Ion Exchange Chromatography−II

Note: This lecture is continuation of previous lecture. Before starting this one should have clear understanding of relationship of charge on protein and pH of buffer explained in last class.

Anion Exchange Chromatography: Anion exchange chromatography exploits difference in surface negative charge of protein or other molecules for separation. Anion exchange matrix has positively charged functional group. Few common examples of anion exchanger functional groups are as follows:

As explained in cation exchange chromatography, solid support with these functional groups can be prepared with various beads. They differ in few properties like flow rate etc. Anion exchangers based on dextran (Sephadex), agarose (Sepharose) or cross-linked cellulose (Sephacel) are few common options. Anion exchange Chromatography are performed using buffers at pH between 7 and 10 and running a gradient from a solution containing just this buffer to a solution containing this buffer with 1M NaCl. The surface charge of the molecule (proteins, nucleic acids etc) which bind will be net negative, thus to get binding of a specific protein one should perform purification above the pI of that protein.

The salt in the solution competes for the binding to the column matrix and releases the protein from its bound state at a given concentration. Proteins separate because the amount of salt needed to release the protein varies with the external charge of the protein.