p. 5
2. Selection of ion exchanger
and conditions
Ion exchange chromatography is based on adsorption and reversible
binding of charged sample molecules to oppositely charged groups
attached to an insoluble matrix. The pH value at which a biomolecule carries
no net charge is called the isoelectric point (pI). When exposed to a pH below
its pI, the biomolecule will carry a positive charge and will bind to a cation
exchanger (SP). At pH’s above its pI the protein will carry a negative charge
and will bind to an anion exchanger (Q). If the sample components are most
stable below their pI’s, a cation exchanger should be used. If they are most
stable above their pI´s, an anion exchanger is used. If stability is high over a
wide pH range on both sides of pI, either type of ion exchanger can be used
(Figure 1).
Selection of buff er pH and ionic strength
Buffer pH and ionic strength are critical for the binding and elution of
material (both target substances and contaminants) in ion exchange
chromatography. Selection of appropriate pH and ionic strength for
the start and elution buffers allows the use of three possible separation
strategies.
Fig 1. The net charge or a protein as a function of pH.
