Centrifugation

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Centrifugation
A centrifuge is used to separate particles or
even macromolecules -Cells -Sub-cellular
components -Proteins -Nucleic acids Basis of
separation -Size -Shape -Density
Methodology -Utilizes density difference
between the particles/macromolecules and the
medium in which these are dispersed -Dispersed
systems are subjected to artificially induced
gravitational fields
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Densities of biological material
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Principle of centrifugation
Induced gravitational field
Supernatant
Precipitate
Suspension
In process
Separation complete

• Centrifuges are classified into two categories
• Laboratory centrifuges
• Preparative centrifuges

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Laboratory centrifuges
Centrifuge tube
Supernatant
Precipitate
Suspension
Rotor

• Used for small-scale separation and particle free
  sample preparations
• Typical liquid volumes handled is about 1 5000
  ml
• The material to be centrifuged is distributed in
  centrifuge tubes
• Tubes are attached rotor in a symmetric manner
• Two types of rotors fixed rotors and swing out
  rotors
• Induced gravitational field move particles
  towards the bottom of the tubes
• Typical rotation speeds1,000 15,000 rpm
• Induced gravitational field is measured in terms
  of the G value
• G value depends on the rotation speed as well as
  the manner in which the centrifuge tubes are held
  by the rotor

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Laboratory centrifugation

• G value will depend on the location
• Highest bottom of tube
• Lowest top of tube
• Particle experience variable G values during
  their motion
• Average value is frequently used
• Typical G values 1,000 20,000

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Laboratory centrifugation

• Difficult to make very exact calculations
• Due to
• Acceleration times
• Deceleration time
• Different G values
• Empirical correlation is commonly used for
  estimating the precipitation time (t)

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Rotors

• Fixed angled rotors have a lower k-factor
  smaller difference between rmax and rmin
• Time required for precipitation is less with the
  fixed angled rotor
• Distance travelled by particles is less with the
  angled rotor
• Fixed angled rotors are heavier and require much
  higher energy to operate
• Swing out rotors are preferred for centrifuging
  substances with high S such as cells and coarse
  particles
• Precipitated macromolecules and finer particles
  are centrifuged using fixed angled rotors

Fixed angled rotor
Swing-out rotor
Rmax - Rmin
Rmax - Rmin
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Preparative centrifugation

• Handle larger liquid volumes (i.e. 1 to several
  thousand litres)
• Range of designs
• Common feature rotating chamber into which the
  suspension is fed from one end while the
  supernatant and precipitate is collected from the
  other end in a continuous or semi-continuous
  manner
• Most common type tubular centrifuge
• Typical rotating speed 500 - 2000 rpm

Supernatant collection
Precipitate collection
Developing precipitate layer
Rotating tubular bowl
Annular space
Supernatant
Feed
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Preparative centrifugation
(A)
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Preparative centrifugation
ra
z
rt
Q
(B)
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Preparative centrifugation
Dividing (A) by (B) and integrating
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Ultracentrifugation

• Rotates at high speeds e.g. 30000 rpm
• Ultracentrifuges
• Analytical ultracentrifuge (AUC) is mainly used
  for studying properties of macromolecules
• Preparative ultracentrifuges are used to separate
  macromolecules such as proteins and nucleic acids
• The high speeds used in such devices generate
  considerable amounts of heat
• Therefore cooling arrangements are required in
  ultracentrifuges