GENETICS | GENETIKA 244

CENTRIFUGATION

BACKGROUND: Centrifugation
Particles suspended in a fluid move, under the influence of gravity towards the bottom of a
tube at a rate that depends in general on their size, shape, and density. Centrifugation is a
technique designed to utilize centrifugal forces which are greater than the force of gravity
to speed up the sedimentation rate (pellet forming) of particles (separation of a mixed
solution into supernatant and pellet). This is achieved by spinning the tube containing the
fluid and particles about an axis of rotation so that the particles experience a force acting
from the axis.
The particles in the solution are separated according to their size and shape, the density
and viscosity of the medium and rotor speed. During this process, materials with a higher
particle density as the solvent will sediment towards the axis of centrifugation (sink to the
bottom of tube), while materials with a lower particle density than the solvent will sediment
away from the axis of centrifugation (float to the top of the tube). Centrifugation takes
advantage of even minute differences in density to separate particles within a solution. For
example, bacterial cells in suspension have a high particle density and will tend to settle to
the bottom (Figure 1).

Before After Aqueous

centrifugation centrifugation phase

Lower phase

Figure 1: Centrifuge tubes showing separation.

In biological analysis centrifugation is commonly used for 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 as found in extraction and clean-up protocols
for DNA analysis and cell culture.

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 GENETICS | GENETIKA 244

Principle of centrifugation rpm versus g

The force experienced by particles in a solution acting from the axis is measured in multiples
of the Earth’s gravitational force and is known as the relative centrifugal field (RCF) or more
commonly the “g” force. Thus, g is the unit used to measure relative gravitational force and
is determined by the speed at which the rotor spins in revolutions per minute (rpm) and the
radius of the rotor (the distance from the axis).
This distinction is important because two centrifuge rotors with different diameters running
at the same rotational speed will subject samples to different accelerations. The higher the
rotational speed, the higher the g force exerted on the particles and the faster the particles
will accumulate at the bottom of the centrifuge tube.
Protocols for centrifugation typically specify the rotational speed as revolutions per minute
(rpm). To convert rpm to RCF, and vice versa is as follows:
RCF = 11.18 x r (rpm/1000)2
Rpm = 299.07 √RCF/r
r = radius (cm)

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.

Instruments Components
Centrifuge Unit
The centrifuge unit houses the motor that drives your centrifuge (Figure 3). It must be
placed on a work surface that is level and firm. Do not use the centrifuge on an uneven or
slanted work surface.

Figure 3: Centrifuge body.

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 GENETICS | GENETIKA 244

Safety Lid
Safety lids can have a transparent design or might be made of a solid material, but all
safety lids protect the rotor bucket from aerosol leakage and spills (Figure 4). It is extremely
important to remember to fix the safety lid in place before starting your centrifuge
otherwise a high pitch whistling sound will be heard.

Figure 4: Three different types of safety lids.

Rotor
Most centrifuges can accept rotors of different sizes and capacities, although most rotors
are now capable of accepting a large range of tube sizes by making use of tube adaptors.
Rotors have three basic designs: horizontal or swinging bucket (Figure 5A), in which the
tubes are carried in buckets that can swing outwards; and vertical (Figure 5B), in which the
tubes are fixed in the vertical position; and fixed angle (Figure 5C), in which the tubes are
held at a fixed angel to the vertical position. The fixed angle rotors are what you will
encounter in the benchtop centrifuges you will use in your practical’s.

A B C

Figure 5: A: Swinging-bucket rotor; B: Vertical rotor; C: Fixed angle rotor.

Copyright © Department of Genetics, Stellenbosch University, 2024

 GENETICS | GENETIKA 244

The load on the rotor should ALWAYS be balanced before operating the centrifuge (Figure
6.1). Small differences in the mass of the load at different positions on the rotor can result in
a large, unbalanced force when the rotor is at high speed. This force can put strain on the
equipment, which eventually can lead to failure of the rotor. This will cause serious
damage to the equipment and may lead to personal injury. The load must be balanced
both by equal mass and tube type.

How do I know if my rotor is imbalanced?

The centrifuge will make a high pitch whistling sound and can even begin to shake or
wobble excessively. You should stop it immediately and rebalance the samples!

How do I avoid centrifuge imbalances in the first place?

At high speeds, a centrifuge can easily become unbalanced if equal masses are not
located opposite each other in the rotor:
For fixed-angle rotors, balance your tubes according to their weight. Load the rotor
symmetrically and ensure the opposing tube is not only the same type of tube, but that it is
also filled with the same mass. If the number of tubes with samples is uneven,
counterbalance using water in an additional tube. Thus, the minimum number of centrifuge
tubes you can use is two as this will allow the load to be evenly balanced.
Remember to balance the mass (weight) of the tubes, not the volume (size). If you are
spinning more than two tubes, only the tubes directly opposite each other must be equal in
mass (Figure 6). Also watch the video on how to balance a centrifuge (Figure 6.2).

Figure 6.1: Balanced rotor vs unbalanced rotor.

Figure 6.2: Balanced rotors.

Copyright © Department of Genetics, Stellenbosch University, 2024

 GENETICS | GENETIKA 244

Operating a Lab Centrifuge

When using a centrifuge, it is very important that you follow the correct protocol. The table
below summarises the steps needed to be performed to operate a centrifuge correctly.

Operation Action

Open the lid by pressing the lid The lid of the centrifuge can only be lifted if the
button. centrifuge is plugged in, and the rotor has stopped
moving.

Load the centrifuge rotor. Make sure sample tubes are properly balanced in rotor
before use.

Fix the safety lid. Press or pull the attachment on the lid.

Close the lid GENTLY. It will make a ‘click sound when secured.

Set the speed and run time. Remember to check if you need rpm or g and if your
centrifuge is on the correct setting. Use the rotating
knobs on the front to set the speed and time.

Press start. Always wait till centrifuge has reach its set speed before
walking away.

Open lid and remove tubes. Once the centrifuge has spun for the allocated time,
the rotor will slow down and stop.
The lid locking mechanism will then unlock and the lid
can be lifted, and the sample tubes removed.
After centrifugation. Clean the chamber and the rotor after your run.

Safety Considerations When Operating a Lab Centrifuge

1 Make sure the rotor is correctly balanced. If uneven numbers, use a balance tube.

2 Make sure that the centrifuge safety lid is in place and the lid is closed and locked.

3 Wait until the centrifuge has reached its full operating speed and you are sure that it
is operating correctly before you leave it.

4 Wait for the centrifugation process to stop completely before opening the lid.

5 Remove samples carefully to not disturb any particles.

6 Clean up any spills or leaks in the centrifuge chamber and in the rotor.

Copyright © Department of Genetics, Stellenbosch University, 2024