Lec9 - Separation techniques Characteristic Solubility...

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Separation techniques Characteristic Procedure Solubility Salting out Charge Ion exchange chromatography Electrophoresis Polarity Hydrophobic interaction chromatography Size Gel filtration Electrophoresis Ultracentrifugation Binding specificity Affinity chromatography
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Fractionation by salting out
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Ion-exchange Charged molecules bind to opposite charged groups on the column Anion exchange (DEAE) Cation exchange (CM) Affinity depends on the presence of salt and the pH
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Anion exchange Matrix-CH 2 -CH 2 -NH(CH 2 -CH 3 ) 2 + Diethylaminoethyl (DEAE) Cation exchange Matrix-CH 2 -COO - Carboxymethyl (CM)
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Ion-Exchange Chromatography
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Hydrophobic interaction chromatography Column contains hydrophobic groups (phenyl or octyl) Non-polar patches on proteins are excluded from the solvent Interactions are increased in high salt Elution with low salt buffer, detergens or changes in pH
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Gel-filtration (size exclusion) chromatography The beads contain pores The buffer runs along the beads Small molecules enter pores Large molecules do not Larger molecules move faster
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Size exclusion Chromatography
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Lec9 - Separation techniques Characteristic Solubility...

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