LEARNING AND DEVELOPMENT


Centrifugation: a brief guide to equipment use and maintenance


Increased emphasis on work-based learning means that biomedical science trainees and support workers require a solid grounding in laboratory techniques. This month The Biomedical Scientist once again goes back to basics, the focus here being on centrifugation and the use and maintenance of laboratory centrifuges.


The centrifuge is ubiquitous in biomedical laboratories and a basic knowledge of the theory of centrifugation is very useful. Centrifuge performance can be classified as low-speed, high-speed and ultra-speed. Usual applications include the separation of serum or plasma from red blood cells, the separation of precipitated solids from the liquid phase of a mixture, or the separation of liquids of varying density.


Principles of centrifugation Particles suspended in a fluid move, under the influence of gravity, towards the bottom of a vessel at a rate that depends, in general, on their size and density. Centrifugation is a technique designed to utilise centrifugal forces, which are greater than the force of gravity, to speed up the sedimentation rate of particles. This is achieved by spinning the vessel containing the fluid and particles about an axis of rotation so that the particles experience a force acting away from the axis. The force is measured in multiples of the Earth’s gravitational force and is known


Table 1. Calculations to convert rpm to RCF, and vice versa.


RCF = 11.18 x r (rpm/1000)2 rpm = 299.07 √RCF/r


r: radius (cm) 238


as the relative centrifugal field (RCF) or, more commonly, the g force.


Relative centrifugal field The RCF generated by a rotor depends on the speed of the rotor in revolutions per minute (rpm) and the radius of rotation (ie the distance from the axis of rotation). The equations that permit calculation of the RCF from a known rpm and radius of rotation and calculation of the rpm from a known RCF and radius are shown in Table 1. The RCF value can also be obtained using a nomogram (Fig 1). Using a straight-edged ruler, line up the known rotating radius (distance from the centre of rotor to the bottom of the centrifuge bucket) on the left with the known rpm on the far right and read the RCF value where the line crosses the graph in the centre. Most manufacturers include a nomogram in the instruction manual; however, most modern centrifuges now have the facility to swap the figure displayed on the control panel between rpm and RCF, making manual calculation unnecessary.


Low-speed instruments Low-speed centrifuges have maximum rotor speeds of less than 10,000 rpm, which do not require the rotors to be run in a vacuum, and there are instruments with a temperature control facility. Most instruments now include a sensor that will


20


18 16 14


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30000 20000


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Inches cm Relative centrifugal field (xg)


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200


Revolutions per minute


Fig 1. Nomogram used to convert revolutions per minute to relative centrifugal field (xg), and vice versa.


detect any imbalance when the rotor is running and cut off the drive mechanism if imbalance is present. Low-speed instruments are used to separate serum or plasma from red blood cells, and to harvest and purify chemical precipitates, intact cells, nuclei, large mitochondria and large plasma-membrane fragments.


High-speed instruments In general, high-speed centrifuges are capable of rotor speeds up to 21,000 rpm, although the new generation of super-speed instruments are capable of rotor speeds of 30,000 rpm, in which RCFs of 120,000 xg are possible. These instruments require


MAY 2016 THE BIOMEDICAL SCIENTIST


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