Ion Exchange Chromatography in FPLC Explained

Ion exchange chromatography, abbreviated to IEX, is a popular technique in the protein purification pipeline. It is a unique chromatography technique that exploits the competitive binding of molecules in solution based on their relative binding to an ionic solid phase.  

In this blog post, KNAUER explains the principles of ion exchange chromatography for FPLC applications in detail.

Introduction to Ion Exchange Chromatography

Ion exchange chromatography is used to separate proteins based on their charge. Different ion exchangers are used to exploit the adsorptive properties of charged proteins to a stationary phase with an opposing charge.  

The first stage of ion exchange chromatography is equilibration with an appropriate buffer, the pH level of which affects the protein’s net charge. At a certain pH value, all charged molecules in solution carry a net charge close to zero. This is defined as their isoelectric point (pI). Generally speaking:

Ion exchange chromatography exploits this property to separate distinct biomolecules using a buffer to maintain a constant pH (thus constant net charge) and an ion exchanger with either a positively- or negatively-charged stationary matrix. For example: A cationic column is used to separate target proteins with a negative net charge. Vice versa with an anionic column positively-charged biomolecules are purified.  

The next key parameter is the ionic strength, or salt gradient that is used to create the right separation conditions. Initial binding takes place under low ionic strength conditions and hence low salt concentrations. The elute proteins from the column one must increase the salt concentration gradually. The first proteins to elute from the column have the weakest binding, while more strongly bound proteins require higher salt concentration before eluting. Precise gradient formation is subsequently essential for successful separation of proteins via either anion or cation exchange chromatography.  

If you would like to read a more detailed case study about using ion exchange chromatography for protein purification, follow the link below:

Ion Exchange Chromatography with KNAUER

KNAUER is one of the leading European manufacturers and suppliers of anionic and cationic columns for ion exchange chromatography. With experience in the development of liquid chromatography solutions for a wide range of applications, we are uniquely equipped to help researchers and developers with their ion exchange chromatography needs. Contact us today to learn more.

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