PEGylation, used to mitigate some of the problems that affect the effectiveness of therapeutic proteins, often results in a heterogeneous population of conjugated species and unmodified protein that presents a protein separations challenge. This study presents the use of a mildly amphiphilic support, Tris-capped CH Sepharose 4B as an alternative for separating PEGylated proteins from their unmodified counterparts. The effects of parameters such as pH, salt type and salt concentration upon the chromatograph is behavior of native, mono-PEGylated and di-PEGylated ribonuclease A on this media were characterized. The separation of the native protein from the PEGylated species was achieved using a gradient elution between a high ionic strength mobile phase (3 M ammonium sulfate in 25 mM potassium phosphate, pH 7.0 or 2 M potassium phosphate, pH 7.0) and a low ionic strength phase (25 mM potassium phosphate, pH 7.0). The pH of the mobile phases as well as the addition of PEG(600) (as a potential mobile phase modifier) to the low ionic strength phase had no significant influence on chromatographic behavior of the species. This media provides a simple and practical chromatographic method for the separation of unmodified proteins from their corresponding PEG conjugates. (C) 2008 Elsevier B.V. All rights reserved.