How Do You Pick the Right Chromatography Method?

Chromatography is the most powerful tool for protein separation, yet the appropriate method depends on the specific purpose of separation and the unique properties of your protein.

Here’s an overview of the most common techniques:

Size Exclusion Chromatography (SEC)

This technique separates proteins based on their size. Larger molecules elute first, while smaller ones are retained longer as they pass through porous resin.
Ideal for studying protein aggregation, complex formation, or size distribution.

Ion Exchange Chromatography (IEX)

It separates proteins based on their charge or charge interaction with a charged resin.
It has 2 types:
1. Cation Exchange Chromatography (CEX) where Negatively charged resin binds to positively charged proteins.

Most effectively used at low pH, where proteins carry a positive charge.

2. Anion Exchange Chromatography (AEX) where positively charged resin binds to negatively charged proteins.

Most effectively used at high pH, where proteins carry a negative charge.
IEX is excellent for proteins with slight charge differences or those that require high purity.

Affinity Chromatography

It uses a highly specific binding interaction between the protein and the stationary phase such as antibody-antigen interaction.
This method provides high purity in a single step, making it ideal for antibodies, enzymes, and recombinant proteins.

Reverse-Phase Chromatography (RPC)

This method separates molecules based on hydrophobicity. The stationary phase is nonpolar, while the mobile phase is polar, increasingly nonpolar over time to elute bound molecules.
Used mainly for peptides, small molecules, and protein fragments.

How to Choose?
The selection of method depends on your aim of separation and protein properties.