Ion Exchange Chromatography is based on the electrostatic interaction of charged surfaces. Ion Exchange Chromatography is of Two types: Cation Exchange Anion Exchange Factors affecting Ion Exchange Chromatography pH of mobile phase Ionic Strength Mobile Phase Modifiers 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 Ligand Density Spacer Arm 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. Got something to say about this post? Leave a comment...your comments are valuable for improving the posts.