Ion-exchange chromatography exploits differences in the sign and magnitude of the net electric charge or proteins at a given pH.
- a. The column matrix is a synthetic polymer (resin) containing bound charged groups; those with bound anionic groups are called cation exchangers, and those with bound cationic groups are called anion exchangers. The affinity of each protein for the charged groups on the column is affected by the
- pH(which determines the ionization state of the molecule) and the concentration
- of competing free salt ions in the surrounding solution. Separation can be
- optimized by gradually changing the pH and/or salt concentration of the mobile
- phase so as to create a pH or salt gradient. In cation-exchange chromatography,
- the solid matrix has negatively charged groups. In the mobile phase, proteins
- with a net positive charge migrate through the matrix more slowly than those with a net negative charge, because the migration of the former is retarded more by interaction with the stationary phase.
b. Proteins move through the column at rates determined by their net charge at the pH being used. With cation exchangers, proteins with a more negative net charge move faster and elute earlier.
c. In ion-exchange columns, the expansion of the protein band in the mobile phase the protein solution) is caused both by separation of proteins with different properties and by diffusional spreading.