Centrifugation 3.

Centrifuge
Acentrifuge is a device for separating particles from a solution
accordingtotheir size, shape, density, viscosity of the medium and rotor
speed.In a solution, particles whose density is higher than that of the
nlvent sink (sediment), and particles that are lighter than it float to the
ton The greater the differencein density, the faster they move. If there is
steady.
no difference in density (isopycnic conditions), the particles stayputs
This equipment is generally driven by an electric motor that called an
object to rotate around fixed axis, and a perpendicular force
according
oentrihogal force is applied to axis. The particles get separated speed.
rotor
to their size, shape, density, viscosity of the medium and from milk.
Examples include separation of liquids from solids like cream
Principle ofcentrifuge
of sedimentation, where the
The centrifuge involves principledenser substances to separate out
acceleration at centripetal force causes
tube. By the same concept
along the radial direction at the bottomn of the
objects will tend to move to the top of the tube; in the rotating
lighter greater the difference in density, the
picture, move to the centef. The
they move. If there is no difference in density (isopycnic
faster take advantage of even tiny
conditions), the particles stay steady. To
density to separate various particles in a solution, gravity
differences in powerful "centrifugal force"
more
can be replaced with the much technique plays an important role and is
centrifuge. This
provided by a
part of all scientific experiments involving separation of
an indispensable mixtures.
cellorganelles from complex
Principleofsedimentation r suspension tosettle out Y
Sedimentation is the tendency for particles in
which they are entrained, and come to rest against a
of the fluid in through the fluid in response to the
barrier. This is due to their motion
them: These forces can be due to gravity, centrifugal
forces acting on Settling is the falling of suspended
acceleration or electromagnetism.
the liquid, whereas sedimentation is the termination of
particles through
the settling process.
Centrifugal force "center", and
Centrifugal force, word from Latin centrum, meaning rotating body
fugere, means "to flee", is thc apparent force that draws a
aWAy from the center of rotation. Centrifugation is a technique used for
the separation of particles using a centrifugal field. The particles are
suspended in liquid medium and placed in a centrifuge tube. The tube is
then placed in arotor and spun at a definitive speed. Rotation of the rotor
3.2 Components of a centrifuge
The basic componentsof a centrifuge are tubes to hold the
holes to accommodate the
Ametal rotor with
liquid.
rotor at selective
A motor or alternative means of spinning the
speeds.
Other accessories for maintaining the environment - Sample
containers.
 gtadhtEamfll
Centrifugation M
3.6
Desígnand|Types of Rotors:
The different types of rotors uscd for low speed centrifugation are
made up of brass or steel because they experience a much Jower degree
nf stress. Rotors made of aluminium or titanium are used in bigh speeda a
centriíttgation. More recently some rotors have been made of light
weight carbon fibre composite material, which are up to 60% lighter,
resultingin faster acceleration/deceleration rates. Carbon fibre composite
rotors also are comosion-resistant, eliminating amajor cause of rotor
uilures. The various rotors used are swinging bucket rotors, fixed angle
rotors, vertical tube rotors, zonal rotors,elutriator rotors etc.
1. Swinging Bucket Rotors:
The swinging bucket rotor has buckets that start off in a vertical
position but during acceleration of the rotor swing out to a horizontal
position so that during centrifugation the tube and hence the solution in
the tube, is aligned perpendicular to the axis of rotation and parallel to
the applied centrifugal field, the tube returing to its original position .
mess and
during deceleration of the rotor. densa stace
2. Ficed Angle Rotors:
In fixed angles the tubes are located in holes in the rotor body set at a
influence
fixed angle between 14° and 40° to the vertical. Under the
short
centrifugal field, particles move radially outward and have only a
the
distance to travel before colliding with, and precipitating ison,formed
concentration that
wall of the centrifuge tube. A region of high that the
medium, with the result
has a density greater than surrounding
pellet at the outemost
precipitate sinks and collects as a smallcompact
point of the tube. haaie maso1denscy
3. Vertical Tube Rotors: Large pasdieles)
They are considered as zero angle fixed angle
rotors in which the
all times.
tubes are aligned vertically in the body of the rotors at
4. Zonal Rotors:
The
The zonal rotors may be of the batch or continuous flow type.
former being more extensively used than the latter,and are designed to
minimize the wall effect that is encountered in swinging- bucket and
fixed angle rotors, and to increase sample size.
5. Elutriator Rotors:
The elutriator is a kind of continuous flow rotor that contains
recesses to hold a single conical shaped separation chamber, the apex of
which points away from the axiS of rotation, and a bypass chamber on
3.7
Bio-Instrumentati,
de oppOsite side of the rotor that serves as a counter balance and
provide the fuid outlet.
Sample Containers
are available in different rangeofof size
Centrifuge tubes
thickness and rigidity
and bottles
rig from different variety polyethylene,
of materials incuding
cellulose, esters, polymer, polycarbonate, polypropylene
steel. The type of container used will
Kynar, nylon and stainless
upon nature and volume of sample to be centrifuged along wi
depend
centrjtugal forces to be withstood.
 amponents win ngn sedimentation rate are at the 1ower end of the
column. of the molecule also sthe
sedimentation, as sedimentation coefficient is afunction of the mass of
the paticle. The bands are formed at yarious positions_which can be
separatedby puncturing the tube and bands are collected separately.
3.4.1.2 Differential Centrifugation
In this type of centrifugation, the components of a mixture
senarated according to their size, shape and density. Generaly bigger
molecules reach the lower part of the columnn before a smaller one gets
sedimented. These particles which have the same size get sedimented
aRonding to their density difference whereas those particles which have
the same density are separated by density gradient centrifugation. This
method is used to purify subcellular organclles and macro molecules
with density gradients generated by placing layer after layer of gradient
media. In this method a homnogenous mixture of compounds is taken an
centrifuged at a fixed duration in a fixed centrifugal field. After a certain
period of time, itis found that some components are sedimented and they
form a supernatant solution.
Time of centrifugation

Sovent

Smalf-sized particles

Medium-sized particies
Lage sied patticies

sedimentation rate of the biologicalparticles of

Based on theditferences in the hase

ditferent size, shape and density

Diferential Centrifugation
centrifuged. For a fixed
The pellet part is taken out and again centrifugation
duration under fixed centrifugal field. Because the first
the pellet portien
does not give a pure pellet by repeated centrifugation of fluid
alone, a pure pellet can be obtained. The supernatant is separated
from which the particles having lowest concentration rate can be
separated out. In the end, bands of sample components are formed, the
large sized particles At the bottom of the tune and the small sized
particles present at the top of the tube. Thus the different components of
the mixture are separated.
3.13 Bio-Instrum taion
34.1.3 Density Gradient centriftugation 1203|
(As against differential centrifugation where a homogenous
is used for separation, density gradient employs mediums whËchmedi.
h
gradientsDensity centrifugation is based on the buoyant den_ities
pot on the velocities or shape or size of the pariOC and s al
independent of time. The sample is loaded into tube with gradie
forming solution (on top of or below pre-formed gradient, or mixed:
with self-forming gradients under centrifugal force). Particles move
point where their bouyant density equals that part of gradient and for
CsCl, Nalgradients are used for macromolecules and nucleotides. Oth
examples of gradient materials are :Simple sugars (sucrose, sorbitoi
glycerol), Polysaccharides (dextran and glycogen), Proteins (bovina
senumalbumin) and for inorganic salts (CsCI_ etc. - Particle move to
point, where their buoyant densities become equals that part of
gradient and forms a band. At this point of is odensity, no the
further
sedimentation occurs irrespective of how time further the sedimentation
occurs. This method is also called as is opycnic
method is much better when compared to differential centrifugation) This
centrifugation.
Sample containing
particles of
ditferent denalties

Centrltugal torce acting

on theparticle
tisher danaty :acts

(A (B)
Density Centrifugation
The process is used for simple
pelleting, for the separation of sub
cellular organelles like golgi apparatus,
other macromolecules. First, the sampleperoxisomes, mitochondria and
must be homogenised
centrifugation- sedimentation
specific volume of a depends on mass,
- ultra
shape and partial
macromolecule,
size, rate of rotation. - Usually as well as solvent density, rotor
uses fixed angle rotor. The sample is
mixed in a dense solution possessing a
diffusing property. The mixture is spun low
in a
concentration and have fast
the prOcess, the sample is to be in centrifuge but before starting
a uniform mixture. After
centrifugation, the solution forms a density gradient
components occupy these positions in the density and the sample
gradient which