Microscopic analysis is one of the most precise particle size determination methods, since individual particles are measured by direct viewing of the particles, instead of statistically averaging the diameter of a group of particles. Microscopy is often used to verify another particle sizing method, during development and validation of a particle sizing method.
Microscopy measures the projection of the particles, rather than the particles themselves. For spherical particles, the particle size descriptor is the diameter; for irregularly shaped particles, the diameter of a sphere, which has the same projected area, is used to describe the size of the particles. Other parameters describing irregular particles, such as the perimeter diameter, Feret’s diameter and Martin’s diameter, can be used to describe the particle size of irregularly shaped particles. A sample size of at least 300–600 particles should be chosen, and, if the size difference between particle-to-particle is comparatively large, the sample size should be increased to get a more significant determination.
The Principle Of Microscopic Analysis
The fundamental principle of microscopic analysis is based on the analysis of two-dimensional images of projected particles on a projection screen. The projected particles are compared with circles that are generated by the graticule of the microscope.
The diameter of the circle, which corresponds most similarly to the projected particle, is used to describe the size of the particle. The projection screen is divided into small sections to achieve simultaneous analysis of multiple particles.
Types of Microscopy Used in Microscopic Analysis
Light Microscopy
Light microscopy can be used to characterize particle sizes between about 1 to 150 microns in diameter. When preparing powder samples for microscopic analysis, light microscopy requires a monodispersed layer on the slide to avoid agglomeration.
Scanning & Transmission Electron Microscopy (SEM & TEM)
Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are used for particle size measurement as well. Both SEM and TEM can analyze particles having a much smaller size range than light microscopy, typically less than 1 micron in diameter.
Additionally, SEM can acquire a three-dimensional image of particles, providing information about particle shape and thickness. SEM samples are placed on aluminum stubs and coated with a thin film of gold (at a nanometer-scale thickness), while TEM samples are often embedded in resin, sectioned using a microtome, placed on a grid, and coated with metal.
Image analysis
Since hundreds of particles must be viewed in the microscopic method, it is very time-consuming. To overcome the diff iculties brought about by manual analysis of particles, a variety of semiautomatic technologies have been incorporated into the microscopic methods to reduce the workload.
One of the most popular techniques, the particle comparator, was introduced. Here, the instrument is designed to accept a photographic image of the particle projection, produced either by a light or an electron microscope. The photograph is projected onto a translucent screen, and the particles are compared with a superimposed spot of light projected from the other side. A computer, connected with the variable aperture iris, automatically ensures that the particle is counted and placed into the appropriate particle size category. These semiautomatic technologies have many advantages, including easier and faster analysis, time savings, and no need for professional operators.
Reference:
- Felton. L. (2013). Remington Essentials of Pharmaceutics. London. UK: Pharmaceutical Press.
