Biochemical Engineering Journal 26 (2005) 12–21 Adsorption of serum albumin and -globulin from single and binary mixture and characterization of pHEMA-based affinity membrane surface by contact angle measurements G¨ ulay Bayramo ˘ glu, Emine Yalc ¸in, M. Yakup Arica ∗ Biochemical Processing and Biomaterial Research Laboratory, Faculty of Science, Kırıkkale University, 71450 Yah¸ sihan, Kırıkkale, Turkey Received 3 November 2004; received in revised form 2 May 2005; accepted 3 June 2005 Abstract In this study, an affinity membrane was synthesized using 2-hydroxyethylmethacrylate (HEMA) via UV-initiated photopolymerization. A dye-ligand (i.e., Procion Red HE-3B; Red-120) was covalently immobilized onto membrane. Human serum albumin (HSA) and human - globulin (HIgG) adsorption onto pHEMA-Red-120 membrane were studied using bare poly(hydroxyethylmethacrylate) (pHEMA) membrane as a control system. The information about surface energy, hydrophobicity and chemical heterogeneity of the affinity membrane was obtained by contact angle measurements. The contact angle values of the affinity membrane were determined by sessile drop method using water, glycerol and diiodomethane as test liquids. Component and parameters of the surface free energy of all the investigated samples were calculated from measured contact angle values using the acid–base method of the van Oss. The adsorption of HSA and HIgG significantly changed both the contact angles and component of surface free energies of the affinity membrane. The reversible HSA and HIgG adsorption on the pHEMA-Red-120 followed the Freundlich and Langmuir–Freundlich isotherm models. Selectivity of the affinity membrane was tested at different pH values to HSA and HIgG and the protein concentration of in the binary system was determined by HPLC. The affinity membrane was stable when subjected to sanitization with sodium hydroxide after repeated adsorption–elution cycles. © 2005 Elsevier B.V. All rights reserved. Keywords: Microporous membrane; Affinity chromatography; Dye-ligand; Adsorption; Separation; Albumin; -Globulins; Contact angle 1. Introduction The recently introduced affinity membrane chromatog- raphy offers some apparent advantages over conventional bead-packed column chromatography in bio-separation such as higher flow rate, low-pressure drops, faster binding kinetics, and simple scale-up [1,2]. Affinity separation relies on the highly specific binding between a target protein in solution and an immobilized ligand to achieve a high degree of protein purification. Different types of affinity ligand molecules have been introduced on the membrane surfaces for protein purification such as protein A [3,4], triazidine dyes [5–7], immobilized metal [8–10] and amino acids [12–14]. The dye-ligand affinity separation has become a popular technique for purifying proteins. For example, Arıca et al. ∗ Corresponding author. Tel.: +90 318 357 2477; fax: +90 318 357 2329. E-mail address: yakuparica@tnn.net (M.Y. Arica). [11] reported the purification of hydroxypyruvate reductase from bacterial homogenised solution using the poly(HEMA- EGDMA) microspheres with Cibacron Blue F3GA as a dye-ligand. Suen et al. [15] reported the effective adsorption of lysozyme from aqueous solution using Cibacron Blue F3GA immobilised cellulose membrane discs. Reactive Red- 120 and Reactive Yellow-86 have been used as dye-ligands for the purification of high-Mr glutenin subunits from wheat flour [16]. This affinity technique makes use of immobilised dye molecules that interact with the protein sites by electro- static and hydrophobic force. The three-dimensional surface properties of the protein and the ligand-polymer structure, the surface ligand density, the type and concentration of salt, pH of the medium and temperature influence the dye-ligand affinity separation of proteins. [17,18]. The performance of an affinity membrane depends on its surface properties. The surface properties of an affinity mem- brane may affect the adsorption capacity and its behaviour by 1369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2005.06.002