pH is an important factor in the operation of ion exchange chromatography, and pH stabilization and changes are usually achieved with buffers, so the choice of buffer is an important factor affecting the separation.
When selecting a buffer, pH and ionic strength are two key factors that affect not only the separation of the target product from the impurities, but also the yield of the product. The pH chosen depends on the isoelectric point, stability and solubility of the target product, not only to make the separated material an ion that can be exchanged, but also to maintain its high activity. The pK value of the ion exchanger should also be considered.
Since the buffer itself is charged, it is also combined with the ion exchange chromatography medium
. This combination will cause two kinds of interference, on the one hand, reducing the concentration of the buffer, thereby reducing the buffering capacity; on the other hand, it is exchanged with the separation medium to compete with the protein for the exchange capacity of the medium. Therefore, when using anion exchange chromatography media, avoid using negatively charged buffers such as phosphate. When using cation exchange chromatography media, avoid using positively charged buffers such as Tris.
Due to the different types of separation media, the initial process is slightly different. Under normal circumstances, when anion exchange chromatography media is used, the initial buffer is higher than the isoelectric point of the target protein by 0.5-1 pH; using a cation exchange layer When the medium is analyzed, the starting buffer is lower than the isoelectric point of the target protein by 0.5 to 1 pH.