Separation principles
•Size:
size exclusion chromatography (= gel filtration, = gel permeation chromatography)
•Charge
anion or cation exchange chromatography; chromatofocusing
•Hydrophobicity
hydrophobic interaction chromatography (HIC)
•Affinity
affinity chromatography
•Solubility
ammonium sulfate precipitation (non chromatographic, rel. imprecise)
Size exclusion chromatography
•Different pore sizes, depending on the size of the proteins
•Separation is based in diffusion slow flow rate
•Pressure sensitive materials
Example for a separation by gel filtration
Noteworthy about chromatography
Gel filtration:
limited sample volume: 2-3mllimited flow rate gel filtraion takes timedelutionof the sample by a factor of about 3low purificatopn factor: 3-6needs column buffer with high ionic strength: min 0.1 M
Ion exchage chromatography:
To remember:Protein binding depents on electrostatic interactions with the column material.Strength of binding depents on pH and ionic strength of the buffer, the pI of the protein and the charge density on the column.
In general:Technical easier than gel filtration: columns could be packed at the FPLC.Sample volume can be multiple times the column volume.Higher flow rates.Purification factor: 3-15Sample gets concentrated.
Don’t use charged detergents!
Ion exchage chromatography
