p. 5
2. Selection of ion exchangerand conditionsIon exchange chromato graphy is based on a dsorption and rev ersible
binding of charged samp le molecules to opp ositely charged g roups
attached to an insolub le matrix. The p H value at which a biom olecule carries
no net charge is called the i soelectric p oint (pI). Whe n exposed to a pH belo w
its pI, the biomolec ule will carry a posi tive charge and wil l bind to a cation
exchanger (SP). At pH ’s above its pI the prot ein will carry a nega tive charge
and will bind to an anion exc hanger (Q). If t he sample compon ents are most
stable below their pI ’s, a cation exchang er should be used. I f they are most
stable above their pI ´s, an anion exchanger i s used. If stabili ty is high over a
wide pH range on both side s of pI, either type of io n exchanger can be us ed
(Figure 1).
Selection of buff er pH and ionic strength
Buffer pH and ionic st rength are critic al for the binding an d elution of
material (both target substances and contaminants) in ion exchange
chromatography. Sel ection of approp riate pH and ionic s trength for
the start and eluti on buffers allo ws the use of three po ssible separat ion
strategies.
Fig 1. The net charge or a pr otein as a functi on of pH.