis methods can be categorized based on several different criteria :-
-Size range of analysis
-Wet or dry methods
-Manual or automatic methods
-Speed of analysis

Example of particle size analysis methods in:-
-Sieve method
-Microscope method
-Coulter counter
-Laser light scattering method
-Dynamic light scattering method
-Sedimentation method

OBJECTIVE
-To able to use the instruments to analyse particle size.
-Able to use different methods of particle size analysis to determine particle size and size distribution of different products.
-Able to analyse and interpret the data obtained in the experiment.
-To determine the best method to measure particle size for each product.

MATERIALS 

Gaviscon Suspension (Sample B)

Cod Liver Oil emulsion (Sample C)

Polystyrene nanoparticles from Malvern, U.K. (Sample D)

Distilled water

METHODS 
1.  The Malvern Zetasizer or Mastersizer  was powered up.

      2.  The software was launched.

      3. The cell was loaded and the measurement was run.  

      4.The results were viewed.

      5. The cell was removed from the machine.

      6. The cell washed

RESULTS

1.  Fill in the table below:

No.	Particle size, µm			Size Distribution
	Cod Liver Oil®	Gaviscon®	Polystyrene nanoparticles	Cod Liver Oil ®	Gaviscon®	Polystyrene nanoparticles
1.	21.105	35.969	105.9	0.645	1.01	0.024

2. Draw the shape of peak(s) obtained for each sample. Give your comments.

Comments:

            As the three samples were analyzed using MalverZetasizer and Metasizer, the result show that the largest particle size is the polystyrene nanoparticles which is the value is 105.9µm. while the smallest particle size is the Cod Liver Oil suspension with 21.105µm.

            In size distribution parameter, the largest value belongs to the Gaviscon suspension with 1.01. While polystyrene nanoparticles has the smallest size distribution with 0.024.

DISCUSSIONS 

1. Describe TWO (2) parameters which should be provided when using Malvern Zetasizer® and         Mastersizer®.

Two parameters that should be provided when using Malvern Zetasizer and Mastersizer are particle size and size distribution. Particles size is important because it will influence stability of suspension, reactivity or dissolution rate and viscosity. While size distribution is important because in light scattering method it will give volume weight distribution. Volume of particle will be contributed by each of the particle which is equivalence to the mass if the density uniform. Thus, relative contribution will be proportional to size of distribution.

2. Discuss the appropriateness of using the above methods to analyse the samples.

The Mastersizeruses the technique of laser diffraction to measure the size of particles. It does this by measuring the intensity of light scattered as a laser beam passes through a dispersed particulate sample. This data is then analyzed to calculate the size of the particles that created the scattering pattern.The method can be used on all powders containing less than 10% fat.A typical system is made up of three main elements which are optical bench, sample dispersion units and instrument software.For the optical bench, a dispersed sample passes though the measurement area of the optical bench, where a laser beam illuminates the particles. A series of detectors then accurately measure the intensity of light scattered by the particles within the sample over a wide range of angles.Then for the sample dispersion units (accessories).Sample dispersion is controlled by a range of wet and dry dispersion units. These ensure the particles are delivered to the measurement area of the optical bench at the correct concentration and in a suitable, stable state of dispersion. Third one is Instrument software which controls the system during the measurement process and analyzes the scattering data to calculate a particle size distribution.

3. Suggest another method to measure particle size, if both methods are not suitable to analyse particle size any of the samples.

Another method to measure particle size is by using microscope. Primary particles are vacuum dispersed in air or liquid on object glass prior to image analysis and magnified up to *1000. Microscopy and digital image analysis can be used for Determination of particle size distribution.Particle Analytical applies  microscopes equipped with objectives for 40 to 1000 times magnification and a macroscope with objectives for 3.5 to 90 times magnification. A digital camera enables live recording of images. The images are processed with Image-Pro Plus software from MediaCybernetics. The measurement of the particles is thus operator independent and reproducible. All images are stored for complete documentation. The primary particles are dispersed in liquid or in air by vacuum dispersion on object glass prior to image analysis.

4. Explain the principle of Malvern Mastersizer® and Zetasizer® operation on measurement of particle size. (Please include the Frounhofer and Mie Theory).

     Laser diffraction measures particle size distributions by measuring the angular variation in intensity of light scattered as a laser beam passes through a dispersed particulate sample. Large particles scatter light at small angles relative to the laser beam and small particles scatter light at large angles. The angular scattering intensity data is then analyzed to calculate the size of the particles responsible for creating the scattering pattern, using the Mie theory of light scattering. The particle size is reported as a volume equivalent sphere diameter.

5. Describe the advantages of using the above methods in analysing particle size.

Malvern Mastersizer uses laser light scattering method. Advantage of using this method are:-

•   It has a wide and dynamic range in measuring particle size, between approximately 0.1 and 2000 microns.
•   This technique allows for the whole sample to be measured by analyzing the diffraction of light when it pass through every particle in the sample.
•   Results can be obtain in a very short time.
•   Technique is repeatable so to obtain the most accurate measurements.

On the other hand, Zetasizer uses dynamic light scattering method. The advantage of this method includes:-

• Provide an accurate, reliable and repeatable particle size analysis in a short time
• Simple or no sample preparation, high concentration, turbid samples can be measured directly.
• Simple set up and fully automated measurement.
• Size measurement of sizes < 1nm.

CONCLUSION

Based on the objectives, we are able to know how to use instruments in analyzing particle size and also use different methods of particle size analysis to determine particle size and size distribution of different products. From the results, we are able to interpret the data obtained.