What Is the Workflow of Cation Exchange Chromatography?

What is cation exchange chromatography?

Cation exchange chromatography is a powerful technique for separation and purification in biochemistry. It belongs to the broader category of ion exchange chromatography (IEX). In cation exchange chromatography, negatively charged ion exchange resins are used. The resin consists of small porous beads that have immobilized negatively charged groups, such as carboxylate (-COO-) or sulfonate (-SO₃-) groups. These negative charges on the resin interact and combine with molecules that have a net positive charge on their surface, thereby achieving separation of the target molecules.

By controlling pH, salt concentration, and resin properties, cation exchange chromatography can separate and purify proteins based on their net surface charge. This technology is widely used in a variety of applications in biochemistry, including protein purification in biopharmaceutical production, research, and analysis.

How to choose a cation exchange column?

Cation Exchange Column Selection List

Workflow of cation exchange chromatography

Below is a general workflow for cation exchange chromatography. It is important to note that specific running conditions may need to be adjusted based on the target protein, buffer system, and selected cation exchange resin.

• Buffer preparation
  • Prepare a buffer with appropriate pH and ionic strength.
  • Make sure the buffer pH is properly titrated and compatible with the resin.
  • Use a counterion in the buffer that has the same charge as the resin (e.g., phosphate buffer for negatively charged cation exchange resins).
• Column equilibration
  • Equilibrate the column with buffer until pH and conductivity readings stabilize.
  • Typically, this requires passing 3-5 column volumes of buffer through the column.
• Sample loading
  • Whenever possible, load protein samples into the same starting buffer used for column equilibration and subsequent wash steps.
  • Ionic strength and pH are key factors affecting protein binding to cation exchange resins.
• Wash the column
  • Wash the column with loading buffer (0% buffer B) until no protein is detectable in the effluent.
  • This typically requires passing ≥5 column volumes of loading buffer through the column.
• Elution
  Protein elution can be performed using gradient elution or step (isocratic) elution.
  • Gradient elution: Adjust the buffer composition by varying the ratio of buffer A (starting buffer) to buffer B (elution buffer) in a linear gradient. Optimize gradient conditions to achieve efficient elution of target proteins.
  • Step elution: Once the elution profile of a protein has been determined (e.g., the specific ionic strength or pH at which the protein elutes), perform a step elution to speed up the purification process.
• Column extraction and equilibration
  • After elution of the target protein, any remaining bound protein is stripped from the column resin by increasing the ionic strength or changing the pH of the elution buffer.
  • Once all remaining protein has been eluted, equilibrate the column in low ionic strength buffer.
  • If this column is to be used in the near future, it is recommended to equilibrate it with the starting buffer used for purification.
  • For long-term storage, replace the column buffer with a 20% solution to prevent microbial growth.