1. Particle Size Sizing Technique 1: Coulter principle
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Particle Size Sizing Technique 1: Coulter principle
Kausar Ahmad
Kulliyyah of Pharmacy, IIUM
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2. 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.
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3. Wallace Coulter - Coulter orifice (1956)
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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!!!
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4. From: http://www.cyto.purdue.edu/flowcyt/educate/ee520/sld008.htm
                                                                                                                                                
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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
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5. 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
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6. 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.
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7. Advantages of Technique
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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….
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8. Converting Signals to Particle Diameter
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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.
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9. Calibration of instrument
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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)
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10. 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
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11. 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?
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12. 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.
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13. 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
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14. 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
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15. 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)
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16. 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)
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