Ion exchange chromatography (IEX) is a common form of chromatography used for the separation and purification of ions and charged molecules, particularly proteins, peptides, and nucleic acids.
Let's delve into the methodology of ion exchange chromatography in the context of instrumental analysis:
1. Principle of Separation:
IEX separates molecules based on differences in their net surface charge. The separation relies on the reversible adsorption of charged molecules (analytes) to oppositely charged stationary phase particles (the resin).
The resin contains covalently attached charged groups. If the resin is negatively charged, it's an anion exchanger. If it's positively charged, it's a cation exchanger.
2. Preparation of the Column:
A chromatographic column is packed with ion-exchange resin, either cationic or anionic.
The resin is pre-equilibrated with a buffer to ensure it is charged and ready for the separation process.
3. Sample Introduction:
The sample containing a mixture of charged molecules is loaded onto the column.
Molecules with a charge opposite to that of the resin will bind, while those of the same charge or neutral molecules will pass through.
4. Elution & Separation:
After loading, the column is washed with the starting buffer to remove any unbound or weakly bound molecules.
Bound molecules are then eluted from the column using a gradient of increasing ionic strength or by changing the pH. This is typically done by gradually increasing the concentration of a salt (like NaCl) in the mobile phase.
As the ionic strength or pH changes, the interactions between the analyte and the resin are disrupted, causing the analyte to be released from the column.
Different molecules will be eluted at different salt concentrations or pH levels based on their charge and their affinity for the resin.
5. Detection & Analysis:
As molecules elute from the column, they can be detected, often using UV-Vis absorbance, especially when analyzing proteins or nucleic acids.
The resulting chromatogram shows peaks for different analytes, allowing for both qualitative (identification) and quantitative (concentration) analysis.
6. Regeneration & Cleaning:
After a run, the column is typically regenerated to its original state by washing with a strong salt solution or a solution at a pH that ensures all bound molecules are released.
This is followed by equilibrating the column with the starting buffer, making it ready for another round of separation.