Lecture 10 Chromatography

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Lecture 10Chromatography
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• Techniques for purification of molecules
• solubility
• chromatography
• electrophoresis
• ultracentrifugation
• ultrafiltration

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• Chromatography
• name given to a particular family of separation
  techniques
• method described in 1903 by Tswett used it for
  the separation of coloured substances
• most chromatographic separations performed on
  mixtures of colourless substances, including gases

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• Common feature use of two phases, one
  stationary and the other mobile
• Separations depend on the relative movement of
  these two phases

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• Chromatographic methods classified according to
  the nature of stationary phase, which may be a
  solid or liquid
• Adsorption stationary phase is a solid
• Partition stationary phase is a liquid

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• Four main types of chromatography
• 1. Liquid-solid
• Classical adsorption chromatography
• Thin layer chromatography
• Ion-exchange chromatography
• 2. Gas-solid
• Gas-solid chromatography
• 3. Liquid-liquid
• Classical partition chromatography
• Paper chromatography
• 4. Gas-liquid
• Gas-liquid chromatography
• Capillary-column chromatography

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• Separation depends on the fact that the
  substances to be separated distribute themselves
  between the mobile and the stationary phases in
  proportions which vary from one substance to
  another

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• Adsorption Chromatography
• columns packed with active solid (the stationary
  phase)
• small sample applied to the top of column forms a
  band of adsorbed material
• solvent flows thru column carries components of
  mixture
• rate of movement depends on how much it is
  retarded by adsorption on the column packing

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• Partition Chromatography
• principal difference column packing is a porous
  material coated with a layer of liquid (water)
• rate of movement depends on its solubility in the
  stationary phase more soluble substances travel
  down the column than the less soluble
• substances undergo partition between the two
  phases, separation occurs because of the
  differences in the partition coefficients

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• Separation Techniques
• Elution analysis
• Frontal analysis
• Displacement development analysis

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• Advantages of chromatography
• Comparatively gentle
• decomposition of substances being separated is
  less likely than in other methods. Important in
  the case of labile substances, often of
  biological origin.
• Only a small quantity of mixture is required
• Simple and rapid

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• Techniques for protein purification of molecules
• CHROMATOGRAPHY
• gel filtration
• ion exchange
• paper
• affinity

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• Techniques for protein purification of molecules
• ELECTROPHORESIS
• paper
• gel
• isoelectric focussing

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• Techniques for protein purification of molecules
• SOLUBILITY
• salt concentration
• organic solvents
• pH

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• Some characteristics utilised by procedures
• charge
• ion exchange chromatography
• electrophoresis
• isoelectric focussing
• size
• gel filtration chromatography
• gel electrophoresis
• ultracentrifugation
• ultrafiltration

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• Example of protein purification
• solubility of proteins depends on ionic strength