CENTRIFUGATION

Centrifugation is the most popular technique used in biological sciences for separation
of particles / microorganisms by centrifuge.  A centrifuge is a device for separation of
microorganisms from the suspended fluid using centrifugal force (g-force).  Particles
separate according to their size, shape, density, viscosity of the medium and rotor speed.
 Centrifugation can only be used when the dispersed material is denser than the
medium.  In a solution, higher density particles sediment in influence of gravitational
field.  Movement of particle under the gravitational force is called sedimentation.

GRAVITATIONAL FORCE:
Newton’s Law of Universal Gravitation is used to explain gravitational force. This law
states that every massive particle in the universe attracts every other massive particle
with a force which is directly proportional to the product of their masses and inversely
proportional to the square of the distance between them. The gravitational force on
Earth is equal to the force the Earth exerts on you. At rest, on or near the surface of the
Earth, the gravitational force equals your weight.

CENTRIFUGAL FORCE:
When centrifugal force applied by the centrifuge, particles move faster (> g). For
example, when sand particles added in the water filled bucket it travels slower but it
sediment faster when bucket is swung around in a circle. The biological materials show
a drastic increase sedimentation when they undergo under acceleration in centrifugal
force.

CENTRIPETAL FORCE
A centripetal force is a net force that acts on an object to keep it moving along a circular
path. Its direction is always orthogonal to the motion of the body and towards the fixed
point of the instantaneous center of curvature of the path.
  Fc = Centripetal force
 m = mass
 v = velocity
 r = radius

STROKE’S LAW:

The force of viscosity on a small sphere moving through a viscous fluid is given by:

where:

 F is the frictional force – known as Stokes' drag – acting on the interface

between the fluid and the particle
 μ is the dynamic viscosity (some authors use the symbol η)
 R is the radius of the spherical object
 v is the flow velocity relative to the object.

PRINIPLES OF SEDIMENTATION:
The centrifuge involves principle of sedimentation, where the acceleration at centripetal
force causes denser substances to separate out along the radial direction at the bottom
of the tube. By the same concept lighter objects will tend to move to the top of the tube;
in the rotating picture, move to the center. 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.

Sedimentation is the tendency for particles in suspension to settle out of the fluid in
which they are entrained, and come to rest against a barrier. This is due to their motion
through the fluid in response to the forces acting on them: these forces can be due to
gravity, centrifugal acceleration or electromagnetism. Settling is the falling of
suspended particles through the liquid, whereas sedimentation is the termination of the
settling process.

Svedberg Equation:
The single most important advance in the use of centrifugal force to separate
biologically important substances was the coupling of mechanics, optics and
mathematics by T. Svedberg and J.W. Williams in the 1920’s. They initiated the
mathematics and advanced the instrumentation to a point where it was possible to prove
that proteins were large molecules that could be weighed in a centrifuge. In honor of
that work, the value for a molecule’s (or organelle’s) sedimentation.

RPM:
Revolutions per minute is the number of turns in one minute. It is a unit of rotational
speed or the frequency of rotation around a fixed axis. 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. Basically, it is telling us how fast the rotor is spinning.
Centrifuges will have a speed range that they are capable of achieving and will vary
depending on the centrifuge. A low-speed centrifuge might spin at as low as 300 RPM,
while a high-speed centrifuge could spin up to 15000 RPM. Ultracentrifuges are also
available and are the most powerful type of centrifuge, they can spin in excess of
150,000 RPM.

ROTOR:
The centrifuge is composed of a rotor, which is used to house the tubes where separation
occurs. There are two main types of centrifuge rotors: fixed-angle or swinging bucket.
Fixed-angle rotors hold tubes at a stable angle (typically 45°) relative to the axis of
rotation. A swinging-bucket rotor swings out when centripetal force is applied and holds
the cells at an approximate 90° angle relative to the angle of rotation. Recall that more
dense materials will separate towards the angle of the centripetal force. Therefore in
swinging-bucket rotors, the pelleted material will form at the bottom of the conical
centrifuge tube, while tubes processed in a fixed-angle rotor will form sedimentation on
the side. This could be problematic if the solids get caught in the angle of the tube.

PREPARATIVE Vs ANALYTICAL CENTRIFUGATION

TYPES:
Differential Centrifugation
 It is the most common type of centrifugation employed.
 Tissue such as the liver is homogenized at 32 degrees in a sucrose solution that
contains buffer.
 The homogenate is then placed in a centrifuge and spun at constant centrifugal
force at a constant temperature.
 After some time a sediment forms at the bottom of a centrifuge called pellet and
an overlying solution called supernatant.
 The overlying solution is then placed in another centrifuge tube which is then
rotated at higher speeds in progressing steps.

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
ultracentrifuges.
 The particles travel through the gradient until they reach a point at which their
density matches the density of surrounding sucrose.
 The fraction is removed and analyzed.

APPLICATIONS OF CENTRIFUGATION
 To separate two miscible substances
 To analyze the hydrodynamic properties of macromolecules
 Purification of mammalian cells
 Fractionation of subcellular organelles (including membranes/membrane
fractions) Fractionation of membrane vesicles
  Separating chalk powder from water
 Removing fat from milk to produce skimmed milk
 Separating particles from an air-flow using cyclonic separation
 The clarification and stabilization of wine
 Separation of urine components and blood components in forensic and research
laboratories
 Aids in the separation of proteins using purification techniques such as salting
out, e.g. ammonium sulfate precipitation.

APPLICATIONS IN BIOLOGICAL SCIENCES

 To separate cellular and subcellular components
 Separating one cell type from another.
 Removing cells or other suspended particles from their surrounding milieu on
either a batch or a continuous-flow basis.
 Isolating viruses and macromolecules, including DNA, RNA, proteins, and
lipids or establishing physical parameters of these particles from their observed
behaviour during centrifugation.
 To study the effects of centrifugal forces on cells, developing embryos, and
protozoa.
 These techniques have allowed scientists to determine certain properties about
cells, including surface tension, relative viscosity of the cytoplasm, and the
spatial and functional interrelationship of cell organelles when redistributed in
intact cells.