Effect of mobile phase and ligand arm on protein retention in hydrophobic interaction chromatography

The retentive properties of a series of hydrophobic interactin chromatography packings with six different lignd arms (SynChropak Hydroxypropyl, Methyl, Propyl, Butyl, Pentyl, and Benzyl) were investigated with mobile phases of different ionic compositions and pH. Substitution of ammonium acetate for ammonium sulfate resulted in decreased retention for most combinations of proteins and ligands, although the retention of some proteins, such as lysozyme on the pentyl ligand, was unchanged by the salt substitution. Generally, lower pH resulted in reduced retention, but the elution of lysozyme was more affected by pH than that of ovalbumin.