Ion exchange chromatography

Proteins are separated on the basis of their whole (net) charge in ion exchange chromatography if a protein has a net negative charge at pH 7, it will connect to a column containing positively-charged beads, while a protein with no charge or a net positive charge will not connect. The negatively-charged proteins bound  to such  a column  can  then  be eluted  through washing  the  column  with  an rising  gradient  (rising  concentration)  of a solution  of sodium chloride (Na⁺ Cl^–  ions) at the appropriate  pH. For the positively-charged groups on the column the Cl^– ions compete with the protein.  Proteins having a low density of negative charge elute ?rst, followed through those with a higher density of negative charge.   Columns   containing   positively-charged diethylaminoethyl (DEAE)   collection   (like as DEAE-cellulose or DEAE-Sephadex) are used for separation of negatively-charged proteins (anionic proteins).

Figure: Ion exchange chromatography.  (a) Schematic illustration of ion exchange chromatography; (b) elution diagram indicating the separation of a protein of net positive charge that does not bind to the positively-charged beads and passes straight through the column, and of two proteins with different net negative charges that bind to the positively-charged beads and are eluted on increasing the concentration of NaCl applied to the column. The protein with the lower density of negative charge elutes earlier than the protein with the higher density of negative charge.

This is known as anion exchange chromatography. Columns containing negatively-charged CM  (carboxymethyl) groups  (like as CM-cellulose  or CM-Sephadex)  are used for  the  separation   of  positively-charged  proteins  (cationic  proteins).  This is known as cation exchange chromatography. As  an  instead  to  elution  with  a gradient  of  NaCl  proteins  can  be  eluted  from  anion  replaced  columns  through decreasing   the   pH   of  the   buffer   and   from   cation   exchange   columns   through increasing  the pH of the buffer,  therefore altering  the ionization  state of the amino acid side-chains and therefore the net charge on the protein.