Interference due to non-specific adsorption in ion exchange chromatography of proteins: The role of initial salt concentration in the separation and analysis of lysozyme

In this paper the influence of NaCl concentration in the binding buffer on the analysis of lysozyme in chicken egg white using MonoQ® anion exchange column is examined. When using a mobile phase having pH 9.5, lysozyme (being positively charged at that pH) is not expected to bind to the anion exchange column. However, when the NaCl concentration in the binding buffer is lower than 70 mM, lysozyme is found to bind to the column, probably due to non- specific adsorption. This results in lysozyme splitting into two peaks; a primary peak in the column void volume, and a secondary lysozyme peak in the NaCl salt gradient. The proportion of lysozyme in the secondary peak decreases with an increase in NaCl concentration in the binding buffer. No secondary peaks are observed when the NaCl concentration in binding buffer is greater than 70 mM. This observation is confirmed by experiments carried out with pure lysozyme.