Polarized Microscope Lab Report

TITLE :

Examination of Pharmaceutical Formulations Using A Polarised Microscope

OBJECTIVES :

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.

INTRODUCTION :

The optical microscope is used extensively in pharmaceutical development with the primary application being solid-state analysis. The applications range from simple images of drug substance to illustrate particle size and shape to full optical crystallography. The range of utility of the microscope is considerably extended by the use of polarized light which allows us to obtain crystallographic data on small individual crystals.

Polarized light microscopy provides us a unique window into the internal structure of crystals and at the same time is aesthetically pleasing due to the colors and shapes of the crystals. The use of polarized light on the optical microscope allows us to determine the optical crystallographic properties of the crystal. Optical crystallography is related to but different from X-ray crystallography. Each technique provides unique information about the crystal structure and the combination of the two is powerful indeed. Based on optical crystallographic measurement it can yield as many as 20 different characteristics. The applications of this polarised microscope range from simple images of drug substance to illustrate particle size and shape to full optical crystallography.

So, in this experiment, we will use polarised microscope to observe. Gaviscon suspension and Scott’s emulsion cod liver oil. In which by using this polarised microscope we can observe crystal structure and properties of both substances.

MATERIALS :

·         Gaviscon suspension

·         Scott’s emulsion cod liver oil

·         Microscope slides and cover slips

·         Application sticks

·         Polariser

EXPERIMENTAL METHOD :

1)      On a clean and dry microscope slide, spread a thin layer of Gaviscon suspension (as thin as possible for best observation) using an application stick.

2)      Cover the sample gently with a coverslip.

3)      Observe and give a detail microscopic description of the suspension under bright light at x10.

4)      Under bright light, take snapshots and measure the average particle size. Take an average of at least 10 readings from different snapshots.

5)      Place the polarizer on top of the light source.

6)      Turn the polarizer until a crossed polar field was obtained. Observe and give a microscopic description of the suspension under the polarized light at x10.

7)      Repeat step 1-6 with Scott liver emulsion.

RESULTS :

1. Images of suspension and emulsion under bright field at Mag x10. (Print and label)

Suspension

Emulsion 

2. Images of suspension and emulsion under polarised field at Mag x10. (Print and label)

Suspension

Emulsion

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

Suspension under bright light is observed. There are many brown spots. But the figure is still doubted whether it is solid particles or dust. It is confirmed by using polarized light. A glitter-like spots are observed. The glitter-like spots are actually crystals as polarized microscope can detect crystallised particles.

Emulsion under bright light showed droplets of oil. Even it is observed under polarized light, the droplets still seen. Cause emulsion does not contain particles instead of droplet. So under polarized light the figure shown even darkness.

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	17.600	23.500
2	9.800	17.600
3	20.400	20.600
4	18.500	24.200
5	12.800	16.500
6	15.200	12.000
7	26.500	27.300
8	20.700	19.800
9	21.100	30.300
10	18.200	14.200
Average	18.090	20.600
Standard Deviation	4.684	5.790

DISCUSSION :

1.      Explain what is plane polarized light.

        Plane polarized light is the polarization state of the source of light used in polarizing microscope. Polarized light is light that vibrates in one single direction due to the passage through a polarizer. As the normal light vibrating in all direction perpendicular to the path of motion of the light ray while the polarized light vibrate in only one direction. Light is polarized when all of the light waves are vibrating in the same parallel direction. The polarizer use the principle of plane polarized light to differentiate the optical active substance and optical inactive substance.

                                                     

2.      Describe the differences between a normal and a polarized microscope.                   

	NORMAL MICROSCOPE	POLARIZED MICROSCOPE
Uses	View objects by illuminating normal light only.	Object can be viewed using both normal and polarized light.
Properties	Characterized by magnification and numerical aperture. Magnification ranges 5x to 100x while numerical aperture range 0.14 to 0.7	Numerical aperture range 0.9 to 1.35 and has same magnification as normal microscope.
Components	No polarizer and analyzer	Has polarizer and analyzer
Applications	Used to observe small microscopy objects and mostly used in biology field.	Used to determine optical crystallographic properties of crystals. Also used in geological sciences to study rocks and minerals, medicine, metallurgy and biology fields.

3.      From your results, explain how polarized light microscope is useful in examining pharmaceutical formulations.  

Polarized light microscope is useful in examining pharmaceutical formulations in which it can be used to determine mainly to differentiate between crystalline and non crystalline structure.  In this experiment, polarized light is used to differentiate the presence of liquid droplets in an emulsion and solid structure in a suspension. This is because polarized light cannot pass through the liquid droplet as it is amorphous structure. The emulsion is seen in a darkness as no polarized light able to pass through it. On the other hand, in Gaviscon suspension, a pitch black with yellow spots were observed. This is mainly due to crystal structure in solid particles of Gaviscon suspension that allow the passage of polarized light to pass through it. This is due to the crystal lattice of the solid particle that have space that enable polarized light to pass through it. Polarized light also used to observe directly the shape of solid particles and liquid droplets in suspension and emulsion respectively. Instead of using bright light microscope, we can use polarized light microscope which has more advantages in examining pharmaceutical formulations. Polarized light microscope can shows more clear and accurate shape of any particles or droplets in order to do the particle size analysis for a particular formulation.

4.      Describe the advantages and disadvantages of a microscope in determining droplet and particle size in pharmaceutical formulations.

Advantages of using microscope are they are relatively inexpensive, each particle can be examined individually by observing particles shape, 2D structure, colour, photograph can be taken for permanent record and small sample size is required. It gives convenience to the user as it provide a software to allow more accurate examination and adjustment for desired snapshot. Crystal like-structure of particle also can be examined and give very brief differentiation between crystal like-structure and amorphous structure. As for the disadvantages are every single particle need to be examined so this can cause error in examiner due to fatigue plus it is time consuming. It also give no information in 3D and very low throughput. Moreover, software use required manual.

CONCLUSION :

            Polarized microscope is best use to examine and determine the type of pharmaceutical formulation that cannot be differentiated by naked eye. Polarized microscope able to differentiate the crystalline and non-crystalline structure. The advance in technology of polarize microscope made the process to measure the diameter of particle or droplet become easy as there is camera and analyzer. 

Particle Size Analysis Lab Report

TITLE :

EXAMINATION OF PHARMACEUTICAL FORMULATION USING LASER LIGHT SCATTERING METHOD PARTICLE SIZE ANALYSIS (METASIZER AND ZETASIZER)

OBJECTIVE :

1. To study how to use instruments to analyse particle size.

2. To determine particle size and size distribution of different products using different methods of particle size analysis.

3. Analyse and interpret the data obtained.

4. Determine the best method to measure particle size for each product.

INTRODUCTION :

To determine the size distribution and particle size of small particles in suspension or polymers in solution instrument that can be used is Zetasizer. Zetasizer instrument apply Dynamic Light Scattering technique in order to determine particle size and size distribution of nanoparticles, dissolve emulsion or suspension. Dynamic Light Scattering technique apply for small size particles because small size particles possess thermal motion known as Brownian motion. As these molecules move around in the liquid, they are also bouncing off any suspended particles in a random manner. When the laser beam shine the particles, the beam may scattered in all direction results in scattering-angle-dependent intensity pattern because the particles are experiencing Brownian motion. This make the intensity pattern also fluctuates randomly. The rate of fluctuation depends on the size of particles. Smaller size of particles, the higher rate of fluctuation.

The Malvern Mastersizer uses 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 principle used is laser light scattering and the analysis is Mie and Fraunhofer scattering. A typical system is made up of three main elements which are optical bench, sample dispersion units and instrument software. Optical bench is 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. 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. The Mastersizer software controls the system during the measurement process and analyzes the scattering data to calculate a particle size distribution. The usual particle size used is between 0.02 - 2000 µm. It has broad particle size range suitable for many different applications. Besides that, it has wide range of sample dispersion options for emulsions, suspensions and dry powders and fully automated simple SOP operation for ease of use and method transfer.

MATERIALS :

Gaviscon Suspension (Sample B)

Cod Liver Oil emulsion (Sample C)

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

PROCEDURES :

1. Malvern Zetasizer was powered up.

2. The software was launced up.

3. Cell was loaded and measurement was ran.

4. The resut was reviewed.

5. The cell was removed from the cell.

6. The cell was washed.

RESULTS :

1.      Fill in the table below:

Result for Metasizer:

No.	Particle size, µm			Size Distribution
	Cod Liver OilⓇ	GavisconⓇ	Polystyrene nanoparticles	Cod Liver OilⓇ	GavisconⓇ	Polystyrene nanoparticles
1.	36.797	21.997		0.444	0.537
2.	36.865	23.302		0.442	1.000
3.	39.919	0.000		0.678	0.000
Mean ±SD	37.774 ± 1.459	22.650 ± 0.653		0.523 ± 0.111	0.769 ± 0.232

Result for Zetasizer:

No.	Particle size, µm			Size distribution
	Cod Liver Oil	Gaviscon	Polystyrene nanoparticles	Cod Liver Oil	Gaviscon	Polystyrene nanoparticles
1.	1.662		212.000	1.000		0.048
2.	1.559		212.900	0.400		0.021
3.	1.674		212.500	0.106		0.047
Mean SD	1.632 0.052		212.470 0.368	0.502 0.372		0.039 1.563 X 10 - 4

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

Gaviscon ( No. 1)

Gaviscon (No. 2)

Cod liver oil (No. 1)

Cod liver oil (No. 2)

Cod liver oil (No. 3)

Cod liver oil (Average)

DISCUSSION :

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

The parameters that should be provided when using Malvern Zetasizer® and Mastersizer® are time and  temperature of dispersant material. The time for the Malvern Mastersizer® is less 10 seconds and its operating temperature (°C) is from 5°C to 40°C.While for the Malvern Zetasizer®,the time is 2 minutes and the  operating temperature range (°C) is from  10°C – 35°C.

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

By using the above method to analyse the samples, the accurate, reliable and repeatable particle size analysis can be determined. This can be proved by Mastersizer 3000 which is the latest generation of the world’s most popular particle sizing instrument that can deliver rapid, accurate particle size distributions with minimum effort  for both wet and dry dispersions .It can measures over nanometer to millimeter particle size ranges. It delivers class-leading particle sizing performance in a compact footprint and bring the operator-independent measurements that every user can rely on. For Malvern Zetasizer® , particles and molecules from less than a nanometer in size to several microns can be analyzed by a range of variants to suit your applications and budget. Next, the apparatus of above method is easy to set up as it is fully automated measurement.

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

If both methods are not suitable to analyse particle size any of the samples, the other method that can be used to analyse the particle size is microscope method which involve using of the polarized microscope .This method involve observation of the material (Eg: Gaviscon suspension, Scott's emulsion cod liver oil) under the bright light and polarized light. Detail microscopic description under bright light and polarized light at 10x was analysed for determination of particle size distribution.

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

Mastersizer uses the technique of laser diffraction to measure particle sizes  by measuring the intensity of light scattered as a laser beam passes through a dispersed particulate sample. To calculate the size of the particles that created the scattering pattern this data is then analyzed.

Large particles scatter light at small angles relative to the laser beam. Meanwhile,  small particles scatter light at large angles. To calculate the size of the particles responsible for creating the scattering pattern the angular scattering intensity data is then analyzed. Laser diffraction uses Mie theory of light scattering to calculate the particle size distribution, assuming a volume equivalent sphere model.

Mie theory requires knowledge of the optical properties which are refractive index and imaginary component of the sample being measured, along with the refractive index of the dispersant. Optical properties of the dispersant are easy to find. Mie theory completely solves the equations for interaction of light with matter. This theory assumes the volume of the particles as opposes to Fraunhofer which is a projected area prediction.

Fraunhofer approximation is more simplified which does not require knowledge of the optical properties of the sample. This can provide accurate results for large particles. However it should be used with caution when working with samples which might have particles below 50µm or particles are relatively transparent.

Zetasizer uses two techniques in a single compact unit which are Dynamic Light Scattering and Static Light Scattering and has a range of options and accessories to optimize and simplify the measurement of different sample types.

Dynamic Light Scattering is used to measure particle and molecule size. This technique measures the diffusion of particles moving under Brownian motion, and converts this to size and a size distribution using the Stokes-Einstein relationship. Typical applications of dynamic light scattering are the characterization of particles, emulsions or molecules, which have been dispersed or dissolved in a liquid. The Brownian motion of particles or molecules in suspension causes laser light to be scattered at different intensities.

Static Light Scattering is used to determine the molecular weight of proteins and polymers. In this technique, the scattering intensity of a number of concentrations of the sample is measured and used to construct a Debye plot. Thus, average molecular weight and second virial coefficient can be calculated.

This measurement is very demanding on the stability of the whole system, and means that every element of the design has been optimized to ensure accuracy and repeatability.

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

Advantages of Laser light scattering method are it does not require calibration against a standard. The sample also measured entirely. It is also simple and fast data collection. It has very broad dynamic range(< 0.1 μm up to millimeter sizes). It can also measure powders and fluid suspensions. Testing is non-destructive. Thus, samples can be recovered if necessary. The method is widely used and many people are familiar with the method.

Dynamic light scattering technology from Malvern Instruments have the following advantages. It is accurate, reliable and repeatable particle size analysis in one or two minutes. The mean size only requires knowledge of the viscosity of the liquid. It is simple in which no sample preparation required and  high concentration and turbid samples can be measured directly. The set up is simple and fully automated measurement. Size measurement of sizes is less than 1nm. Furthermore, a minimum amount of information about the sample is needed to run an analysis. Even mixtures of different materials can be accurately measured. Except for the viscosity of the medium must be known accurately. Besides, only a tiny sample is needed. The analysis also fast and simple. Lastly, testing is non-destructive, so samples can be recovered if needed.

CONCLUSION :

One of the theory use in Mastersizer is the Fraunhofer model. This model can predict the scattering pattern that is created when a solid, opaque disc of a known size is passed through a laser beam. Other than that, the Mie theory was developed to predict the way light is scattered by spherical particles and deals with the way light passes through. This theory is more accurate but it require some specific information about your particle such as its refractive index and its absorption.

If you know the size of the particle and other details about its structure, you can accurately predict the way it will scatter light. Each size of particle will have its own characteristic scattering pattern. The Mastersizer works backwards from the above theories by using the Mastersizers’ optical unit to capture the actual scattering pattern from a field of particles. Then using the theories above it can predict the size of particles that created that pattern.

Zetasizer provides the ability to measure three characteristics of particles or molecules in a liquid medium which are particle size, zeta potential and molecular weight. The Zetasizer system also enables determination of the protein melting point and perform Trend measurements.

REFERENCES :

1.http://www.malvern.com/en/products/technology/laser-diffraction/default.aspx

2.http://www.cpsinstruments.eu/pdf/Compare%20Sizing%20Methods.pdfhttp://www.cpsin

struments.eu/pdf/Compare%20Sizing%20Methods.pdf

3.http://www.ceic.unsw.edu.au/centers/partcat/facilities/Mastersizer.pdf

4.http://www.biophysics.bioc.cam.ac.uk/files/Zetasizer_Nano_user_manual_Man0317-1.1.pdf