Ion Exchange Chromatography is based on
the electrostatic interaction of charged surfaces.
Ion Exchange Chromatography is of Two
types:
types:
-
Cation Exchange
-
Anion Exchange
Factors affecting Ion Exchange
Chromatography
Chromatography
-
pH of mobile phase
-
Ionic Strength
-
Mobile Phase Modifiers
-
Temperature
Operating pH of Ion Exchange
Chromatography (IEX) is mostly 2-9.
Chromatography (IEX) is mostly 2-9.
Buffers Used in Ion Exchange
Chromatography
Chromatography
Use Cationic Buffers in Anion Exchanger
: Eg: Alkyl Amines, Tris, Amino Ethyl Alcohol
: Eg: Alkyl Amines, Tris, Amino Ethyl Alcohol
Use Anionic Buffers in Cation
Exchanger: Eg: Phosphate, Acetate, Citrate, Barbiturate
Exchanger: Eg: Phosphate, Acetate, Citrate, Barbiturate
Ionic Strength:
Care should be taken while choosing
ionic strength since the use of >1M salt concentration can induce
hydrophobic interaction.
ionic strength since the use of >1M salt concentration can induce
hydrophobic interaction.
Mobile Phase Modifiers
-
EDTA – For Chelation
-
Chaotropic Salts – Eg: Urea for
stabilization -
PEG – For Enhanced Selectivity
-
DTT – To Prevent Oxidation
Stationary Phase
Selectivity is Based on the following
parameters
parameters
-
Ligand Density
-
Spacer Arm
-
Backbone of the resin material
Particle size generally used is in the
range of 20 – 200um.
range of 20 – 200um.
Ion Exchange Functional group
Stationary phase in Ion Exchange
chromatographic matrix is functionalized with Basic (Anion Exchange)
or Acidic (Cation Exchange) groups.
chromatographic matrix is functionalized with Basic (Anion Exchange)
or Acidic (Cation Exchange) groups.
Strong or Weak Ion Exchanger
Strong Ion Exchangers retain their
charge over a wide range of pH
charge over a wide range of pH
Weak Ion Exchanger retain their charge
only in a narrow range of pH.
only in a narrow range of pH.
Strong Ion Exhangers will have very low
or very high pKa.
or very high pKa.
Example for Strong Exchanger –
Sulfopropyl, Quarternary Ammonium
Sulfopropyl, Quarternary Ammonium
Binding Capacity
Total no of charges per unit stationary
phase volume or the mass of protein adsorbed per unit volume.
phase volume or the mass of protein adsorbed per unit volume.
Pore Size of Ion Exchange resin can
range from 10 – 1500A.
range from 10 – 1500A.
Dynamic Binding Capacity can be found
out by doing breakthrough Curve.
out by doing breakthrough Curve.
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