Principle of Centrifugation

A note on the principle of centrifugation (how centrifuge machines work?)

1. SYED MUHAMMAD KHAN ( BS HONS. ZOOLOGY)

pg. 1

Principle of Centrifugation
Centrifugation is a technique that involves the application of centrifugal force to
separate particles from a solution according to their size, shape, density, viscosity of
the medium, and rotor speed. A centrifuge is a device for separating particles from a
solution via centrifugation.
PRINCIPLE OF CENTRIFUGATION
In a solution, particles whose density is higher than that of the solvent sink (sediment),
and particles that are lighter than it float to the top. The greater the difference in
density, the faster they move. If there is no difference in density (isopycnic conditions),
the particles stay steady. To take advantage of even tiny differences in density to
separate various particles in a solution, gravity can be replaced with the much more
powerful “centrifugal force” provided by a centrifuge. There is a correlation between
the size and density of a particle and the rate that the particle separates from a
heterogeneous mixture when the only force applied is that of gravity. The larger the
size and the larger the density of the particles, the faster they separate from the
mixture. By applying a larger effective gravitational force to the mixture, like a
centrifuge does, the separation of the particles is accelerated. The remaining solution
(supernatant) may be discarded with a pipette. Centrifugation of protein solution, for
example, allows the elimination of impurities into the supernatant. The rate of
centrifugation is specified by the angular velocity usually expressed as revolutions per
minute (RPM), or acceleration expressed as g. The particles' settling velocity in
centrifugation is a function of their size and shape, centrifugal acceleration, the volume
fraction of solids present, the density difference between the particle and the liquid,
and the viscosity.
TYPES OF CENTRIFUGAT ION
The following are the types of centrifugation:

2. SYED MUHAMMAD KHAN ( BS HONS. ZOOLOGY)

pg. 2

Micro-centrifuges: These are used to process small volumes of biological molecules,
cells, or nuclei. Micro-centrifuge tubes generally hold 0.5 to 2.0 mL of liquid and are
spun at maximum angular speeds of 12,000–13,000 rpm. Micro-centrifuges are small
enough to fit on a table-top and have rotors that can quickly change speeds. They may
or may not have a refrigeration function.
High-speed Centrifuges / Super-speed Centrifuges: These can handle larger sample
volumes, from a few tens of milliliters to several liters. Additionally, larger centrifuges
can also reach higher angular velocities (around 30,000 rpm). The rotors may come
with different adapters to hold various sizes of test tubes, bottles, or micro-titer plates.
Ultracentrifugation: This makes use of high centrifugal force for studying the properties
of biological particles. Compared to micro-centrifuges or high-speed centrifuges,
ultracentrifuges can isolate much smaller particles, including ribosomes, proteins, and
viruses. Ultracentrifuges can also be used in the study of membrane fractionation. This
occurs because ultracentrifuges can reach maximum angular velocities over 70,000
rpm. Additionally, while micro-centrifuges and super-centrifuges separate particles in
batches (limited volumes of samples must be handled manually in test tubes or
bottles), ultracentrifuges can separate molecules in batch or continuous flow systems.
In addition to purification, analytical ultracentrifugation (AUC) can be used for the
determination of the properties of macromolecules such as shape, mass, composition,
and conformation. Molecular properties can be modeled through a sedimentation
velocity analysis of sedimentation equilibrium analysis. During the run, the particle or
molecules will migrate through the test tube at different speeds depending on their
physical properties and the properties of the solution, and eventually form a pellet at
the bottom of the tube, or bands at various heights.
Density Gradient Centrifugation: This is considered one of the more efficient methods
of separating suspended particles. Density gradient centrifugation can be used both
as a separation technique and as a method of measuring the densities of particles or
molecules in a mixture. A tube, after being centrifuged by this method, has particles in

3. SYED MUHAMMAD KHAN ( BS HONS. ZOOLOGY)

pg. 3

order of density based on height. The object or particle of interest will reside in the
position within the tube corresponding to its density. This method was used in
Meselson and Stahl’s famous experiment in which they proved that DNA replication is
semi-conservative by using different isotopes of nitrogen. They used density gradient
centrifugation to determine which isotope or isotopes of nitrogen were present in the
DNA after cycles of replication.
Differential Centrifugation: Differential Centrifugation is a type of centrifugation in
which one selectively spins down components of a mixture by a series of increasing
centrifugation forces. This method is commonly used to separate organelles and
membranes found in cells. Organelles generally differ from each other in density in
size, making the use of differential centrifugation, and centrifugation in general,
possible. The organelles can then be identified by testing for indicators that are unique
to the specific organelles.