At its core, the mechanism of ion exchange chromatography is the reversible exchange of ions between a liquid phase (mobile phase or eluent) and a solid phase (stationary phase or resin).
The resin has fixed charged groups attached to it and a counter-ion which can be exchanged with ions of the same charge present in the solution.
Ion Exchange Mechanism:
1. Equilibrium Process:
When the mobile phase (containing ions to be separated) is introduced to the column packed with the ion exchange resin, the ions from the mobile phase will displace the counter-ions on the resin based on their affinities.
The exchange process will continue until an equilibrium is reached, where the rate of ion association is equal to the rate of ion dissociation.
2. Selectivity:
Not all ions will bind with equal affinity to the resin.
This selectivity is governed by the properties of the resin (like type and density of functional groups), the type of ion, and the conditions of the eluent (like pH and ionic strength).
As a result, different ions will elute from the column at different times, enabling their separation.
3. Elution:
Once the ions of interest are bound to the resin, they can be eluted (or washed out) using a mobile phase with a different composition (e.g., increased ionic strength or pH).
The eluting ions will compete with the bound ions, leading to their release from the resin.
Binding and Exchange:
1. For Cation Exchange Resins:
The resin has negatively charged sites. Let's consider a resin with sulfonic acid groups (-SO3^-).
The resin may start with H+ ions as counter-ions.
When a solution containing, for instance, Na+ ions is passed through, the Na+ ions displace the H+ ions:
SO3^–H + Na+ → -SO3^–Na + H+
2. For Anion Exchange Resins:
The resin has positively charged sites.
For a resin with quaternary ammonium groups (-N(CH3)3^+), if it starts with Cl- as the counter-ion and a solution containing Br- ions is passed through, the exchange can be:
N(CH3)3^+Cl− + Br− → -N(CH3)3^+Br− + Cl−
Factors Affecting Ion Exchange
1. Nature of the Resin:
The type of functional group and degree of crosslinking can influence the resin's selectivity, capacity, and rate of exchange.
2. pH of the Mobile Phase:
Especially important for ionizable compounds, as it affects the charge state. For proteins, the pH can influence the overall charge, thus impacting its binding to the resin.
3. Ionic Strength:
Higher ionic strength solutions can promote the elution of bound ions due to increased competition.
4. Temperature:
Can affect the kinetics of ion exchange as well as the capacity of the resin.
5. Particle Size of the Resin:
Smaller particles can offer better resolution but might also result in increased back pressure in the column.
6. Flow Rate:
Too fast, and there might not be enough time for equilibrium to be achieved, affecting separation efficiency. Too slow, and the process becomes time inefficient.
7. Concentration of Sample Ions:
Higher concentrations might saturate the resin quickly, affecting the efficiency of separation.
