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What buffers are commonly used in exchange chromatography?

Release time:

2022-07-13

It is very important to keep the pH constant during the ion exchange process. The change of pH will cause changes in the number and distribution of protein charges, which directly affects whether the protein can bind to the exchanger and the strength of the binding force. Therefore, buffers must be used for the mobile phase in ion exchange chromatography.

There are many types of buffers, and the substances that can act as buffers can be divided into two categories: the first type is a system composed of weak acids (or weak bases) and corresponding salts; the second type is zwitterionic compounds. For the first type of buffer substances, during ion exchange, if the charge of the buffer ion is opposite to the functional group on the ion exchanger, it will participate in the ion exchange process and may affect the local pH, so it should be used as much as possible. Buffer ions with the same charge as the functional group, namely: when using an anion exchanger, choose a buffer ion with a positive charge; when using a cation exchanger, choose a buffer ion with a negative charge.

Table 1 Commonly used buffers for cation exchange chromatography

Of course, this is not absolute. For example, phosphate buffer is often used in the anion exchange process, but in this case, special attention should be paid to fully equilibrating before loading the sample to ensure that the pH and ionic strength of the chromatographic system are consistent with the initial value. Buffers are the same. The second type of buffer substances can be used in both anion and cation exchange. Commonly used buffers for cation exchange chromatography and anion exchange chromatography are listed in Tables 1 and 2, respectively.

Table 2 Commonly used buffers for anion exchange chromatography

The mobile phase of ion exchange chromatography must be a solution with a certain ionic strength and a certain pH buffering capacity. In order to avoid the inactivation of the target protein, the use of buffer can stabilize the pH of the mobile phase, so that it does not change significantly during the chromatography process, and at the same time, it can stabilize the charge on the target molecule to ensure the importance of the chromatographic results.

The selection of buffer is generally based on the following principles: cation exchangers should be anion buffers, such as citrate, phosphate, acetate, glycinate, etc.; anion exchangers should be cation buffers, such as alkylamine, Tris, amino Ethanolamine, ethylenediamine, imidazole, etc.; the concentration of the starting buffer should be as low as possible (<100mmol/L) so that more substances can be adsorbed and separated on the chromatographic column;

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