J. Sep. Sci. 2012, 00, 1–7 1 Nat ´ alia K ´ aˇ navov ´ a 1 Anna Kosior 1 Monika Anto ˇ sov ´ a 1 Ren ´ e Faber 2 Milan Polakovi ˇ c 1 1 Department of Chemical and Biochemical Engineering, Institute of Chemical and Environmental Engineering, Faculty of Chemical and Food Technology, Slovak University of Technology, Bratislava, Slovakia 2 Sartorius Stedim Biotech AG, G¨ ottingen, Germany Received April 25, 2012 Revised June 4, 2012 Accepted June 4, 2012 Research Article Application of a micromembrane chromatography module to the examination of protein adsorption equilibrium A micromembrane chromatography module based on a 96-well plate design and enabling fast and simple separation of small amounts of proteins was used for the determination of binding capacities of lysozyme, bovine serum albumin, ovalbumin, bovine -globulin, and human immunoglobulin G on a hydrophobic membrane Sartobind R  Phenyl. Depen- dence of the binding capacity of the proteins on the ammonium sulfate concentration was examined in the salt concentration range of 0.5–2.0 mol L −1 . An exponential increase of the binding capacity was observed for all proteins. Simple Langmuir one-component isotherm was found suitable for the characterization of the effect of protein concentration in all cases. A combined effect of protein and salt concentrations was expressed via the Langmuir exponential isotherm and fitted the adsorption data for three of the investigated proteins well. Keywords: Adsorption isotherm / Hydrophobic interaction / Membrane chro- matography / Micromembrane / Protein-binding capacity DOI 10.1002/jssc.201200396 1 Introduction Microcolumn and micromembrane liquid chromatography is a separation technique having a large potential that has so far been utilized mainly in analytical applications. For example, it was successfully used for determination of fat-soluble vita- mins and provitamins in milk or bovine blood plasma [1, 2]. Henzel et al. [3] combined this technique with mass spec- trometry as a rapid method for separation and identification of proteins separated by gel electrophoresis. Other authors applied microcolumn liquid chromatography as a powerful separation and purification technique for very small amounts of proteins and peptides [4, 5]. It is also attractive technique for enantiomeric separations because it allows the use of ex- pensive stationary and mobile phases as their consumption is very low and chromatographic efficiency and selectivity is quite high [6]. Protein separation and purification are very important industrial processes in biotechnology. Particle packed bed is a dominant form of chromatographic stationary phase in this field but membranes and monoliths have gained impor- tance in the recent period. Considering the variety in texture, chemical structure of supports and ligands of all three main forms it is not easy to choose a chromatographic stationary Correspondence: Dr. Milan Polakoviˇ c, Department of Chemical and Biochemical Engineering, Faculty of Chemical and Food Tech- nology, Slovak University of Technology, Radlinsk´ eho 9, 812 37 Bratislava, Slovakia E-mail: milan.polakovic@stuba.sk Fax: +421-2-52493198 Abbreviation: HIC, Hydrophobic interaction chromatography phase material with the best process performance. Also the search for optimal chromatographic conditions can be very time consuming and expensive. The whole development pro- cess can become more effective when microcolumns and/or micromembranes are employed in the screening of suitable stationary phase materials. Since these techniques require only small amounts of proteins, chemicals, and simple equip- ment, they can be efficiently used to generate rich adsorption equilibrium data beneficial for optimal design of chromato- graphic processes. Hydrophobic interaction chromatography (HIC) is a sep- aration and purification technique that separates proteins by differences in their hydrophobicity. Adsorption is based on hydrophobic interaction between ligands immobilized on the stationary phase and nonpolar domains on the protein sur- face. The key advantage of HIC is the use of relatively mild conditions so that the conformation and biological activity of proteins are maintained [7–9]. Significant properties of hy- drophobic adsorbents are the type, density, and length of the ligand. The most frequently used ligands are linear chains of alkanes with or without terminal amino group, phenyl, or other aromatic groups. Hydrophobicity of n-alkanes increases with their chain length but the selectivity of adsorption can decrease [8, 9]. High ligand density is also favorable only to some extent since it can result in rather irreversible binding. Hydrophobic interactions are also significantly enhanced by the presence of kosmotropic ions that are added into the mo- bile phase in relatively high concentrations [10, 11]. Membrane chromatography has been introduced to over- come some limitations of particle bed chromatography. The key advantage of membrane chromatography is convective mass transfer to adsorbent-binding sites that makes its C  2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com