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Hydrodynamic Techniques
Types of Centrifuges
• Ultracentrifuge
-Analytical
-Preparative
• High Speed
• Table Top
• Clinical
• Microfuges
• electrophoresis
• centrifugation
Fc = m2r
Fb = -mo2r
Ff = -fv
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.
Sedimentation Coefficient (s)
• is the velocity per Fc, or
• s = v/2r = m(p - s)/f
• units are the Svedberg (S),
where 1 S = 10-13 sec
Analytical vs Preparative Centrifugation
• collect material
• cells
• precipitated
macromolecules
• subcellular
fractionation
Differential Centrifugation
• prepare cell lysate
• subject to centrifugation
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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 = 1.119 x 105 (rpm)2r
[radius in cm]
Problems
• contamination
• 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®
Density Gradient Centrifugation
Two Types:
1) Rate Zonal  p > s
• separates primarily by mass
2) Isopycnic  p < s
• equilibrium
• separates by density
Example of
Isopycnic Separation
• Percoll, self-forming
gradient
• light vs. heavy fraction
General Procedures
1) prepare gradient 2) apply sample 3) centrifuge
• step
• continuous
• self-forming
(isopycnic)
• layer on top
• bottom (isopycnic)
• dispersed (self
forming)
4) collect and analyze fractions
• manually aspirate ‘bands’
• fraction collector
Subcellular Fractionation
and Marker Enzymes
nuclei
mitochondria
lysosome
peroxisome
Golgi
plasma membrane
cytosol
DNA
cytochrome oxidase
hydrolases
catalase
-mannosidase
adenylate cyclase
lactate dehydrogenase
Measuring Density
• marker beads
• refractometer