Centrifugation Handout

Sedimentation Theory
A particle in a centrifugal field will experience 3
major forces:
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!
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centrifugal force (Fc) = m" r

2
buoyant force (Fb) = -mo" r
frictional force (Ff) = -fv

where m = mass of the particle, " = angular

velocity, r = distance from the axis, mo = the mass
of the displaced solution, f = frictional coefficient
and v = velocity of the particle.

Centrifugal Force

The particle will move at a velocity such that the total force equals 0, therefore:
m"2r - mo"2r - fv = 0
substituting m#$s = mo, where # = partial specific volume of the particle (i.e., the reciprocal of the
density of the particle or $p) and $s = density of the solvent, and solving for v results in:
v

" rm(1 - #$s)/f

" rm#($p - $s)/f

This equation tells us several things about sedimentation:

1.
2.
3.
4.
5.
6.

The greater the centrifugal force ("2r) the faster the particle sediments.
The more massive a particle (m), the faster it moves in a centrifugal field
The denser a particle ($p) the faster in moves in a centrifugal field.
The denser the solution ($s), the slower the particle will move in a centrifugal field.
The greater the frictional coefficient (factors such as viscosity, particle shape, etc. influence
this parameter), the slower the particle will move.
The particle velocity is 0 when the solution density is greater than the particle density.

Analytical Centrifugation
The velocity per unit force will be defined as the sedimentation coefficient (s), or:
s = v/"2r = m#($p - $s)/f
When mass is expressed in g and f in g/sec s ranges from 10-13 to 10-11 sec. This is normally
expressed in Svedberg (S) units where 1 S = 10-13 sec.
Molecules and subcellular structures can be defined by a sedimentation coefficient which reflects
there size, shape, and density.

Differential Centrifugation
Preparative Centrifugation
o takes advantage of the fact that more massive
particles sediment faster than less massive
particles
o for example, organelles and other subcellular
components can be isolated by differential
centrifugation.

Differential Centrifugation
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prepare cell lysate

subject lysate to centrifugation and separate
supernatant and pellet fractions
! centrifugal force expressed as relative centrifugal force
(RCF) or 'x g' (see appendix)
! product of time and g-force determines sedimentation
! tube size, shape and angle also affect sedimentation
re-centrifuge supernatant at higher g-force
problems
! contamination (larger particles contaminated with smaller
particles)
! resolution (particles with similar properties difficult to
separate)
! vibrations and convection currents

Density Gradient Centrifugation

Centrifugation through a dense medium, or density gradient
centrifugation, can increase the resolution and solve some
problems associated with differential centrifugation. The two
types of density gradient centrifugation are rate zonal and
isopycnic (or equilibrium).
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rate zonal centrifugation: density of the particles being

separated are greater than the density of the solvent.
Separation is based upon mass (i.e., larger particles will
sediment faster)
isopycnic centrifugation: solvent density encompasses density
of particles. The separation is based upon particle density.