Ion Exchange Chromatography.pptVIP

Ion Exchange Chromatography Useful at all stages of purification and at all scales Scope of this presentation Some typical applications Mechanism and principles Practical aspects What is ion exchange chromatography? Ion exchange chromatography is a form of adsorption chromatography which separates molecules on the basis of their charge. Why use ion exchange? Useful at all stages of purification and at all scales Controllable High selectivity High capacity Concentrating High recovery Capture Concentration Versatility: DNA Binding Protein Purification Versatility: Membrane Protein Purification Controllable separation Small scale What happens in ion exchange? What happens in ion exchange? What happens in ion exchange? What happens in ion exchange? What happens in ion exchange? Separation by charge Interaction between opposite charges Charged groups on the proteins interact with charged groups on the ion exchanger. Different proteins have different charges and interact differently. Anion or cation exchange When the protein is negatively charged, it is an anion - anion exchange When it is positively charged, it is a cation - cation exchange Basis for selectivity Some of the charged regions which will influence ion exchange Different proteins have different charges and different patterns of surface charge Effect of pH on charge Titration curves Controlling selectivity by pH Practical Considerations What equipment do we need? What ion exchanger should we use? How are we going to run the separation? The right ion exchanger There is no single perfect ion exchanger! Different ion exchangers are needed for different sample loads and different purposes Functional properties of ion exchangers Charged groups Type Density Matrix Porosity Particle size Chemistry Selectivity Capacity Capacity, speed Peak width, speed Useful life, recovery Charged groups Anion exchangers -Diethylaminoethyl (DEAE) -OCH2CH2N+(CH2CH3)2 -Quaternary aminoethyl (QAE) -OCH2CH2N+(C2H5) 2CH2CHOHCH3