How buffer pH and NaCl affect size exclusion chromatography?

Size exclusion chromatography (SEC) is a non-invasive technique that’s used to separate proteins based only on their hydrodynamic radius (size). In this process, NaCl (sodium chloride) is added to the eluent to minimize secondary interactions between the sample components and the resin.

The presence or absence of NaCl in aqueous elements alters the hydrodynamic radius of proteins, thereby affecting the elution volume of the proteins. The presence of NaCl in the mobile phase prevented any protein-matrix interactions. This results in more defined protein peaks and more accurate determination of molecular size.

Optimum buffer pH for size exclusion chromatography is between 6.0 and 8.0, which mimics biological conditions. Most proteins are stable in that pH range. Proteins are usually less soluble at their pI value, which is the pH at which the protein has a zero net charge. If the pH is not within the optimum range, it needs to be adjusted in order to keep the protein in solution.