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Centrifugation

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Supernatant. Separation complete. Centrifuges are classified into ... Supernatant ... Supernatant collection. Rotating tubular bowl. Developing ... – PowerPoint PPT presentation

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Title: Centrifugation


1
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
2
Densities of biological material
3
Principle of centrifugation
Induced gravitational field
Supernatant
Precipitate
Suspension
In process
Separation complete
  • Centrifuges are classified into two categories
  • Laboratory centrifuges
  • Preparative centrifuges

4
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

5
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

6
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)

7
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
8
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
9
Preparative centrifugation
(A)
10
Preparative centrifugation
ra
z
rt
Q
(B)
11
Preparative centrifugation
Dividing (A) by (B) and integrating
12
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
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