Journal of Chromatography A, 1216 (2009) 6716–6727 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Effects of thermal heterogeneity in hydrophobic interaction chromatography Renata Muca, Wojciech Pi ˛ atkowski, Dorota Antos ∗ Chemical and Process Engineering Department, Rzeszow University of Technology, W. Pola Str., 2, 35-959 Rzeszow, Poland article info Article history: Received 23 April 2009 Received in revised form 30 July 2009 Accepted 5 August 2009 Available online 11 August 2009 Keywords: HIC Heat transfer Radial temperature gradient Salt gradients abstract Manipulating temperature and salt concentration can have a powerful effect on the separation effective- ness in hydrophobic interaction chromatography (HIC). However, use of temperature as an operating variable in large-scale applications may involve undesirable consequences such as radial heterogeneity of the column temperature. In this study non-ideal effects of heat transfer in HIC columns were ana- lyzed. The radial temperature gradients were measured by thermocouples immersed in a bed packed into a preparative column. The column wall was either thermostatted by a water jacket or left under ambient conditions. The inﬂuence of ineffective column thermostatting and of heat losses on the radial temperature proﬁles was demonstrated and predicted by a model of heat dispersion in a packed bed. To analyze possible positive or negative effects of thermal heterogeneity on band propagation, non- isothermal chromatographic elution of a model protein (-chymotrypsinogen A) was recorded under salt gradient conditions as well as at constant salt concentration. To predict temperature and concen- tration proﬁles a model of the column dynamics was used. The model accounted for kinetics of mass and heat transfer. A good agreement between experimental and simulated proﬁles was achieved. It was shown that by proper selection of the process conditions undesirable temperature effects can be avoided or controlled. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Hydrophobic interaction chromatography (HIC) is a useful tool for preparative and industrial scale puriﬁcation of proteins [1–6]. It provides an alternative or supplementary chromatographic tech- nique to the most widely used mode in preparative bioseparations, i.e., ion exchange chromatography [2,7]. Hydrophobic interactions that control the separation selectiv- ity are affected by a number of the process variables, among which salt type and its concentration and temperature are of major importance [8–13]. Protein adsorption is induced by high concen- tration of structure-enhancing salts (cosmotrope) such as those used in precipitation (e.g., ammonium sulfate). High salt concen- tration enhances hydrophobic interactions between immobilized hydrophobic ligands and non-polar patches on the protein sur- face while elution is promoted at low salt concentration. Similar effect can be achieved by manipulation of temperature. In general, increasing temperature enhances hydrophobic interactions and the protein retention and lowering temperature facilitate the protein elution [14,15]. Therefore, both the operating variables the salt concentration as well as temperature might be used to alter the efﬁciency of HIC processes. ∗ Corresponding author. Tel.: +48 17 865 1853. E-mail address: dorota.antos@prz.edu.pl (D. Antos). In our last study [16] these two variables were combined in step gradients to separate few model proteins and a procedure for the process design has been suggested. The separation was realized using an analytical small-diameter column immersed in water bath. Because the column wall was thermostatted properly undesirable effects of thermal heterogeneity could be neglected and the process temperature was controlled solely by the column wall. However, the scaling up such a process is not straightforward. Because heat transfer in the packed bed is slow when the mobile phase enters the column at a temperature different from that of the column wall a radial temperature gradient exists, which can be sig- niﬁcant for a large-diameter column. This phenomenon is affected by the ﬂowrate, the column dimensions and by temperature differ- ences between the wall and the mobile phase [17–19]. Temperature heterogeneity of the column may also result from the viscous friction of the mobile phase generating heat in the col- umn. Such a phenomenon is particularly pronounced for columns, which operating requires very high pressure [20,21]. For low- pressure systems heat exchange caused by the viscous friction is insigniﬁcant. Nevertheless, the temperature heterogeneity in non- isothermal low-pressure systems cannot be neglected. Poor eluent preheating or ineffective thermal equilibration of the mobile phase in column may cause band broadening and distortion of chromato- graphic peaks, which results in drastic reduction of the column performance [17–23]. On the other hand, as reported in literature [24,25], the separation efﬁciency can be increased by proper manip- ulation of the difference between temperature of eluent and the 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.08.007