ppt on ultracentrifugation

4. Ultracentrifuge

6. What happens to a particle in a centrifugal field
The particle (m) is acted on by three forces:
FC: the centrifugal force
FB: the buoyant force
Ff: the frictional force between the particle and the liquid
Equation that describes the motion of this particle as follows:
F = ma
where m is the mass of the particle and a is the acceleration.

7. M: mass of particle
r: radius of rotation (cm) (ie distance of particle from axis of
rotation)
ω :Average angular velocity (radians/sec)

8. Centrifugal field :-
• field where centrifugal force is experiended.
• Depends on the radical distance of the particle from the
rotation axis and the square of the angular velocity.
Angular Velocity:-
• Rate of rotation around an axis
• Detect to revolution per minute (r.p.m)

9. 2.Sedimentation rate:- This force acts on the suspended particles
pushing them towards the bottom of the tube at a rate determined by
the velocity of the spinning rotor.
Rate of Sedimentation:
Where,
r = radius at which the organelle is located
t = time
M = molecular weight
ν = partial specific volume of the molecule; inverse of the
density
ρ = density of the solvent
f = translational frictional coefficient
ω = angular velocity
N = Avagadro’s numberA

10. 3.Sedimentation coefficient:- Centrifugation separates
particles in a suspension based on differences in size, shape and
density that together define their sedimentation coefficient.
Sedimentation Coefficient:
 This is known as the Svedberg equation and is usually
expressed in Svedberg units,
S (= second).
 This equation indicates that ‘S’ is dependent upon the molecular
weight, the density and the frictional coefficient.

11. Frictional coefficient,
f = 6πηr
where, r : particle radius
η : viscosity of solution
• f is minimal when particle is a sphere
• Non spherical particle has larger surface area and thus
greater value of f
p
p

12. Types of Ultracentrifugation:

13. • Used for performing physical measurements on sample during
sedimentation.
• Sedimentation coefficient used to charecterise changes in the
size and shape of macromolecules with changing experimental
conditions.
Two kinds of experiments are commonly performed on
these instruments:
1. Sedimentation velocity experiments
2. Sedimentation equilibrium experiments

14. 1. Sedimentation velocity experiments:-
• to estimate sample purity
• Aim of SVEs to interpret the entire time-course of
sedimentation, and report on the shape and molar mass of
the dissolved macromolecules, as well as their size
distribution.
• Components observed as peaks.
2, Sedimentation equilibrium experiments:-
• SEEs are concerned only with the final steady-state of the
experiment, where sedimentation is balanced by diffusion
opposing the concentration gradients, resulting in a time-
independent concentration profile.

15. • Designed for sample preparation
• Lack sample observation facility
Types of preparative centrifugation:
1. Differential ultracentrifugation:-
• used to separate certain organelles from whole cells for further
analysis of specific parts of cells.
• Based on differences in sedimentation rate of particles.
• Crude tissue homogenate divided into different fractions by
stepwise increase in applied centrifugal field.
• Largest sediment faster followed by smaller particles.
• Rpm gradually increased to sediment particles.

16. 2. Density gradient ultracentrifugation:-
• Based on denstiy difference.
• Sample layered on top of preformed density gradient.
• Cesium chloride density gradient is widely used for DNA,
isolation of plasmids, nucleoproteins and viruses.
• Sodium bromide and sodium iodide for fractionation of
lipoproteins.
• Max density of gradient must exceed density of most dense
particle of the sample.
• Step wise gradient and continuous gradient applied.
• There are two types of density gradient ultracentrifugations
under preparative ultracentrifugation such as.
1. ZONAL or RATE
2. ISOPYCNIC

17. 1} ZONAL or RATE Centrifugation:
• Mixture to be separated is layered on top of a gradient
(increasing concentration down the tube).
• Provides gravitational stability as different species.
• Move down tube at different rates.
• Sucrose gradient is commonly used to create zones of
different gradient.
• Separation based on molecular masses.
• Fractionation achieved by puncturing bottom of celluloid
centrifuge tube.

19. 2} ISOPYCNIC Centrifugation:
• Isopycnic means “of the same density”.
• Molecules separated on equilibrium position.
• Sample dissolved in relatively concentrated solution of dense, fast
diffusing substance and spun at high speeds until solution
achieves equilibrium.
• Cesium chloride or cesium sulphate used.
• High centrifugal field causes low molecular mass solute to form a
steep density gradient in which the sample components band at
positions where their densities are equal to that of solution.
• Bands collected as separate fractions.
• Used for separating sample whose components have a range of
densities.
• Used for nucleic acids, viruses and certain subcellular organels.
• Not used for proteins as they have similar densities.
• Used to show semi conservative replication of DNA.

21. Schematic presentation of a Ultracentrifuge:
Fig; A Beckman Ultracentrifugation.

22. Functions of analytical and preparative
Ultracentrifugation:
Analytical
 Uses small sample size (less than 1 ml).
 Built in optical system to analyze progress of molecules during
centrifugation.
 Uses relatively pure sample.
 Used to precisely determine sedimentation coefficient and MW
of molecules.
 Beckman Model E is an example of centrifuge used for these
purposes.
.

23. Preparative
 Larger sample size can be used.
 No optical read-out collect fractions and analyze them
after the run.
 Less pure sample can be used.
 Can be used to estimate sedimentation coefficient and
MW.
 Generally used to separate organelles and molecules. Most
centrifugation work done using preparative
ultracentrifuge

24. 1. Fixed-angle rotor,
2. Swinging-bucket rotor,
3. Vertical rotor and
4. Near-vertical rotor.

25.  Titanium rotors are stronger and more chemical resistant than
the aluminum rotors.
 Exterior surfaces of titanium and composite rotors are finished
with black polyurethane paint.
 Titanium buckets and lids of high-performance rotors are
usually painted red for identification.

30. Common Centrifuge Classes and Their Applications
( ) = can be done but not usually used for this purpose.

31. Tube Type and Rotor Compatibility
Rotor Types
Tube Types Fixed-Angle Swinging-bucket Vertical
Thin wall open top No Yes No
Thick wall open top Yes Yes No
Thin wall sealed Yes Some tubes Yes
Oak ridge Yes No No
Types of Rotors and Theirs Applications
Rotor Types Pelleting R or Z-Sedimentation Isopycnic
Fixed-angle Excellent Limited Variable
S-bucket Inefficient Good Good
Vertical Not suitable Good Excellent
N-vertical Not suitable Excellent Good

34. Some High Speed Centrifuges and Rotors

35. Some Ultra Speed Centrifuges and Rotors