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CENTRIFUGATION (lab scale (example (Eppendorf centrifuge, Falcon tube…

- - - - contain multiple coned discs in a stack which are spun
      - operated semi-continuously @ using centripetal pressurising pump to force the sludege out through a valve continuously
      - large capacity and radius
      - small thickness of liquid due large effective surface area
      - restricted angular velocity (max acceleration factor 8000 g
      - self-cleaning bowl is essential for solid-liquid separation
    - - an Archimedes' screw collects the precipitate so that fluid and solids leave at opposite ends of the apparatus
      - low acceleration (3000 g); only for collection of large particles
    - - the entry nozzle placed at the bottom of the base
      - liquid accelerates and move upwards due to the centrifugal force; separates the liquids according to specific gravities
      - lighter liquid forms the inside layer and heavier liquid forms the outer layer
      - mixture entering continuously in the bowl
      - 2 phase are discharged continuously from 2 separate outlets on he top portion
      - one discharge of clarified liquid is possible
      - solid accumulated inside the bowl is remove manually
      - sediment small particles of cell debris and precipitated protein
      - operate semi-continuous , long and thin for rapid acceleration and deceleration , minimizing down-time required for removal of sedimented solids
      - small radius and effective liquid thickness; allowing high angular velocity high centrifugal force (16,000- 50,000 g)
- - - - From the equation
        
        sedimentation velocity is proportional to the difference between the particle and liquid density and is zero when these two densities are
        equal
        
        sedimentation velocity decreases as the viscosity of the liquid medium increase
        
        sedimentation velocity is proportional to the square of the sphere size.
        
        sedimentation velocity increases as the centrifugal force (ω2R) increases
    - - Types of slurry and solid particles contained in it
        
        Liquid viscosity
        
        Liquid density
        
        Solids concentration
        
        Particle size distribution
        
        Surface charge of particles
  - - - RCF = 1.12 x r x (rpm /1000)²
- - - - particles will not sediment at bottom of centrifugal tube
      - particle have specific density
        
        the particle will sediment until reach gradient media that have same density with it, the particles will stay at the equilibrium density gradient medium part
        
        this condition called ISOPYCNIC
        
        the particles separate according to their buoyancy
    - - uniform mixture of macromolecules in sample dissolve in density gradient medium (example: Caesium Chloride, CsCl)
      - during centrifugation, the caesium chloride form the density gradient
      - by having the density gradient, the particles will starts to sediment until reach their equilibrium density in which part of the density gradient medium
      - the separate particles can be collected and select the desired product of centrifugation
    - - Sufficient solublilty to produce the range of densities required
        
        Does not form solutions of high viscosity in the desired density range
        
        Is not hyperosmotic or hypoosmotic when the particles to be separated are osmotically sensitive
        
        Solutions of the gradient should be adjustable to the pH and the ionic strengths that are compatible with the particles being separated
        
        Does not aff ect the biological activity of the sample
        
        Nontoxic and not metabolized by cells
        
        Does not interfere with assay procedures or react with the centrifuge tubes
        
        Exhibits a property that can be used as a measure of concentration
        
        Easily removed from the purified product
        
        Autoclavable
        
        Reasonable cost
  - - - equal volume of appropriate concentration of gradient media solution should be used
      - layered in a centrifuge tube using pasture pipette that can reach the bottom of the tube
      - the lightest concentration should place in the tube first, then higher concentration gradient will layered below the lighter concentration gradient
      - do not disturb interface between layer: holding tip of pipette to the wall when removing or introducing media
      - sample will then layered on the top of the gradient media
      - Results after centrifugation
        
        Particles having larger size or mass will sediment faster than the smaller one
        
        Since density of the particles is greater than the density of the gradient, all the particles will eventually form pellet at the bottom after long period of time
        
        Faster sedimenting particles are not contaminated by the slower particles due to the presence of the gradient media
    - - sucrose
      - polysaccharide
      - alkali metal salt
    - - Stabilize the zone during recovery
      - Prevent mixing of resolved zone
    - - separation of cellular organelles: endosomes
      - separation of proteins: antibodies
- - - - pellet
        
        mixture of sedimenting particles
      - supernatant
        
        contains purified component (slowest sedimenting component)
  - - - *yield of light particles is not very high (particles trapped in pellet)
  - - - Example 2
        
        1) cells first disrupted to release internal content
        
        2) crude disrupted cell mixture can be centrifuged for few times to generate pellets of partially purified organelles
        
        *serves as preliminary step for purification