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Determination of Particle Size by Microscopy

Learn how to determine the Particle Size of any substance using a microscope.
This method is suitable for counting and characterising particles of 1µm and greater. With increased resolving power of the microscope particles smaller than 1 micron can be detected and characterised. Although alternative techniques are available the method is particularly useful for characterising particles that are not spherical.


A microscope with sufficient magnification to allow adequate characterisation of the smallest particles to be classified in the sample under examination, polarising filters in conjunction with analysers and retardation plates, and colour filters of relatively narrow spectral transmission for photomicrography. Condensers, corrected for spherical aberration are required in the microscope substage and with the lamp. The lamp should provide uniform and adjustable intensity of light over the entire field of view. The numerical aperture of the substage condenser should match that of the objective under the conditions of use.
All the elements of the optical system should be aligned and focussed in accordance with the recommendations of the equipment manufacturer. Critical axial alignment is essential.
Before use, it must be ensured that the microscope is stable and is placed on a surface protected from vibration.

Visual characterization

The magnification and numerical aperture must be sufficiently high to allow adequate resolution of the images of the particles. The actual magnification should be determined using a calibrated stage micrometer to calibrate an ocular micrometer. If the magnification is such that the image ofthe particle is at least 10 ocular divisions, errors can be minimised. Each objective should be calibrated separately.

To calibrate the ocular scale, the stage micrometer and the ocular scale should be aligned in order to enable a precise determination of the distance between ocular stage divisions. The use of different magnifications may be necessary to characterise materials of varying particle size in a sample. Photographic characterisation. It must be ensured that the object is sharply focussed at the plane of the photographic emulsion. The actual magnification may be determined by photographing a calibrated stage micrometer, using photographic film of the right speed, resolving power and contrast. Exposure and processing must be identical for the photographs of both the test sample and the determination of magnification.

Limit test of particle size

Weigh a suitable quantity of the powder under examination (10-100 mg) and suspend it in 10 ml of a suitable medium in which sufficient contrast between the sample and the medium is obtained to ensure adequate detail of the sample edge. The medium should be such that the powder will not dissolve in it. Add, if necessary, a wetting agent in order to obtain a homogeneous suspension and provide adequate agitation to achieve uniform distribution of the powder. Introduce a portion of the homogeneous suspension into a suitable mounting cell. It must be ensured that the particles rest in one plane and are adequately dispersed to distinguish individual particles of interest. It is very important that the particles on the mount are representative of the distribution of sizes in the material and have not been altered during preparation of the mount.

Scan under a microscope an area corresponding to not less than 10 µg of the powder. Count all the particles having a maximum dimension greater than the prescribed size limit. Particle size characterization. Determine the size of the sample, measure the particle sizes and analyse the data as detailed in International Standard ISO 9276. For spherical particles define the size by diameter and for irregular particles, by the definitions of the different types of diameter stated in the Standard.

Particle shape characterization

This may be done for irregularly shaped particles. The homogeneity of the powder must be checked using appropriate magnification. Commonly used descriptions of shape are:
- acicular: slender, needle-like particle of similar width and thickness,
- columnar: long, thin particle with a width and thickness that are greater than those of an acicular particle,
- equant: particle of similar length, width, and thickness; both cubical and spherical particles are included,
- flake: thin, flat particle of similar length and width,
- lath: long, thin blade-like particle,
- plate: flat particle of similar length and width but with greater thickness than a flake particle.
Crystallinity characterisation. Unless otherwise specified in the individual monograph, mount a few particles of the sample in mineral oil on a clean glass slide. Examine the mixture using a polarising microscope; the particles should show birefringence (interference colours) and extinction positions
Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
Email: .moc.enilediugamrahp@ofni Need Help: Ask Question

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