Introduction

Chromatography is a technique used in biotechnology for the purification of biomolecules. It allows a mixture of molecules to be separated based on their physical and chemical properties, with the goal of isolating the desired molecule(s) from the mixture. There are two main types of chromatography: traditional and advanced. In this blog post, we will compare the strengths and weaknesses of these techniques.

Traditional Chromatography

Traditional chromatography techniques have been around for decades and are still widely used today. They include techniques such as ion exchange, size exclusion, and affinity chromatography. These techniques are relatively simple to set up and can be performed with relatively inexpensive equipment.

Ion Exchange Chromatography

Ion exchange chromatography is used mainly for the separation of charged molecules. It uses a resin that has either positively or negatively charged groups on its surface, which bind to molecules of the opposite charge in the sample. The bound molecules are then released from the resin by altering the pH or salt concentration of the buffer.

Advantages:

• Relatively cheap
• High capacity
• Broad range of applications

Disadvantages:

• Poor resolution
• Non-specific binding
• Challenging to recover protein with high purity

Size Exclusion Chromatography

Size exclusion chromatography, also known as gel filtration chromatography, separates molecules based on their size. It uses a column filled with beads that have different-sized pores. Smaller molecules can enter the pores of the beads, slowing them down and causing them to elute later than larger molecules.

Advantages:

• Good resolution
• Mild conditions preserve protein activity
• Easy to use

Disadvantages:

• Low capacity
• Slower flow rates
• Requires large amounts of buffer

Affinity Chromatography

Affinity chromatography uses a resin that has a ligand (a molecule that binds specifically to a target protein) attached to its surface. When the sample is applied to the column, the target protein binds to the ligand. The bound protein is then released by altering the buffer conditions.

Advantages:

• High specificity
• High resolution
• Large-scale production

Disadvantages:

• Expensive
• Low capacity
• Limited ligands available for different proteins

Advanced Chromatography

Advanced chromatography techniques have been developed more recently and utilize advanced materials and methods to improve molecule separation. Two examples of advanced chromatography techniques are magnetic bead chromatography and membrane chromatography.

Magnetic Bead Chromatography

Magnetic bead chromatography uses magnetic beads coated with a ligand to capture and purify target molecules. The beads can be easily separated from the rest of the mixture using a magnet.

Advantages:

• High specificity
• Rapid binding
• Scalable

Disadvantages:

• High cost
• Low capacity

Membrane Chromatography

Membrane chromatography uses a membrane with a specific ligand attached to its surface. The sample is passed through the membrane, and the target molecules are bound to the ligand. The bound molecules are then released by altering the buffer conditions.

Advantages:

• High capacity
• High resolution
• Easy to scale up

Disadvantages:

• Expensive
• Membrane fouling can occur
• Limited types of membranes available

Conclusion

As with many techniques in biotechnology, the choice of chromatography technique depends on the specific needs of the experiment. Traditional chromatography techniques are still widely used in labs, but advanced chromatography techniques are becoming increasingly popular due to their higher resolution and specificity. It is important to evaluate the advantages and disadvantages of each technique when selecting the best option for your purification needs.

References

• Skoog, Douglas A.; West, Donald M.; Holler, F. James (1996). Fundamentals of Analytical Chemistry (7th ed.). Saunders College Publishing. ISBN 978-0-03-005938-9.
• Jungbauer, Alois (2008). "Chromatography of Proteins". Springer. ISBN 978-3-540-72803-9.
• Sandle, Tim (2017). "Membrane Chromatography: From Lipid Bilayers to Biochemical Separation". William Andrew. ISBN 978-0-12-811992-3.