Principle of Chromatography: Ion Exchange, Size Exclusion and Affinity Chromatography

Ion exchange chromatography

Principle: 
Ion exchange chromatography is a technique for separating compounds based on their net charge. Ion exchange chromatography resin contains negatively or positively charged functional groups covalently bound to a solid support, yielding either a cation or anion exchanger, respectively. Charged compounds are adsorbed and retained by an ion exchanger having the opposite charge, whereas compounds that are neutral or have the same charge as the media pass through the void volume and are eluted from the column. The binding of the charged compounds is reversible, and adsorbed compounds are commonly eluted with a salt or pH gradient. Ion exchange media are available in various particle sizes, ionic forms, and purity ranges. Ion exchange chromatography is used both for preparative and analytical purposes can separate a large range of molecules from amino acids and nucleotides to large proteins. 

Cation exchange chromatography: uses a negatively charged ion exchange resin with an affinity for molecules having net positive surface charges.
Anion exchange chromatography:  uses a positively charged ion exchange resin with an affinity for molecules having net negative surface charges. 


Affinity Chromatography

Affinity chromatography is a separation method based on a specific binding interaction between an immobilized ligand and its binding partner. Examples include antibody/antigen, enzyme/substrate, and enzyme/inhibitor interactions. The degree of purification depends on the specificity of the interaction.




Hydrophobic Interaction Chromatography

Hydrophobic interaction chromatography (HIC) separates molecules based on their hydrophobicity. HIC is a useful separation technique for purifying proteins while maintaining biological activity due to the use of conditions and matrices that operate under less denaturing conditions. 



Size Exclusion Chromatography

Size exclusion chromatography (SEC), or gel filtration chromatography, separates molecules based on their sizes. SEC resins are gels that contain beads with known pore size. When a complex protein mixture is passed over SEC resin, small molecules move through the bead pores, whereas molecules too large to fit into the pores move around the beads and through the void space between beads.







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