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Friday, July 19, 2013

An Insight on Ion Exchange Chromatography


Ion Exchange Chromatography is based on the electrostatic interaction of charged surfaces.



Ion Exchange Chromatography is of Two types:
  • Cation Exchange
  • Anion Exchange


    Ion Exchange Chromatography Principle

Factors affecting Ion Exchange Chromatography
  1. pH of mobile phase
  2. Ionic Strength
  3. Mobile Phase Modifiers
  4. Temperature
Operating pH of Ion Exchange Chromatography (IEX) is mostly 2-9.

Buffers Used in Ion Exchange Chromatography

Use Cationic Buffers in Anion Exchanger : Eg: Alkyl Amines, Tris, Amino Ethyl Alcohol

Use Anionic Buffers in Cation 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.

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
  1. Ligand Density
  2. Spacer Arm
  3. Backbone of the resin material
Particle size generally used is in the 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.

Strong or Weak Ion Exchanger

Strong Ion Exchangers retain their charge over a wide range of pH
Weak Ion Exchanger retain their charge only in a narrow range of pH.

Strong Ion Exhangers will have very low or very high pKa.

Example for Strong Exchanger – Sulfopropyl, Quarternary Ammonium

Binding Capacity

Total no of charges per unit stationary phase volume or the mass of protein adsorbed per unit volume.

Pore Size of Ion Exchange resin can range from 10 – 1500A.

Dynamic Binding Capacity can be found out by doing breakthrough Curve.