If secondary interactions are suspected, the mobile phase can be modified to minimize
secondary interactions. While some buffer salt (20-100 mM) can help maintain pH
in the system and column, there are other additives that can be used to modify secondary
interactions. For example, varying NaCl concentration between 0-0.3 M can be
used to modulate any ionic interactions between the protein and the stationary phase.
Although higher salt results in less ionic interactions between protein and stationary
phase, it may also increase hydrophobic interactions.
To reduce hydrophobic interactions, salt can be reduced and small amounts of organic
(e.g. acetonitrile or isopropanol) can be added to the mobile phase. Alternatively,
adjusting the mobile phase pH to either affect silanol interactions (e.g. running under
acidic conditions) or affect the net charge of a protein based on protein isoelectric
point (pI) can reduce these interactions.