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