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2} ISOPYCNIC Centrifugation:
Isopycnic means of the same density.
Molecules separated on equilibrium position.
Each molecule floats or sinks to position where
density.
Schematic presentation of a Ultracentrifuge:
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.
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.
Rotors are made from either aluminum 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 aluminum 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 aluminum alloy.
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.
1.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.
Centrifugal Force:
Where,
M: mass of particle
r: radius of rotation (cm) (ie distance of particle from axis of rotation)
:Average angular velocity (radians/sec)
Centrifugal field :- Depends on the radical distance of
the particle from the rotation axis and the square of the
angular velocity.
OR
Rate of Sedimentation:
Where,
r = radius at which the organelle is located
t = time
M = molecular weight
= partial specific volume of the molecule; inverse of the
density
= density of the solvent
f = translational frictional coefficient
= angular velocity
NA = Avagadros number
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: