Hydrodynamic Techniques

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• Hydrodynamic Techniques
• electrophoresis
• centrifugation

• Types of Centrifuges
• Ultracentrifuge
• -Analytical
• -Preparative
• High Speed
• Table Top
• Clinical
• Microfuges

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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.

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• Sedimentation Coefficient (s)
• is the velocity per Fc, or
• s v/?2r
• units are the Svedberg (S), where 1 S 10-13 sec

m?(?p - ?s)/f
Analytical vs Preparative Centrifugation
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• collect material
• cells
• precipitated macromolecules
• subcellular fractionation

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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 1.119 x 105 (rpm)2r
• radius in cm

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• 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

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Density Gradient Centrifugation

• Two Types
• 1) Rate Zonal ? ?p gt ?s
• separates primarily by mass
• 2) Isopycnic ? ?p lt ?s
• equilibrium
• separates by density

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Example of Isopycnic Separation

• Percoll, self-forming gradient
• light vs. heavy fraction

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General Procedures

• 2) apply sample
• layer on top
• bottom (isopycnic)
• dispersed (self forming)

3) centrifuge

• 1) prepare gradient
• step
• continuous
• self-forming (isopycnic)

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• 4) collect and analyze fractions
• manually aspirate bands
• fraction collector

• Measuring Density
• marker beads
• refractometer