Download Centrifuge A centrifuge is a device for separating particles from a

Centrifuge
A centrifuge is a device for separating particles from a solution according to their size, shape,
density, viscosity of the medium and rotor speed. Centrifugation is a separation process which
uses the action of centrifugal force to promote accelerated settling of particles in a solid-liquid
mixture.Centrifuge is used to separate particles or macromolecules:Cells, Sub-cellular
components, Proteins,Nucleic acids, etc. The separations of particles arebased on mainly Size,
Shape and Density. The technique of separation utilizes density difference between
theparticles/macromolecules and the medium in which these aredispersed. The dispersed systems
are subjected to artificially inducegravitational fields for separation.
[In filtration the gravitational force is at work, where the fluid flows down through the filtration
medium (cloth or paper) leaving behind the suspended particles on the medium. An artificially
created centrifugal force is employed in the equipment called centrifuge to achieve the same
objective. In molecular biology and biochemistry work, the centrifuge is used to separate cells,
organelles and different biomolecules from suspensions and from one another.]
Principles of centrifugation
The centrifuge works using the sedimentation principle, where the centripetal acceleration
causes denser substances to separate out along the radial direction (the bottom of the tube). Many
particles or cells in a liquid suspensionwill eventually settle at the bottom of a container due to
gravity (1 x g). However, the length of time required for such separations is impractical. Other
particles, extremely small in size, will not separate at all in solution, unless subjected to high
centrifugal force. When a suspension is rotated at a certain speed or revolutions per minute
(RPM), centrifugal force causes the particles to move radially away from the axis of rotation.
The force on the particles (compared to gravity) is called Relative Centrifugal Force (RCF). For
example, an RCF of 500 x g indicates that the centrifugal force applied is 500 times greater than
Earth’s gravitational force.
Centrifugal force F = Mω2r
M: mass of particle, r: radius of rotation (cm) (iedistance of particle from axis of rotation)
ω:Average angular velocity (radians/sec)
ω = 2π revmin-1 / 60 Rev: revolution per minute (r.p.m.), 1 revolution = 2π radians =360
[Centrifugal forces: Solid particles denser than the liquid
migrate radially outward toward the wall of the rotating
cylinder (sedimentation). Centripetal forces: Particles less
dense than the liquid migrate radially inward toward the
axis until they reach at air liquid interface (floating).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
(isopyknic 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.]
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Relative Centrifugal Force,
RCF = fc / fg = Mω2r / Mg = ω2rg-1
RCF = (2π rpm / 60)2r x g-1
i.e. RCFvalue is "No. x g"(multiples of earth's gravitational force).
Laboratory scale centrifuge : up to 106g and Industrialscale centrifuge : up to 18000 g
Centrifuge Instrument: The basic component of a centrifuge is a rotorthat holds the centrifuge
tubes. The rotor is spun at very high speed via a drive shaft connected to an electric motor. This
causes the suspended particles to sediment at the bottom of the tube in a compacted state called
the pellet with the clear fluid at the top called the supernatant.Protocols for centrifugation
typically specify the amount of acceleration to be applied to the sample, rather than specifying a
rotational speed such as revolutions per minute. This distinction is important because two rotors
with different diameters running at the same rotational speed will subject samples to different
accelerations.
Broadly, three types of centrifuges are known:
Clinical centrifuge can attain 4000 – 6000 revolutions per minute (rpm) spinning speed, which
is adequate for separation of cells from suspension fluids (e.g. microbial cells from urine
samples).
High speed centrifuge is employed for isolation of cell organelles, such as nuclei, insoluble
biomolecules such as the precipitated protein etc as an rpm of up to 25,000 can be reached in it.
Ultracentrifuge can reach spinning speeds around 75,000 rpm which enables the separation of
the biomolecules such as DNA, RNA, plasmids etc. Owing to such high speed of operation, the
rotor is housed in a refrigerated chamber under vacuum.
Centrifuge: Its Use and Safety
Mechanical stress
1. Always ensure that loads are evenly balanced before a run.
2. Always observe the manufacturers maximum speed and sample density ratings.
3. Always observe speed reductions when running high density solutions, plastic adapters,
or stainless steel tubes.
Centrifuge machine
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Corrosion
Many rotors are made from either titanium or aluminum alloy, chosenfor their advantageous
mechanical properties. While titanium alloys arequite corrosion-resistant, aluminum alloys are
not. When corrosionoccurs, the metal is weakened and less able to bear the stress from
thecentrifugal force exerted during operation. The combination of stress andcorrosion causes the
rotor to fail more quickly and at lower stresslevels than an uncorroded rotor.
Analytical Ultracentrifugation
An analytical ultracentrifuge spins a rotor at an accuratelycontrolled speed and temperature. The
concentrationdistribution of the sample is determined at known timesusing absorbance
measurements.
Isolation of organelles using centrifugation
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