Chromatography

Desalting and Affinity

Chromatography

• Technique to separate components of a mixture by passing them through a matrix.– A solvent is used for carrying sample mixture

through the matrix– Separation occurs because each compound

in a mixture interacts differently with the matrix.

Gel-Filtration

• Ammonium sulfate interferes with affinity chromatographic step.

• We have to desalt our extract by using either dialysis or gel filtration.

• Gel filtration chromatography is a separation based on size. It is also called molecular exclusion or gel permeation chromatography.

Gel-Filtration

• Separation mechanism is not based on adsorption.

• It is independent of the eluent system

• Has high recoveries both on basis of mass and biological activity.

• Since it is iso-cratic, samples are diluted.

Gel- Filtration

• The volume between the beads is called as void volume – accessible to all molecules.

• The volume in the pore is the pore volume• The non-porous part of the beads is the

backbone volume not accessible to samples.• Partition occurs between the molecules that

have access to the void volume and molecules that have access to void and pore volume.

Gel-Filtration

• Three steps– Equilibration– Sample loading– Elution

Picture from Amersham website

• Pores in the gel

Desalting

• Group Separation Mode: Used in separating small molecules from large molecules in a mixture– Macromolecules have access only to the void volume and

therefore grouped and eluted together.– Small molecules (neutral salts, buffer salts, low mw additives)

are separated as a single unit – as late as possible. They have access to pore volume.

• The large difference in the elution volumes allow sample volumes up to 30% of the column volume.

• High flow rates can be applied and broad or narrow columns can be used.

Sephadex

• Sephadex -50 is the matrix which exploits both physical and chemical properties of the mixture.

• These highly specialized gel filtration and chromatographic media are composed of macroscopic beads synthetically derived from the polysaccharide, dextran(polymer of glucose).

• The organic chains are cross-linked to give a three dimensional network having functional ionic groups attached by ether linkages to glucose units of the polysaccharide chains.

• Available forms include anion and cation exchangers, as well as gel-filtration resins, with varying degrees of porosity; bead sizes fall in discrete ranges between 20 and 300µ.

http://web.siumed.edu/~bbartholomew/images/protein_methods/sephadex.gif

Affintiy Chromatography• Affinity chromatography (AC) is a technique enabling purification

of a biomolecule with respect to biological function or individual chemical structure.

• The substance to be purified is specifically and reversibly adsorbed to a ligand (binding substance), immobilized by a covalent bond to a chromatographic bed material (matrix).

• Samples are applied under favourable conditions for their specific binding to the ligand.

• Substances of interest are consequently bound to the ligand while unbound substances are washed away.

• Recovery of molecules of interest can be achieved by changing experimental conditions to favour desorption. AC media are commonly used for applications such as purification of fusion proteins, mono- and polyclonal antibodies, and glycoproteins.

Affinity Chromatography (AC)

• AC involves an inert matrix coupled with a affinity ligand specific for a binding site on the target molecule.

• Under suitable binding conditions this affinity matrix will bind molecules according to its specificity only.

• All other sample components will pass through the medium unadsorbed.

Affinity Chromatography

• After washing, condition are changed to disassociate or displace the molecules from the ligand.

• simple "on-off" mode of chromatography is applied by switching abruptly from full binding to complete release conditions.

• Thus AC fishes out the target molecule by way of highly specific binding and release, rather than removing contaminants by fine-tuned isocratic or gradient elution techniques.

Affinity Chromatography

• Two important things to consider;– Finding a ligand specific enough to allow

step elution.– Finding conditions for safe binding and

release within the stability window of the target molecule and the ligand.

Affi-Gel Blue

• Agrose gel covalently attached Cibacron Blue F3GA dye.

• ≥1.9mg dye per mL of gel• We are using 50-100 mesh (150-300 µm)• Separates nucleotide dependent enzymes

(dinucleotide fold biospecifically bind to the dye)

• Enzyme can be eluted from the dye with a specific nucleotide co-factor

Sigma-Aldrich site