Journal of Chromatography A, 1018 (2003) 155–167 Investigation of particle-based and monolithic columns for cation exchange protein displacement chromatography using poly(diallyl-dimethylammonium chloride) as displacer Beata Schmidt, Christine Wandrey, Ruth Freitag ∗ Laboratory of Chemical Biotechnology, Institute of Chemical and Biological Process Sciences, Faculty of Basic Sciences, Swiss Federal Institute of Technology Lausanne, Ecublens, Lausanne CH-1015, Switzerland Received 3 March 2003; received in revised form 21 July 2003; accepted 22 July 2003 Abstract The overall topic of the investigation was the separation of basic proteins by cation exchange displacement chromatography. For this purpose two principal column morphologies were compared for the separation of ribonuclease A and -chymotrypsinogen, two proteins found in the bovine pancreas. These were a column packed with porous particles (Macro-Prep S, 10 m, 1000 Å) and a monolithic column (UNO TM S1). Both columns are strong cation exchangers, carrying –SO 3 - -groups linked to a hy- drophilic polymer support. Poly(diallyl-dimethylammonium chloride) (PDADMAC), a linear cationic polyelectrolyte composed of 100–200 quaternary pyrrolidinium rings, was used as displacer. The steric mass action (SMA) model and, in particular, the operating regime and dynamic affinity plots were used to aid method development. To date the SMA model has been applied primarily to simulate non-linear displacement chromatography of proteins using low molar mass displacers. Here, the model is applied to polyelectrolytes with a molar mass below 20 000 g mol -1 , which corresponds to a degree of polymerization below 125 and an average contour length of less than 60 nm. The columns were characterized in terms of the adsorption isotherms (affinity, capacity) of the investigated proteins and the displacer. © 2003 Elsevier B.V. All rights reserved. Keywords: Displacement chromatography; Steric mass action model; Stationary phases, LC; Monolithic columns; Poly(diallyl-dimethylammonium chloride); Proteins 1. Introduction Displacement chromatography has been suggested as a powerful mode for preparative biochromatogra- Abbreviations: GPC, gel permeation chromatography; PDAD- MAC, poly(diallyl-dimethylammonium chloride); RPC, reversed- phase liquid chromatography; SMA, steric mass action ∗ Corresponding author. Tel.: +41-21-693-6108; fax: +41-21-693-6030. E-mail address: ruth.freitag@epfl.ch (R. Freitag). phy, which may have significant advantage over the currently preferred (overloaded) elution mode [1]. In ion exchange displacement chromatography, the col- umn is initially equilibrated with a relatively low ionic strength carrier buffer, assuring strong binding. The feed mixture is then loaded onto the stationary phase followed by the so-called displacer. The displacer has a higher stationary phase affinity than the molecules of interest. Hence, as the displacer front advances, the number of available stationary phase binding sites is 0021-9673/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0021-9673(03)01326-8