# Particle Size Sizing Technique 1: Coulter principle

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Particle Size Sizing Technique 1: Coulter principle
Physical Pharmacy 2 Particle Size Sizing Technique 1: Coulter principle Kausar Ahmad Kulliyyah of Pharmacy, IIUM Physical Pharmacy 2 KBA

Electrical Sensing Zone Method A.k.a. the Coulter principle
Basic method of counting and sizing based on the detection and measurement of changes in electrical resistance, produced by a particle or biological cell, suspended in a conductive liquid, traversing through a small aperture. Physical Pharmacy 2

Wallace Coulter - Coulter orifice (1956)
Physical Pharmacy 2 Wallace Coulter - Coulter orifice (1956) (as early as 1948) - measured changes in electrical conductance as cells suspended in saline passed through a small orifice Cells are relatively poor conductors Blood is a suspension of cells in plasma which is a relatively good conductor Previously it was known that the cellular fraction of blood could be estimated from the conductance of blood As the ratio of cells to plasma increases the conductance of blood decreases Because cells are poor conductors….. Presence of a cell in the electrical sensing zone disturbs the electrical current. This in turn will give rise to a change in voltage. As a result, a signal indicating the passing of a cell through the aperture is given to the analyst. ONE cell is thus counted!!! Physical Pharmacy 2 KBA

From: http://www.cyto.purdue.edu/flowcyt/educate/ee520/sld008.htm
Physical Pharmacy 2 From: Why use direct current? A signal or a pulse, can be generated only from DC Alternating current applicable for appliances that use simple conversion of energy: heater, iron, light, motor Other appliances such as radio, TV and computer, must use DC because of the need to give signals/pulses Physical Pharmacy 2 KBA

Principle A current, between two electrodes, create a sensing zone around the aperture When passing through the aperture, the magnitude of the current is ca. I mA When a particle passes through the aperture, it causes changes in electrical impedance EACH particle will trigger a voltage pulse indicating a depression in current flow The magnitude of the decrease depends on size of particle Physical Pharmacy 2

Interpreting the results
Amplitude of the pulse is proportional to the volume of the particle Hence, can determine diameter of particle Each pulse represents one particle Hence, can determine the number of particle And therefore, Coulter counter………. The voltage pulses will be counted, amplified and allocated to the right size class. Physical Pharmacy 2

Physical Pharmacy 2 Advantages of Technique capable of counting thousands of particles per second Results are not affected by: Colour Composition Refractive index Or other light interaction effects Absolute sample volume. Absolute sample volume is an advantage if you want to monitor multiplication of cells. You may have a reduction in number of particles as a result of coalescence…. Physical Pharmacy 2 KBA

Converting Signals to Particle Diameter
Physical Pharmacy 2 Converting Signals to Particle Diameter Calibrate instrument using 10 or 20 µm polystyrene standards Obtain the ‘Kd’ The Kd is used to convert the amplitude of the pulse in volt to volume of the particle (this is a linear response) From the volume, the diameter can be calculated. Physical Pharmacy 2 KBA

Calibration of instrument
Physical Pharmacy 2 Calibration of instrument A monodisperse standard is used Pulses on oscilloscope of uniform size Concentration used is very low so that coincidence effects are less than 2% Instrument is adjusted/calibrated to give the ‘Kd’ Instrument is adjusted so that height is 40% of maximum by controlling ‘gain’ and ‘currect selector’ settings. This gives the ‘Kd’ Kd = {6WVm1012/VT(nV)}1/3 W =mass of sample in beaker (g) VT = volume of electrolyte solution in which W is diluted Vm= manometer volume (cm3) = immersed density of the particles (g/cm3) n = number of particles in a size interval V = arithmetic mean volume for that particular size interval, in instrument units (e.g. product of threshold value, aperture current, and attenuation) Physical Pharmacy 2 KBA

Relationship between electrical signal and volume of particle
Voltage proportional to volume of particle: U = constant x V constant = r0if / 2R4 U=amplitude of voltage pulse V=particle volume r0=electrical resistivity i= aperture current f= particle ‘shape’ factor R=aperture radius must not be dirty Physical Pharmacy 2

Physical Pharmacy 2 Sample Concentration If more than one particle passes through the aperture at exactly the same time (coincidence effect), the reading is not accurate. Therefore, sample must be reasonably diluted and should be within the specified range as indicated by the instrument. Exercise: For a given volume, the smaller the particles, the lesser is the sample required. WHY? Physical Pharmacy 2 KBA

Sample Condition It is important that only one particle passes through the aperture. There should not be any aggregation or flocculation. For detecting a stable suspension, the particles must exist as discrete individual entities. A dispersant must be used Samples dispersed in the electrolyte must be stirred during measurement, especially if it is a solid dispersion, to prevent settling. Physical Pharmacy 2

Aperture The aperture comes in different sizes
E.g. an aperture of 100 µm can detect particles within 2 to 60 µm Outside the range, the measurement is not accurate Aperture should be clean Physical Pharmacy 2

Results Generated Results can be displayed in terms of number, volume, surface area against particle size. Size axis can be linear, logarithmic scale Distribution can be differential or cumulative data Cumulative data can be oversize or undersize Physical Pharmacy 2

To standardise standards !
Application Counting algae Counting bacteria Counting cells To standardise standards ! In industries To detect contaminant in petroleum Electronic: TV screen, CRT (Dots per inch = DPI) Physical Pharmacy 2

References JZ Knapp, TA Barber & A Lieberman, Liquid and Surface-Borne Particle Measurement Handbook, Marcel Dekker, New York (1996). T Allen, Particle Size Measurement 4th. Ed., Chapman and Hall, London (1990). Beckman-Coulter Multisizer 3 operation manual (lab) Physical Pharmacy 2