Centrifugation Theory and

Practice
Routine centrifuge rotors
Calculation of g-force
Differential centrifugation
Density gradient theory
Centrifuge rotors
Fixed-angle
axis of rotation
At rest
Swinging-bucket
g
Spinning g
Geometry of rotors
b c
r
max
r
av
r
min
r
max
r
av
r
min
Sedimentation path length
axis of rotation
a
r
max
r
av
r
min
k-factor of rotors
The k-factor is a measure of the time taken for a
particle to sediment through a sucrose gradient
The most efficient rotors which operate at a high
RCF and have a low sedimentation path length
therefore have the lowest k-factors
The centrifugation times (t) and k-factors for two
different rotors (1 and 2) are related by:
2
2 1
1
k
t k
t =
Calculation of RCF and Q
2
1000
18 . 11
|
.
|

\
|
=
Q
r x RCF
r
RCF
Q 299 =
RCF = Relative Centrifugal Force (g-force)
Q = rpm; r = radius in cm
RCF in swinging-bucket and
fixed-angle rotors at 40,000 rpm
Beckman SW41 swinging-bucket (13 ml)
g
min
= 119,850g; g
av
= 196,770g;
g
max
= 273,690g
Beckman 70.1Ti fixed-angle rotor (13 ml)
g
min
= 72,450g; g
av
= 109,120g;
g
max
= 146,680g
g
d
v
l p

18
) (
2

=
Velocity of sedimentation of a particle
v = velocity of sedimentation
d = diameter of particle

p
= density of particle

l
= density of liquid
= viscosity of liquid
g = centrifugal force
Differential centrifugation
Density of liquid is uniform
Density of liquid << Density of particles
Viscosity of the liquid is low
Consequence:
Rate of particle sedimentation depends
mainly on its size and the applied g-force.
Size of major cell organelles
Nucleus 4-12 m
Plasma membrane sheets 3-20 m
Golgi tubules 1-2 m
Mitochondria 0.4-2.5 m
Lysosomes/peroxisomes 0.4-0.8 m
Microsomal vesicles 0.05-0.3m
Differential centrifugation of a
tissue homogenate (I)
1000g/10 min
Decant
supernatant
3000g/10 min
etc.
Differential centrifugation of a
tissue homogenate (II)
1. Homogenate 1000g for 10 min
2. Supernatant from 1 3000g for 10 min
3. Supernatant from 2 15,000g for 15 min
4. Supernatant from 3 100,000g for 45 min
Pellet 1 nuclear
Pellet 2 heavy mitochondrial
Pellet 3 light mitochondrial
Pellet 4 microsomal
Differential centrifugation (III)
Expected content of pellets
1000g pellet: nuclei, plasma membrane
sheets
3000g pellet: large mitochondria, Golgi
tubules
15,000g pellet: small mitochondria,
lysosomes, peroxisomes
100,000g pellet: microsomes
Differential centrifugation (IV)
Poor resolution and recovery because of:
Particle size heterogeneity
Particles starting out at r
min
have furthest to
travel but initially experience lowest RCF
Smaller particles close to r
max
have only a
short distance to travel and experience the
highest RCF
Differential centrifugation (V)
Fixed-angle rotor:
Shorter sedimentation path
length
g
max
> g
min

Swinging-bucket rotor:
Long sedimentation path length
g
max
>>> g
min

Differential centrifugation (VI)
Rate of sedimentation can be modulated by
particle density
Nuclei have an unusually rapid
sedimentation rate because of their size
AND high density
Golgi tubules do not sediment at 3000g, in
spite of their size: they have an unusually
low sedimentation rate because of their very
low density: (
p
-
l
) becomes rate limiting.
Density Barrier Discontinuous Continuous
Density gradient centrifugation
How does a gradient separate
different particles?
Least dense
Most dense
g
d
v
l p

18
) (
2

=
When
p
>
l
: v is +ve
When
p
=
l
: v is 0
Predictions from equation (I)
g
d
v
l p

18
) (
2

=
When
p
<
l
: v is -ve
Predictions from equation (II)
Summary of previous slides
A particle will sediment through a
solution if particle density > solution
density
If particle density < solution density,
particle will float through solution
When particle density = solution density
the particle stop sedimenting or floating
Buoyant density
banding

Equilibrium
density banding

Isopycnic
banding
1
5
2
3
4
1
2
3
3 Formats for separation of particles according
to their density
When density of particle < density of liquid V is -ve
Discontinuous
Resolution of density gradients
Continuous Density Barrier
I II
Problems with top loading

p
>>
l
: v is +ve
for all particles
throughout the
gradient
Separation of particles according to size