Techniques of protein purification CHROMATOGRAPHY Chromatography Based on

Techniques of protein purification CHROMATOGRAPHY

Chromatography Based on the migration of the mobile phase against the stationary phase The sample is dissolved in the mobile phase As the mobile phase moves against the stationary phase the sample interacts with the matrix of the stationary phase Stationary phases can be different and therefore can exploit different properties of the sample

Column chromatography

Ion exchange chromatography Separates molecules according to charge Ionizable groups are immobilized on support matrices such as cellulose Commonly used ionizable groups are: CM – cellulose Carboxymethyl DEAE – cellulose Diethylamino ethyl

Ion exchange chromatography

Ion exchange resins

Size exclusion chromatography Separates molecules according to size and shape Stationary phase consists of beads with pores Pores are of a certain size & can only allow only molecules of certain size to enter Smallest molecular mass to enter the pores of the beads is called the exclusion limit Molecules that are too large to enter are excluded first ie do not enter the pores

Size exclusion chromatography The smaller molecules able to enter the beads will be excluded later Fractions collected in different test tubes for further analysis Affected by shape Elongated molecules less likely to penetrate the pores compared to spherical ones

Size exclusion chromatography

Affinity chromatography Exploits different affinities with which materials bind to different stationary phases Can use antigen – antibody system

Affinity chromatography

Affinity chromatography

High Performance Liquid Chromatography Faster than other column chromatography types Requires more expensive equipment Stainless steel columns – to withstand high pressures Pressure generating pump – generates pressure to push mobile phase through stationary phase at higher speeds Solvent degasser – to prevent formation of bubbles which would otherwise damage the column Precolumn – column before separating column prevents any impurities reaching the separating column Can use all types of stationary phases

HPLC device set up

Schematic HPLC diagram

HPLC Columns

Fraction collector

Chromatogram

Thin layer chromatography

Paper chromatography