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Guide To Centrifugation
Lab and Pathology
By Jaykishan Parekh
16th December 2021
Centrifuge is device that applies centrifugal force to separate mixture in the fluid by putting an object in rotational movement around fixed axis there by separating fluids of different densities or liquid from solids.
Centrifuge is relatively simple instrument but yet tricky to maintain. Most of the centrifuge related blunders are due to user handling error and many machines have been destroyed by the scientist who are in it for simple pelleting. Therefore, it is crucial to understand what we do during centrifugation and avoid damaging the instrument and it’s in for the prolonged stay in the lab
- Balance
Centrifugation produces centrifugal force to effectively separate and sediment the sample component based on size and density. Rotors spin very rapidly generating extreme force and therefore it is crucial to properly balance rotors during spin especially when rotors are loaded partially with tubes and plates. Any imbalance in the rotor including placement of the centrifuge on an uneven or slanted work surface can cause abnormal vibration that can in turn damage the rotor. An unbalanced centrifuge is as dangerous as unstable chemical reaction and can start domino effect of catastrophes all the way from instrument failure, to sample breakage, user injury and overall large-scale destruction in the laboratory and can be major safety hazard in the laboratory environment
Precise balancing at high speed become more crucial as the centrifugal force generated during centrifugation is directly proportional to sample mass and acceleration. At higher RCF even slight change in sample mass can lead to substantial force imbalance which in turn could be catastrophe.
It doesn’t matter if centrifuge has a fixed angle or swing bucket rotor, two important factors to consider when balancing includes sample volume and symmetrical arrangement of tubes. It is important to ensure that the sample volumes are equal in all tubes and the tubes are placed directly opposite each other in the centrifuge. If the solution are not of similar density (eg acetone and water), tubes should be equal mass rather than volume to be correctly balanced. In case of swing bucket rotor it is to make sure that all slots contain rotor bucket appropriate to the machine and are of same weight.
- Time, Speed and centrifugation
The choice of centrifugation speed depends on the size of the particle in the sample, smaller the particle size higher centrifugation speed for example bacterial cells are pelleted at higher speeds (2000-10000 g) than mammalian cells. Relative centrifugal force (RCF) generated by spinning rotor is expressed relative to earths gravitational force and therefore is also known as G-force . The G force acting on particle is exponential to the speed of rotation due to this square dependence of RCF on RPM, centrifugation speed of 10000RPM for 5 minutes and 500 RPM for 10 minutes are not interchangeable. The former produces a much larger RCF than latter. RCF is proportional to the radius of the rotor and to the square of RPM
- RPM and RCF are not the same
RPM (Revolution per minutes) and RCF (Relative centrifugal force or g-force) are commonly used to describe centrifugal speed. RCF refers to the acceleration applied to your sample for example 10,000 g means 10,000 times earth’s gravitational force. RPM is not useful unit because the force varies with radius of the machine (Bigger the radius, the more acceleration is applied to your sample for the same RPM) therefore RPM speed setting need to be converted to RCF to ensure that the correct centrifugal force is applied.
- Centrifugation and the temperature
Centrifugation generates heat because of the movement molecules and friction with air which in turn can increase the temperature within a centrifuge which can affect stability of the sample. Temperature inside the centrifuge is primarily influenced by three factors: Rotor material, Rotor shape and speed of rotation.
Rotor material such as steel and aluminium have a high density and high thermal conductivity which means it can transfer heat efficiently and get chilled quickly. Material like polymer and carbon fibre are low density material (Heat Insulator) and helps in maintaining constant temperature.
Rotor Shape determines the airflow within a centrifuge. Optimizing the airflow within a centrifuge through rotor shape is essential to maintaining the temperature.
Speed of Centrifuge, Centrifugation is proportional to the rise in temperature – at higher speed, more heat is generated. The degree of warming is very much depending on the maximum speed and shape of the rotor. It is important to understand the maximum speed of the centrifuge and the range of speed that maintain temperature range that will not change the outcome of the experiment. This information is supplied by manufacturer to help user understand the limitation and work around it.
- Acceleration and Deceleration
Many Centrifuge offer the option to control deceleration setting (brakes) to bring it to stop faster and what is the effect on sample outcome.
Braking can be particularly useful during centrifugation involving Nucleic acid extraction or bacterial cell pelleting which are not affected by sudden stopping. Experiments that more sensitive to abrupt deceleration such as isolation of peripheral blood mononuclear cells and gradient centrifugation, braking can cause separated layers to remix.
The Interphase separation of peripheral blood mononuclear cells (PBMC) is very sensitive to vibration and too fast acceleration or deceleration would disturb the gradient and lead to a milky/Non-existent interphase, making difficult to extract PBMC. To prevent remixing the protocol requires acceleration/deceleration be shutoff completely so that centrifuge accelerates slowly and high forces cannot disturb the gradient and accumulate in the appropriate phase according to their density. When the brake is shutoff, the centrifuge simply runs down until the rotor stops by itself.
Thus, centrifuge with different Acceleration/Deceleration ramps offers users the option of adjusting acceleration and deceleration speed and optimizing their protocols quite easily
- Know where is your pellet?
One of the applications of centrifuge is to pellet down bacterial cells, mammalian cells or nucleic acid. Angle of rotor determines the position of pellet. To Ensure and know where your pellet is, a good lab practise is to always spin the tube with lid hinges placed in same orientation (for example lid hinges facing outward). In case of swing rotor, the pellet are formed at the bottom of the tube
This blog is taken from Guide To Centrifugation - Neuation
Courtesy of Neuation Technologies
