Fast protein liquid chromatography (FPLC) is a powerful technique used to analyze and purify large biomolecules like proteins or DNA. However, during the purification process, proteins can become denatured or aggregated due to the presence of dissolved gases in the mobile phase. This can lead to a decrease in protein yield and purity and can even result in irreparable damage to the protein. To prevent these problems, online degassing is an essential step in FPLC.
Degassing is the process of removing dissolved gases from a liquid. In FPLC, online degassing is achieved by passing the mobile phase through a degasser unit before it enters the column. The degasser unit uses vacuum pressure to remove dissolved gases such as oxygen and carbon dioxide, which can interfere with the protein purification process. This allows for the mobile phase to be free of any dissolved gases, resulting in improved protein yield and purity. Degassing the mobile phase prevents bubble formation. An online degasser is the preferred method.
One of the main benefits of online degassing is that it helps prevent protein denaturation. Denaturation is the process by which a protein loses its native structure and function. This can be caused by several factors, including changes in pH, temperature, and the presence of denaturants such as detergents or salts. However, dissolved gases can also cause denaturation by altering the pH of the mobile phase. When carbon dioxide dissolves in water, it forms carbonic acid, which can lower the pH of the mobile phase. This can cause proteins to become denatured, leading to reduced yield and purity.
In addition to preventing denaturation, online degassing also helps prevent protein aggregation. Aggregation is the process by which proteins form clumps (aggregates). Which can reduce the yield and purity of the protein sample. Dissolved gases can cause aggregation by altering the electrostatic interactions between protein molecules. For example, oxygen can react with sulfhydryl groups on proteins to form disulfide bonds, which can lead to protein aggregation. By removing dissolved gases, online degassing helps prevent these unwanted interactions and ensures a higher yield and purity of the protein sample.
Online degassing is also important for maintaining the accuracy and reproducibility of FPLC experiments. When dissolved gases are present in the mobile phase, they can cause fluctuations in the baseline of the chromatogram, making it difficult to accurately measure the absorbance of the protein sample. This can lead to inaccurate quantification of the protein yield and purity and can even result in errors in downstream applications such as protein structure determination. By removing dissolved gases, online degassing helps ensure that FPLC experiments are more accurate and reproducible.
Another benefit of online degassing is that it helps improve the stability of the protein sample. Dissolved gases can cause oxidative damage to proteins, leading to reduced stability and increased susceptibility to proteolysis. This can be particularly problematic for labile proteins or proteins that are sensitive to oxidative stress. By removing dissolved gases, online degassing helps protect the protein sample from oxidative damage, improving its stability and shelf life.
Online degassing is critically important for single pump gradient FPLC systems. In these systems the gradient is formed before the pump. Degassing before the valve inlets enables buffers to be bubble free. Bubbles in the valve inlet create inaccurate and irreproducible gradients.
Finally, online degassing can help reduce the cost and time associated with FPLC experiments. When dissolved gases are present in the mobile phase, they can cause column fouling, leading to a decrease in the efficiency of the chromatography. This can result in longer run times and increased use of mobile phase, which can be costly and time-consuming. By removing dissolved gases, online degassing helps prevent column fouling and ensures a more efficient and cost-effective FPLC experiment.
In conclusion, online degassing is an essential step in FPLC that helps prevent protein denaturation and aggregation, maintains the accuracy and reproducibility of experiments, improves the stability of the protein sample, and reduces the cost and time associated with FPLC experiments. By removing dissolved gases from the mobile phase, online degassing increases the reliability of the FPLC system.
ICON Scientific, Inc
