Affinity adsorption

1
Affinity adsorption

• Stereo-specific recognition of molecules by
  ligands
• Combination of
• Hydrogen bonding
• Hydrophobic interaction
• van der Waals forces
• Specific and general affinity adsorption

Ligand
Adsorbent
Molecules
2
Affinity adsorbent

• Support material
• Cross-linked dextran
• Cross-linked agarose
• Cross-linked cellulose
• Synthetic resins
• Ligands
• Dye ligands
• Antibodies
• Antigens
• Protein A and protein G
• Substrate analogues
• Enzyme inhibitors
• Complimentary base-pair sequence
• Receptors
• Amino acids
• Lectins
• Concanavalin A
• Immobilized metal ions

3
Affinity binding and desorption

• Binding
• Physiological conditions
• Conditions that favour
• Hydrophobic interactions
• Hydrogen bonding
• Van der Waals forces
• High anti-chaotropic salt concentrations
• Desorption
• pH
• Chaotropic salts

4
Affinity separation Monoclonal antibody
Monoclonal antibody
Impurities
Cell culture supernatant
low pH buffer
Protein-A affinity adsorbent
Protein-A affinity adsorbent
Adsorbent monoclonal antibody
5
Reverse phase adsorption

• Partition type behaviour
• Adsorption of non-polar molecules
• Partition into thin immobilized layer of
  hydrocarbon C4 to C18
• Support silica
• Adsorption Polar solvent e.g. water
• Elution Non-polar solvent e.g. acetonitrile and
  isopropanol

Polar media
Non-polar media
Adsorbent
Immobilized hydrocarbon layer
Adsorption
Desorption
6
Reverse phase separation Insulin
Insulin
Filtered fermentation media acetonitrile
Impurities
80 acetonitrile 20 isopropanol
C-18 reverse phase adsorbent
C-18 reverse phase adsorbent
Adsorbent Insulin
7
Hydrophobic interaction

• Interaction between the hydrophobic patches on
  molecules and those on the adsorbent
• Mainly used for protein separation.
• Hydrophobic amino acids shielded by structured
  layer of water molecules
• Hydrophobic surface shielded by structured layer
  of water
• Anti-chaotropic salts make water molecules in
  bulk solution structured
• Less shielding of hydrophobic interactions

Molecule
Structured water layer
Adsorbent
Structured water in bulk solution
Hydrophobic layer
High salt concentration
Low salt concentration
Desorption
Adsorption
8
Hydrophobic interaction Adsorbent, adsorption
and desorption
Adsorbent Support Agarose most widely Groups
Butyl, octyl and phenyl Adsorption High salt
concentrations 1 M or higher Salt concentration
depends on the type of salt Less sodium sulphate
than ammonium sulphate or sodium
chloride Desorption Lowering the salt
concentration pH change
9
Affinity separation rEGF
rEGF
Filtered fermentation media Ammonium sulfate
Impurities
Ammonium sulfate free solution
Hydrophobic interaction adsorbent
Hydrophobic interaction adsorbent
Adsorbent rEGF
10
Adsorption isotherms

• Relationship between bound and free solute
  concentrations at equilibrium
• Analogous to equilibrium line in extraction
• Data obtained at constant temperature

Linear
Freundlich
Langmuir
Freundlich
CB
Linear
Langmuir
CU
11
Determination of isotherm constants
Linear Determine K from plot of CB versus
CU Freundlich Determine KF and n from plot of
(log CB) versus (log CU) Langmuir Determine
CBmax and KL from plot of (1/CB) versus (1/CU)
Slope n
log CB
log KF
log CU
1/CB
Slope KL/CBmax
1/CBmax
1/CU