Centrifugation

Muhammad Asif Shaheen

Lecturer Pathology
Department of Pathology
KEMU, Lahore.
• Centrifugation is a technique of separating substances which involves
the application of centrifugal force.
• The particles are separated from a solution according to their
• Size
• Shape
• Density
• viscosity of the medium and
• rotor diamter.
Types
• LOW SPEED CENTRIFUGE
• The low speed centrifuge has a maximum speed of 4000-5000rpm
• These instruments usually operate at room temperatures with no
means of temperature control
• HIGH SPEED CENTRIFUGES
• High speed centrifuges are used in more sophisticated biochemical
applications, higher speeds and temperature control of the rotor
chamber are essential.
• High speed centrifuge has a speed of 6000-30000 RPM
• The operator of this instrument can carefully control speed and
temperature which is required for sensitive biological samples.
• ULTRACENTRIFUGES
• It is the most sophisticated instrument.
• Ultracentrifuge has a maximum speed of 150000 RPM
• Intense heat is generated due to high speed thus the spinning
chambers must be refrigerated and kept at high vacuum.
• It is used for both preparative work and analytical work
• Airfuge™ Air-Driven Ultracentrifuges from Beckman Coulter Life
Sciences
• With the ability to provide a simple, rapid method for removing
chylomicrons (fat particles in lipemic samples), the Airfuge Air-Driven
Ultracentrifuge delivers accurate results for lipemic clarification.
Lipemic samples can be cleaned in 10 minutes, eliminating the need
for sendouts.
• Speed of 80000 rpm
RPM and RCF
• Revolutions Per Minute (RPM)
• Revolutions Per Minute (RPM) in regards to centrifugation is simply a
measurement of how fast the centrifuge rotor does a full rotation in
one minute.
• Relative Centrifugal Force (RCF)
• Relative Centrifugal Force (RCF) or g-force (both are the same, RCF is
expressed as units of gravity) is a measurement of the gravitational force
that a sample is subject to.
• The force is generated from the spinning of the rotor which, in turn, exerts
this force outward on the centrifuge tube.
• Not only does RCF take into account the speed of rotation, it also measures
the distance from the centre of rotation to give us a g-force measurement.
• RCF is the preferred method of measurement as it will remain the same
even if you are using a different centrifuge with a different rotor size.
http://insilico.ehu.es/mini_tools/rcf_rpm.php
Online convertors for RPM to RCF and vice
versa
• http://insilico.ehu.es/mini_tools/rcf_rpm.php
• https://www.fishersci.co.uk/gb/en/scientific-products/centrifuge-
guide/centrifuge-applications-tools/rpm-rcf-calculator.html
Types of rotors in centrifuges
Swinging bucket Fixed bucket
Types of Centrifugation

• Differential Pelleting (differential centrifugation)

• Particles of different densities or sizes in a suspension will sediment at
different rates, with the larger and denser particles sedimenting
faster. These sedimentation rates can be increased by using
centrifugal force. A suspension of cells subjected to a series of
increasing centrifugal force cycles will yield a series of pellets
containing cells of decreasing sedimentation rate.
• Density Gradient Centrifugation
• This type of centrifugation is mainly used to purify viruses, ribosomes,
membranes etc.
• A sucrose density gradient is created by gently overlaying lower
concentrations of sucrose on higher concentrations in centrifuge tubes
• The particles of interest are placed on top of the gradient and centrifuge in
ultra centrifuges.
• The particles travel through the gradient until they reach a point at which
their density matches with the density of surrounding sucrose.
• The the fraction is removed and analyzed.
Safety consideration
• Lid off
• Balance
• Manual manipulation
• Breakage
• Solid surface
•Thanks