Ion exchange chromatography (IEX) is a versatile and widely used technique for separating biomolecules based on charge. It plays a critical role in protein purification in research and biomanufacturing and can deliver high resolution, scalability, and control.

What is ion exchange chromatography?

IEX separates proteins and other charged molecules on the basis of their net surface charge. It does this by taking advantage of some important factors:

• The charged groups within a biomolecule, such as a protein's amino acid side chains, contribute to its net surface charge.
• The net surface charge of a biomolecule is highly pH dependent.
• The relationship between net charge and pH is unique for each protein or molecule.

This means that in IEX, reversible interactions between charged molecules and oppositely charged IEX media can be tightly controlled.

IEX media (e.g., chromatography resin) is composed of charged particles: positively charged anion exchangers and negatively charged cation exchangers. The pH at which a protein has no net charge is called the isoelectric point (pI). Above its pI, a protein will bind an anion exchanger, while below its pI, a protein will bind a cation exchanger. Capto™ Q and SP Sepharose™ resins from Cytiva are strong anion and cation exchanges, respectively.

Ion exchange chromatography is one of the most frequently used techniques for purification of biomolecules and separates the molecules according to differences in their net surface charge. This video describes the principles of the technique.

How it works

1. Equilibration

The first step is equilibration of the stationary phase (IEX media composed of chromatography resin). The porous resin is filled with buffer to ensure all stationary phase charged groups are bound with exchangeable counterions, such as chloride or sodium.

2. Sample application and wash

The goal in this step is to bind the target molecules and wash out all unbound material. Oppositely charged proteins bind to ionic groups of the IEX medium, becoming concentrated on the column. Uncharged proteins, or those with the same charge as the ionic group, pass through the column.

3. Elution

Now that the sample has been loaded, conditions can be changed to elute the bound proteins. Typically, proteins are eluted by increasing the ionic strength (salt concentration) of the buffer. Salt ions (e.g., Na+ or Cl) compete with bound molecules, which are dislodged and begin to elute and move down the column. The proteins with the lowest charge at the selected pH will be the first ones eluted from the column as ionic strength increases. Similarly, the proteins with the highest charge at a certain pH will be most strongly retained and eluted last.

4. Regeneration

A final wash with a high-ionic-strength buffer regenerates the column and removes any molecules that are still bound, ensuring that the stationary phase's full capacity is available for the next run.

When to use IEX

IEX is used to separate proteins or other biomolecules, like nucleic acids, for downstream analysis, characterization, or application. Separation protocols can be broadly categorized based on the purity of the sample required after elution.

Capture: IEX can be used as a capture step to isolate proteins of interest from a crude sample quickly. The focus in a capture step is typically on capacity and speed.

Intermediate purification: When IEX is used for intermediate purification, the objective is to remove most of the significant impurities. The focus is on capacity and resolution to maintain productivity and achieve as high a purity as possible.

Polishing: IEX is used for polishing when most impurities have already been removed. The focus is on achieving the highest resolution possible.

Check out the comprehensive guide from Cytiva about ion exchange chromatography for the complete lowdown on planning, optimizing, and troubleshooting your IEX experiments.