Method:

The amount of sample applied on the column is determined by the dimensions of column and capacity of exchanger used. After packing, column should be washed extensively with distilled water and then equilibrated with starting buffer. Once the molecules are bound, the column is washed to equilibrate it in your starting buffer, which should be of low ionic strength, then the bound molecules are eluted off using a gradient of a second buffer which steadily increases the ionic strength of the eluent solution. Alternatively, the pH of the eluent buffer can be modified as to give your protein or the matrix a charge at which they will not interact and your molecule of interest elutes from the resin. Generally gradient elution is more common than the isocratic elution. Continuous or stepwise pH and ionic strength gradient may be used but continuous gradient gives better results in comparison to stepwise. Generally for cation exchanger both pH and ionic gradient increases whereas with an anion exchanger the pH gradient decreases and the ionic strength increases.

Anion (Cl-) is attached to a positively charged matrix. Negatively charged protein competes with anion and binds to resin. A competing anion concentration (NaCl gradient) replaces the protein and elution takes place. Protein with less negative charge gets eluted early while those with more negative charge get eluted late (Fig.1 and Fig. 2)