1. Hydrodynamic Techniques
electrophoresis
centrifugation
Types of Centrifuges
Ultracentrifuge
-Analytical
-Preparative
High Speed
Table Top
Clinical
Microfuges
Fc = m2r
Fb = -mo2r
Ff = -fv

2. v = 2rm(p - s)/f
The greater the centrifugal force (2r), the faster the particle sediments.
The more massive a particle, the faster it moves in a centrifugal field.
The denser a particle, the faster in moves in a centrifugal field.
The denser the solvent, the slower the particle will move in a centrifugal field.
The particle velocity is 0 when the solvent density is greater than the particle density.
The greater the frictional coefficient (factors such as solvent viscosity, particle shape, etc.), the slower the particle will move.

3. Analytical vs Preparative Centrifugation
Sedimentation Coefficient (s)
is the velocity per Fc, or
s = v/2r
units are the Svedberg (S), where 1 S = sec
= m(p - s)/f
Analytical vs Preparative Centrifugation

4. subcellular fractionation
collect material
cells
precipitated macromolecules
subcellular fractionation

5. Differential Centrifugation
prepare cell lysate
subject to centrifugation
centrifugal force
time (g ·min)
tube size and shape
rotor angle
separate supernatant and pellet
re-centrifuge supernatant
Relative Centrifugal Force
expressed as ‘x gravity’
RCF = Fc/Fg = 2r/980
[ = (rpm)/30]
RCF = x 105 (rpm)2r
[radius in cm]

6. Problems Density Gradients contamination resolution
large particles contaminated with smaller particles
resolution
particles of similar sizes not separated
vibrations and convection currents
Density Gradients
centrifugation in a dense medium
increases stability
provides greater resolution
common media: sucrose, CsCl, Ficoll®, Hypaque®, Percoll®

7. Density Gradient Centrifugation
Two Types:
1) Rate Zonal  p > s
separates primarily by mass
2) Isopycnic  p < s
equilibrium
separates by density

8. Example of Isopycnic Separation
Percoll, self-forming gradient
light vs. heavy fraction

9. General Procedures 1) prepare gradient 2) apply sample 3) centrifuge
step
continuous
self-forming (isopycnic)
2) apply sample
layer on top
bottom (isopycnic)
dispersed (self forming)
3) centrifuge

10. 4) collect and analyze fractions
manually aspirate ‘bands’
fraction collector
Measuring Density
marker beads
refractometer