Separation of different forms of proteose peptone 3 by hydrophobic interaction chromatography with a dual salt system

A panel of four hydrophobic adsorbents (butyl-, octyl-, phenyl- and epoxy-Sepharose) was used to examine the selectivity and fractionation of several proteose peptone 3 (PP3) forms from a freeze-dried extract of whey bovine milk. In parti- cular, the effects of altering the ligand type and salt were investigated. The chromatographic studies suggest that PP3 strongly interacts among the three commercial hydrophobic resins leading to a drop off in selectivity, while a complete binding was achieved at low salt concentrations (below 0.5 M) and total elution only with phosphate buffer and/or water stepwise conditions. Only in epoxy-Sepharose was an appreciably selectivity of the several fractions of PP3 present in the initial feedstock attained. Despite the high salt concentration for a complete binding of PP3 (above 1.5 M ammonium sulfate) onto this support, the dual salt system (ammonium sulfate 1 M and sodium citrate 0.8 M) led to a high separation degree of high and low molecular weight forms of PP3. Copyright © 2008 John Wiley & Sons, Ltd.