A presentation on the principles and uses of the technique of Ultracentrifugation

1. ULTRACENTRIFUGATION
GVM/16– 046
I M.V.Sc
Departmentof VeterinaryPublic Healthand Epidemiology
NTR CVSc,Gannavaram

2. CENTRIFUGE
A centrifuge is a device for
separating particles from a
solution according to
their size, shape, density,
viscosity of the medium
and rotor speed.

3. HISTORY
 Swedish Biochemist
Theoder Svedberg
invented the
Ultracentrifuge in 1923.
 Won the Nobel Prize in
chemistry in 1926 for his
research on colloids and
protein using the
ultracentrifuge.

4. ULTRACENTRIFUGE

5. ULTRACENTRIFUGATION
• It is an important tool in biochemical research.
• Rapid spinning imposes high centrifugal forces on
suspended particles, or even molecules in solution
• Causes separations of such matter on the basis of
differences in weight.
• Its rotational speed up to 150,000 rpm.
• It creates a centrifugal force up to 900,000 g.

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

7. THE PHYSICS OF ULTRACENTRIFUGATION
1.Centrifugal force:-
• The outward force experienced by a particle in circular motion
• The tube containing the suspension of particles is rotated at a
high speed, which exerts a centrifugal force directed from the
centre of the rotor towards the bottom of the tube.

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

9. Centrifugal field :-
• field where centrifugal force is experienced.
• Depends on the radical distance of the particle from the
rotation axis and the square of the angular velocity.

10. Angular Velocity:-
• Rate of rotation around an axis
• Detect to revolution per minute (r.p.m)

11. 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 number

12. 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 (= 10-13 second).
 This equation indicates that ‘S’ is dependent upon the
molecular weight, the density and the frictional coefficient.

13. STOKES EQUATION
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

14. 1. More density  Faster sedimentation
2. More massive  Faster sedimentation
3. Denser biological buffer system slower movement of
particle
4. More frictional coefficient  slower movement
5. More centrifugal force  faster sedimentation
6. Sedimentation rate = 0, if density of particle = density of the
surrounding medium
PRINCIPLESOF SEDIMENTATION

15. 1. Analytical ultracentrifugation:- The aim of Analytical
ultracentrifugation is use to study molecular interactions
between macromolecules or to analyse the properties of
sedimenting particles such as their apparent molecular
weight.
2. Preparative ultracentrifugation:- The aim of Preparative
ultracentrifugation to isolate and purify specific particles such
as subcellular organelles.
TYPES OF ULTRACENTRIFUGATION:

16. ANALYTICAL CENTRIFUGE
• Used for performing physical measurements on sample during
sedimentation.
• Sedimentation coefficient used to characterize changes in the
size and shape of macromolecules with changing experimental
conditions.
• Concentration distributions measured by Schlieren system or
Raleigh interferometric system.

17. ANALYTICAL CENTRIFUGE
Two kinds of experiments are commonly performed on these
instruments:
1. Sedimentation velocity experiments
2. Sedimentation equilibrium experiments

18. 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.
SEDIMENTATIONVELOCITY EXPERIMENTS

19. 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.
SEDIMENTATIONEQUILIBRIUM EXPERIMENTS

20. • Designed for sample preparation
• Lack sample observation facility
PREPARATIVE ULTRACENTRIFUGATION
Types of preparative ultracentrifugation:
• Differential ultracentrifugation
• Density gradient ultracentrifugation

21. • 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.
DIFFERENTIAL ULTRACENTRIFUGATION

22. • Based on density difference.
• Sample layered on top of preformed density gradient.
• Caesium 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.
DENSITYGRADIENT ULTRACENTRIFUGATION

23. • Sucrose – a sugar
• Glycerol
• Ficoll – a polysaccharide
• Percoll – a colloidal silica
• Caesium chloride – chemical
GRADIENTS

24. 1. Zonal or Rate
2. Isopycnic
TYPES OF DENSITYGRADIENT
ULTRACENTRIFUGATION

25. • 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.
ZONAL OR RATE CENTRIFUGATION

27. • 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.
• Caesium chloride or Caesium 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.
ISOPYCNICCENTRIFUGATION

28. • Bands collected as separate fractions.
• Used for separating sample whose components have a range
of densities.
• Used for nucleic acids, viruses and certain subcellular
organelles.
• Not used for proteins as they have similar densities.
• Used to show semi conservative replication of DNA.
ISOPYCNICCENTRIFUGATION

30. Schematic presentation ofa ultracentrifuge:

31. Schematic presentation ofa ultracentrifuge:
Fig; A Beckman Ultracentrifugation.

32. 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.
FUNCTIONSOF ANALYTICAL AND PREPARATIVE
ULTRACENTRIFUGATION

33. 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
Molecular weight.
 Generally used to separate organelles and molecules. Most
centrifugation work done using preparative ultracentrifuge

34. ROTOR
 Four types of rotors are available for ultracentrifugation,
1. Fixed-angle rotor,
2. Swinging-bucket rotor,
3. Vertical rotor and
4. Near-vertical rotor.
.

35. ROTOR
 Rotors are made from either aluminium or titanium, or from
fiber-reinforced composites.
 A titanium rotor is designated by T or Ti, as in the Type 100 Ti,
the SW 55 Ti, or the NVT 90 rotor.
 A composite rotor (fiber) is designated by C, as in VC 53.
 A aluminium rotor is designated by AC, as in VAC 50.
 Rotors without the T, Ti, C, or AC designation (such as the Type
25) are fabricated from an aluminium alloy.

36.  Titanium rotors are stronger and more chemical resistant than
the aluminium 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.

37. FIXED ANGLE ROTOR
 Fixed-angle rotors are general-
purpose rotors that are especially
useful for pelleting subcellular
particles and in short column
banding of viruses and subcellular
organelles.
 Tubes are held at an angle (usually
20 to 45 degrees) to the axis of
rotation in numbered tube cavities.

38. SWINGINGBUCKET ROTOR
 Swinging-bucket rotor are used
for pelleting, isopycnic studies
and rate zonal studies.
 Tubes are attached to the rotor
body by hinge pins or a
crossbar. The buckets swing
out to a horizontal position.

39. VERTICAL ROTOR
 Vertical rotors hold tubes
parallel to the axis of rotation;
therefore, bands separate
across the diameter of the tube
rather than down the length of
the tube.
 Vertical rotors are useful for
isopycnic and, in some cases,
rate zonal separations when
run time reduction is important.

40. NEAR VERTICAL ROTOR
 Near-vertical rotors are designed
for gradient centrifugation when
there are components in a sample
mixture that do not participate in
the gradient.
 Tubes are held at an angle (typically
7 to 10 degrees) to the axis of
rotation in numbered tube cavities.
 In this rotor used only Quick-Seal
and Opti-Seal tubes.

42. Common Centrifuge Classesand TheirApplications
( ) = can be done but not usually used for this purpose.

43. 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

44. ROTOR BALANCE
 The mass of a properly loaded rotor will be evenly distributed
on the ultracentrifuge drive hub, causing the rotor to turn
smoothly with the drive.
 An improperly loaded rotor will be unbalanced; consistent
running of unbalanced rotors will reduce ultracentrifuge drive
life.
 To balance the rotor load, fill all opposing tubes to the same
level with liquid of the same density.
 Weight of opposing tubes must be distributed equally.
 Place tubes in the rotor symmetrically.

45. CARE OF CENTRIFUGESAND ROTORS
 Select the proper operating conditions on the instrument.
 Check the rotor chamber for cleanliness and for damage.
 Select the proper rotor. Many sizes and types are available.
 Be sure the rotor is clean and undamaged.
 Filled centrifuge tubes or bottles should be weighed carefully
and balanced before centrifugation.

46. CARE OF CENTRIFUGESAND ROTORS
 Rotor manufactures provide a max. allowable speed limit for
each rotor. Do nor exceed that limit.
 Keep an accurate record of centrifuge and rotor use.
 If an unusual noise or vibration develops during
centrifugation, immediately turn the centrifuge off.
 Carefully clean the rotor chamber and rotor after
centrifugation.