The centrifugation its principle, terminolgies, formulae, classification, rotors and its types, applications

1. CENTRIFUGATION
&
TYPES OF ROTORS
BY
MAHEK SHARAN
INT.MTECH. BIOTECH
Uttar Pradesh, India

2. Contents
 Centrifugation
 Basis of classification
 Types of centrifugation
 Principle
 Terminologies and formulae
 Parts of centrifugation
 Rotors and its types
 Applications

3. Centrifugation
 A technique which involves the separation of the particles when centrifugal force is applied
 The particles are separated according to their
1. Mass
2. Size
3. Viscosity of the medium
4. Density
5. Rotor speed
 This technique is not only used to separate the particles or miscible liquids but also to analyse
the hydrodynamic properties

4. Basis of classification
 Presence or absence of vacuum
 Temperature manipulation
 Capacity or volume of the solution that could be separated
 Speed of the rotor

5. Two main types of centrifugation
 ANALYTICAL CENTRIFUGATION
 It is the study of the purified macromolecule
or the assemblies of supramolecules
 It is used for the analysis of the
concentration, conformational changes,
ligand binding of the molecules
 3 systems by which it Is analysed
 PREPARATIVE CENTRIFUGATION
 The actual separation of the cells, subcellular
structures, vesicles and other particles
 The preparative is used for the measurement
of the mass, density hydrodyanamic
properties and the sedimentation coefficient
of the particles
Rayleigh interferometric system
Schleiren system
Light absorption system

6. The categories of centrifugation classification
Centrifugation
Preparative
Differential
Density
gradient
Discontinuous
Continuos
Analytical

7. The different types of centrifugation
 Preparative centrifugation
 Analytical centrifugation
 Large scale centrifugation
 Small scale centrifugation
 Large capacity low speed centrifugation
 Refrigerated high speed centrifugation

8. Basic principle of sedimentation
 Whenever the particles undergo acceleration in the centrifugal field( outward force), they tend to
sediment according to their mass, density
 Frictional coefficient which applies on the heavy mass particles just in the opposite direction of
centrifugal acceleration, also plays an important role in the sedimentation of the particles
 The particles when move, the velocity increase which increases the radial distance
 In a nutshell, particles with higher density sinks, thus forming pellet and the particles with lower
density remains In solution, known as supernatant
 The greater is the density difference between the particles, the faster they separate

9. Terminologies and formulae to know
 Centrifugal field
 The average angular velocity of the rigid body that rotate at fixed axis i.e., the ratio of the angular
displacement at a given time
G= ω2r
 By this the speed of rotors (angular velocity) could be determines i.e. Revolution per minute
ω= 2 π (RPM)/60
 Whereas the strength of the fields generated by different rotors at different speeds, i.e., ratio of
centrifugal acceleration at specific radius and speed of standard acceleration due to gravity is called
Relative centrifugal field
RCF=1.12 × 10-5 (RPM)2
Where, G= centrifugal field, ω= angular velocity, r= radial distance, RPM= revolution per minute

10. Terminologies and formulae to know
 Sedimentation coefficient
 it is a characteristic property of the particle
 The ratio of applied sedimentation velocity to the applied centrifugal acceleration
s= v/ ω2r
 The biological molecules have relatively small sedimentation coefficient, thus expressed in
Svedberg Unit (S), it is mainly used for the measurement of the size of the particle based on its
sedimentation rate under acceleration
S= 10-13 s

11. Parts of the centrifuge machine
LID
ROTOR
SAMPLE
LATCH
CHAMBER
CONTROL
MOTOR

12. Rotors and its types
ROTORS
FIXED ANGLE
VERTICAL
ROTOR
SWING
BUCKET
• The rotors are the parts of the centrifuge which
determines the types of centrifuge based on its
speed
• The rotors of different types are made of different
materials
• The down-speed rotor are made up of steel or brass
• The high-speed rotors are made up of either
Aluminium or Fibre

13. Fixed angle
• It is ideal for the differential separation like of
nuclei, mitochondria and microsomes
• It has thick walled tubes as the force on the tubes
increase drastically
• The isopycnic banding is formed during this
centrifugation which means the particles are
separated according to buoyancy and density
• The angle between tube from the centre rod is
14-40°

14. The labelled diagram

15. Vertical rotor
• The tubes on the rotor is parallel to the axis of
rotation
• The isopycnic separation resulting in banding is
shorter than the swing bucket
• The tubes are at a fixed radial distance from the axis
of rotation
• Two subtypes of the vertical rotors are present
True vertical rotor
•The tube angle remains same from all direction
Near vertical rotor
•The radial tube angle reduced to 7-10° for faster run and shorter path length
•Used for the gradient centrifugation

16. The labelled diagram

17. Swing bucket
• This is established in vacuum
• Thin walled tubes made of polymers are
used
• The tubes are loaded in vertical position
• When the acceleration starts, the tubes
swing out horizontally

18. The labelled diagram

19. Application
 Separation of membrane vesicle from cell with organelles of different densities
 Subcellular fractionation
 Affinity purification
 To determine the molecular density and concentration of the particles
 To separate the different organelles
 To identify new molecules and its properties
 To analyse the hydrodynamic properties of the particle