Desh bandhu gangwar


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I pursuing M.Tech in Food Technology from GBU, Greater Noida.

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Desh bandhu gangwar

  1. 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. 2. Densities of biological material Material Density (g/cm 3 ) Microbial cells 1.05 - 1.15 Mammalian cells 1.04 - 1.10 Organelles 1.10 - 1.60 Proteins 1.30 DNA 1.70 RNA 2.00
  3. 3. Principle of centrifugation Suspension In process Induced gravitational field Precipitate Supernatant Separation complete <ul><li>Centrifuges are classified into two categories: </li></ul><ul><li>Laboratory centrifuges </li></ul><ul><li>Preparative centrifuges </li></ul>
  4. 4. Laboratory centrifuges Rotor Centrifuge tube Supernatant Precipitate Suspension <ul><li>Used for small-scale separation and particle free sample preparations </li></ul><ul><li>Typical liquid volumes handled is about 1 – 5000 ml </li></ul><ul><li>The material to be centrifuged is distributed in centrifuge tubes </li></ul><ul><li>Tubes are attached rotor in a symmetric manner </li></ul><ul><li>Two types of rotors: fixed rotors and swing out rotors </li></ul><ul><li>Induced gravitational field move particles towards the bottom of the tubes </li></ul><ul><li>Typical rotation speeds:1,000 – 15,000 rpm </li></ul><ul><li>Induced gravitational field is measured in terms of the G value </li></ul><ul><li>G value depends on the rotation speed as well as the manner in which the centrifuge tubes are held by the rotor </li></ul>
  5. 5. Laboratory centrifugation <ul><li>G value will depend on the location </li></ul><ul><li>Highest: bottom of tube </li></ul><ul><li>Lowest: top of tube </li></ul><ul><li>Particle experience variable G values during their motion </li></ul><ul><li>Average value is frequently used </li></ul><ul><li>Typical G values: 1,000 – 20,000 </li></ul>
  6. 6. Laboratory centrifugation <ul><li>Difficult to make very exact calculations </li></ul><ul><li>Due to: </li></ul><ul><ul><li>Acceleration times </li></ul></ul><ul><ul><li>Deceleration time </li></ul></ul><ul><ul><li>Different G values </li></ul></ul><ul><li>Empirical correlation is commonly used for estimating the precipitation time ( t ): </li></ul>
  7. 7. Rotors <ul><li>Fixed angled rotors have a lower k-factor: smaller difference between r max and r min </li></ul><ul><li>Time required for precipitation is less with the fixed angled rotor </li></ul><ul><li>Distance travelled by particles is less with the angled rotor </li></ul><ul><li>Fixed angled rotors are heavier and require much higher energy to operate </li></ul><ul><li>Swing out rotors are preferred for centrifuging substances with high S such as cells and coarse particles </li></ul><ul><li>Precipitated macromolecules and finer particles are centrifuged using fixed angled rotors </li></ul>Fixed angled rotor Swing-out rotor R max - R min R max - R min
  8. 8. Preparative centrifugation <ul><li>Handle larger liquid volumes (i.e. 1 to several thousand litres) </li></ul><ul><li>Range of designs </li></ul><ul><li>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 </li></ul><ul><li>Most common type: tubular centrifuge </li></ul><ul><li>Typical rotating speed: 500 - 2000 rpm </li></ul>Supernatant collection Rotating tubular bowl Developing precipitate layer Precipitate collection Supernatant Feed Annular space
  9. 9. Preparative centrifugation (A)
  10. 10. Preparative centrifugation r a r t Q z (B)
  11. 11. Preparative centrifugation Dividing (A) by (B) and integrating:
  12. 12. Ultracentrifugation <ul><li>Rotates at high speeds e.g. 30000 rpm </li></ul><ul><li>Ultracentrifuges: </li></ul><ul><ul><li>Analytical ultracentrifuge (AUC) is mainly used for studying properties of macromolecules </li></ul></ul><ul><ul><li>Preparative ultracentrifuges are used to separate macromolecules such as proteins and nucleic acids </li></ul></ul><ul><li>The high speeds used in such devices generate considerable amounts of heat </li></ul><ul><li>Therefore cooling arrangements are required in ultracentrifuges </li></ul>