In fact, sodium, potassium, or ammonium sulfates enhance ligand-protein interactions in HIC while also stabilizing protein structure. As a result, the most prevalent ions are (NH4)2SO4, Na2SO4, NaCl, KCl, and CH3COONH4. Figure 1 depicts how various salts can influence selectivity. The highest resolution of four standard proteins was achieved by starting with 1.7 M ammonium sulfate.
Because each salt has a different capacity to encourage hydrophobic interactions, selecting a salt for an HIC separation can be a trial and error process. When the concentration of a salt rises, the quantity of protein bound increases almost linearly up to a certain salt concentration and then exponentially at greater concentrations.When compared to other salts, ammonium sulfate often provides the greatest clarity at a particular concentration and can be used at ratios up to 2 M.
When using sodium chloride, concentrations of up to 3 M are typically needed.
Although sodium sulfate is an excellent salting-out substance, issues with protein solubility may preclude its use at high amounts.
Working with ammonium sulfate at pH levels above 8.0 is not advised.