Capitol Tech U Doctoral Presentation - April 2024.pptx
Centrifugation
1. CENTRIFUGATION
TEAM MEMBERS : 1.SUBASHREE – 17BBT012
2.SWETHA – 17BBT022
3.AISHWARYA – 17BBT032
4.DN VAISNAVI – 17BBT057
5.AINTHA R – 17BBT062
2. CONTENTS:
■ History
■ What is centrifugation?
■ Basic principle
■ Equations
■ Factors depending upon
■ Types of centrifuge
■ Types of rotors
■ Other centrifuge techniques
■ Applications
■ Reference
3. HISTORY:
■ The first analytical ultracentrifuge –
developed by SVEDBERG in 1920.
■ Centrifuges were first used in the
mid-1400s for processes such as
separation of milk. These early
machines were hand driven and could
reach speeds up to 3000rpm. By
1912, new centrifuges were
developed which ran on electrical
power.
4. WHAT IS CENTRIFUGATION?
■ Biological centrifugation is a process that uses centrifugal forces to
separate and purify mixtures of biological particle in a liquid medium
■ Two distinct major phases are formed in the vessel during centrifugation :
■ The sediment - Usually does not have a uniform structure
■ The centrifugate or centrate which is the supernatant liquid.
5. Basic principle :
■ The basic physics on which the centrifugation works is gravity and generation of
centrifugal force to sediment different fractions.
■ Rate of sedimentation depends upon applied centrifugal field(G) being directed
radially outwards.
■ G depends upon
■ 1. Angular velocity (w in radians / sec)
■ 2. Radial distance (r in cm) of particle from axis of rotation.
■ 𝐺 = 𝑤2r
6. Basic principle:
■ A particle whether it is precipitate a macromolecule or cell organelle
when rotated at high speed is subjected to a centrifugal force .
■ Centrifugal force is defined as the a force, arising from the body's
inertia, which appears to act on a body moving in a circular path and
is directed away from the centre around which the body is moving.
■ Two forces counteract the centrifugal force acting on the suspended
particles:
■ Buoyant force: This is the force with which the particles must
displace the liquid media into which they sediment.
■ Frictional force: This is the force generated by the particles as they
migrate through the solution.
7. Basic principle:
■ Centrifugal force is defined as the 𝐹 = 𝑚𝜔2
𝑟
■ Where F = intensity of centrifugal force
■ m = effective mass of sedimenting particle
■ w = angular velocity of rotation
■ r = distance of migrating particles from central axis of rotation.
8. Difference between RPM and RCF:
RPM - Rotations per minute
■ 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.
■ According to the SI (International System of Units), the RPM is not a unit. Rotations per minute is a
measure of the “frequency” of the rotation whose SI unit is second-1 or per second.
RCF - Relative centrifugal force
■ 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 rotation.
■ Also called as G- Force.
■ (RCF), describes the amount of force of acceleration applied to a sample in a centrifuge.
■ The force is generated from the spinning of the rotor which, in turn, exerts this force outward on the centrifuge
tube.
9. RPM and RCF conversion:
. [ 1.18 x 10-5 is an empirical factor]
10.
11. FACTORS :
■ Mass of particle – density and
volume
■ Density of the medium
■ Shape of the particle
■ Friction
12.
13.
14. Low speed centrifuge (desktop centrifuge)
■ Used to sediment heavy particles.
■ Has a maximum speed of 4000- 5000 RPM.
■ These instruments usually operate at room
temperature with no means of temperature
control.
■ Two types of rotor used – fixed angle and
swinging bucket
■ It is used for sedimentation of RBC until the
particles are tightly packed into a pellet and
supernatant is separated by decantation
15. High speed centrifuge:
■ These are introduced in more biochemical
applications
■ Useful in easily controlling suitable
temperatures for biological samples.
■ Three types of rotor available for this such as
fixed angle , swinging buckets and vertical
rotors.
■ 15000 – 20000 RPM , centrifugal force of
90,000g
■ Noramally refrigerated.
■ Differentiation and separation of nucleus ,
mitochondrial , protein precipitate , large intact
cellular debris , bulky protein aggregates.
16. Ultracentrifuge:
■ Operates at a speed of 75,000 RPM with a
centrifugal force of 500,000g.
■ Stabled refrigerated system.
■ Rotor chamber is sealed and evacuated by a
pump to attain vaccum.
■ It is a high speed centrifuge with fixed head
rotors .
■ Mainly used in separation of lipoproteins.
■ Since the separation is long process there is a
generation of heat and thus provided a internal
cooling system.
■ It is used for both preparative work and analytical
work.
17.
18. Swing Bucket Rotor:
■ Supports sample volume range from 2.2 mL to 36
mL.
■ Two types of separation : Rate zonal and
isopycnic.
■ The swinging bucket rotor allows the tube to
change angle during the run. The buckets are
vertical when stopped and horizontal when
running.
■ Applications include tissue culture processing, high
throughput protocols such as harvesting whole
cells from growth, processing of blood collection
tubes and rate-zonal separations (i.e., based on
size or mass) media.
19. Fixed Angle Rotor:
■ Rotor cavities ranges from 0.2 mL to 1mL.
■ Rotor applications used mainly for pelleting
application ( to collect pellets).
■ The most important aspect in deciding to use
a fixed – angle rotor is the K factor .
■ The K factor indicates how efficient the rotor
can pellet at maximum speed .
■ The lower the K factor , the higher the
pelleting efficiency.
20. Vertical rotor:
■ Vertical rotors are highly specialized.
■ They are typically used to band DNA in cesium
chloride.
■ . Vertical rotors have very low K factors, which
is useful if the particle must only move a short
distance until it pellets.
■ Run time on vertical rotors is short.
centrifugation the clearing factor or k factor represents the relative
pelleting efficiency of a given centrifuge rotor at maximum rotation
speed. ... Since is a constant for a certain particle, this relationship can
be used to interconvert between different rotors.
21.
22.
23. Applications:
In clinical Laboratory , centrifugation is used to:
1. Remove cellular elements from blood to provide
cell free plasma or serum for analysis.
2. Remove chemically precipitated protein from an
analytical specimen.
3. Separate protein bound from free ligand in
immunochemical and other assay.
4. Separation of the subcellular organelle ,DNA ,
RNA.
5. Separate lipid components.
