How can you choose the best elution buffer for chromatography?

1 Types of chromatography

The first step in choosing the best elution buffer is to identify the type of chromatography you are using. There are many types of chromatography, but some of the most common ones in biochemical applications are ion exchange, size exclusion, affinity, and reverse phase chromatography. Each type of chromatography has a different mechanism of separation and requires a different elution buffer to achieve the desired resolution and recovery.

2 Ion exchange chromatography

Ion exchange chromatography separates molecules based on their net charge at a given pH. The column contains either positively or negatively charged groups that bind to the opposite charged molecules in the sample. To elute the bound molecules, you need to use a buffer that either changes the pH or increases the salt concentration. Changing the pH alters the charge of the molecules, making them less attracted to the column. Increasing the salt concentration reduces the electrostatic interactions between the molecules and the column. The best elution buffer for ion exchange chromatography depends on the pI (isoelectric point) of your target molecule, the stability of your molecule at different pH values, and the resolution you want to achieve.

3 Size exclusion chromatography

Size exclusion chromatography separates molecules based on their size and shape. The column contains pores that allow smaller molecules to enter and larger molecules to pass through. To elute the molecules, you need to use a buffer that maintains the stability and solubility of your molecules and prevents non-specific interactions with the column. The best elution buffer for size exclusion chromatography depends on the molecular weight and hydrodynamic radius of your target molecule, the viscosity and osmolarity of the buffer, and the compatibility with downstream applications.

4 Affinity chromatography

Affinity chromatography separates molecules based on their specific binding affinity to a ligand that is attached to the column. The ligand can be an antibody, an enzyme, a metal ion, or any other molecule that recognizes your target molecule. To elute the bound molecules, you need to use a buffer that disrupts the binding affinity between the ligand and the target molecule. This can be done by changing the pH, adding a competitive ligand, or using a chaotropic agent. The best elution buffer for affinity chromatography depends on the type and strength of the ligand-target interaction, the stability and activity of your target molecule, and the purity and yield you want to obtain.

6 Here’s what else to consider

This is a space to share examples, stories, or insights that don’t fit into any of the previous sections. What else would you like to add?