Rotors , centrifugation and its types

1. CENTRIFUGATION
AND
ITS TYPES
DR. KRATIKA PATHAK ​

2. INTRODUCTION
CENTRIFUGATION
AND
ITS
TYPES
2
Definition
Centrifugation is a mechanical process which involves the
use of the centrifugal force to separate particles from a
solution according to their size, shape, density, medium
viscosity and rotor speed.
A dutch
mathematician, by the
name of Christiaan
Huygens
He coined the term
centrifugal force in his
work De vi centrifuga
1659
Benjamin Robins
would use this
idea to
determine drag,
inventing a
whirling arm
apparatus
1700s
Antonin Prandtl
put the idea to
work with a
hand-operated
centrifuge
1864 1925
Theodor
Svedberg created the
analytical ultracentrifuge
capable of reaching
900,000 x g. This early
analytical instrument
helped in his research of
colloids and proteins,
earning him a Nobel

3. PRINCIPLE
If a mix sample is kept steady still the large molecules will settle under the
influence of gravitational force. Thus ;
Rate of sedimentation ∝ force applied
∝ Density of particle and medium
∝ Radius of the particle
∝ Viscosity of the medium
Condition: higher sedimentation rate if force applied is more that than
gravitational force (g).
The principle on which the separation is based on Centrifugal Force (F)
i.e.
F = 𝒓𝝎𝟐 or F = 𝒓𝝎𝟐 /980
r = distance between the particle in the sample holder and rotor axis
𝝎 = 𝐚𝐧𝐠𝐮𝐥𝐚𝐫 𝐯𝐞𝐥𝐨𝐜𝐢𝐭𝐲
F is experienced in rcf (relative centrifugal force) or rpm (rotation per
minute)
𝝎 = 𝝅 (𝒓𝒑𝒎)/𝟑𝟎
OR
-5 2

4. Centrifuges
Small bench
centrifuge
(4000-6000 rpm)
Large capacity
refrigerated
centrifuge
(up to 6000 rpm)
High Speed
(maximum speed
24,000 -90,000g)
Ultracentrifuge
maximum speed
(75,000 -500,000g)
Analytical Ultra
Centrifuge
Preparatory ultra
centrifuge
Differential
Centrifuge
Density Gradient
Rate Zonal
Centrifugatio
n
Isopycnic
Centrifugati
on
• Used to collect small
amount of material that
rapidly sediments
(yeast, erythrocytes)
• Operate at ambient
temperature
• Application: rapid
centrifugation of blood
samples and
synaptosomes used to
study the effect of
drugs
• Refrigerated rotor
chambers
• Inter changeable rotor
• Yeast, nuclei and
chloroplast (Large capacity)
• Microorganisms , cellular
debris , lagers cellular
organelles and protein
precipitated by ammonium
sulphate not for viruses and
smaller organelles such as
ribosomes ( high speed
centrifuges
Continuous flow centrifugation
• They are simple high-speed
centrifuges with 25000 rpm
• The rotors, through which
particles suspended in medium
flow continuously , is long ,
tubular and non-interchangeable
• Medium enters rotating rotors,
particles sedimented against its
wall and excess clarified
medium overflows through an
outlet port
• Can harvest bac and yeast cell
from large volumes of culture
medium

5. • In an analytical ultracentrifuge
(commonly abbreviated as AUC), a
sample’s sedimentation profile is
monitored in real time by an optical
detection system.
• Analytical Ultracentrifugation gives
access to geometric (size, shape,
structure) and thermodynamic
properties (equilibrium constants,
free energies, enthalpies,
entropies)
• Many macromolecules including
proteins have an inherent property
to form aggregates which changes
the molecular weight of the species
formed, and analytical
centrifugation provides valuable
information regarding the molecular
weight of the species.
ANALYTICAL CENTRIFUGES

6. DIFFERENTIALS CENTRIFUGATION
• Separatory method to differentiate particles is based on Size and Density
• Centrifugation will initially sperate the largest particles
• For particles with same mass, one with higher density will sediment fast

7. DENSITY CENTRIFUGATION
Centrifugal
force
Centrifugal
force
Continuously
increasing
Density
Continuously
increasing
Density
Centrifugal force acting
on the particle
Higher density
acts as a
cushions
Rate Zonal
Centrifugation
Isopycnic Centrifugation
Zone of Small
particles
Zone of medium
particles
Zone of large
particles
Position
of
zones
at
the
termination
a b

8. ROTORS
CENTRIFUGATION
AND
ITS
TYPES
8
Fixed
angle
Rotors
• Rotors have holes
in the body where
centrifuge can be
placed
• Holes are placed
at an angle 14-40°
• The particle move
radially outwards
• Particles slide
down the wall
Vertical
Tube
Rotors
• Holes lie parallel to
the rotor shaft and
not at the angle
• Under the
centrifugal field
the solution
reorients 90° to
the axis of rotation
Swinging-bucket
rotors
• The bucket swing
out to a horizontal
position when the
rotor accelerates
• The solution
reorients to lie
perpendicular to
the axis of rotation
and parallel to the
applied centrifugal
field

9. CENTRIFUGATION
AND
ITS
TYPES
9
Fixed
angle
Rotors
Vertical
Tube
Rotors
Swinging-bucket
rotors

10. 1 0
PRESENTATION
TITLE
Sedimentation pattern of homogenised solution under centrifugal field

11. THANK YOU