Ž . Biochimica et Biophysica Acta 1379 1998 303–313 Temperature-responsive size-exclusion chromatography using ž / poly N-isopropylacrylamide grafted silica H. Lakhiari a , T. Okano b , N. Nurdin c , C. Luthi c , P. Descouts c , D. Muller a, ) , J. Jozefonvicz a a Laboratoire de recherche sur les macromolecules, CNRS URA 502, UniÕersite Paris Nord, Institut Galilee, AÕenue J.B. Clement, ´ ´ ´ ´ 93430 Villetaneuse, France b Tokyo Women’s Medical College, 8 Kawada, Shinjuku, Tokyo 162, Japan c Groupe de physique appliquee, UniÕersite de GeneÕe, 20, rue de l’Ecole de Medecine - CH-1211, GeneÕa 4, Switzerland ´ ´ ` Received 12 August 1997; accepted 25 August 1997 Abstract Silica-based packing materials induce non-specific interactions with proteins in aqueous media because of the nature of their surface, mainly silanol groups. Therefore, the silica surface has to be modified in order to be used as stationary phase Ž . for the High Performance Size-Exclusion Chromatography HPSEC of proteins. For this purpose, porous silica beads were Ž . coated with hydrophilic polymer gels dextrans of different molecular weights carrying a calculated amount of diethyl- Ž . aminoethyl groups DEAE . Actually, as shown by HPSEC, these dextran modified supports minimize non-specific adsorption for proteins and pullulans in aqueous solution. Then, in order to change the pore size in response to temperature, Ž .Ž . temperature responsive polymer of poly N-isopropylacrylamide PIPAAm was introduced into the surface of dextran- DEAE on porous silica beads. The structure of these supports before and after modification was alternately studied by Ž . Ž . Scanning Electronic Microscopy SEM and Scanning Force Microscopy SFM . An adsorption of radiolabelled albumin was performed to complete our study. Silica modifications by dextran-DEAE and PIPAAm improve the neutrality of the support and minimize the non-specific interactions between the solid support and proteins in solution. At low temperature, the support having PIPAAm exhibits a high resolution domain in HPSEC and finally permits a better resolution of proteins and pullulans. At higher temperature, hydrophobic properties of PIPAAm produce interactions with some proteins and trigger off a slight delay of their elution time. q 1998 Elsevier Science B.V. Ž . Keywords: Silica; Poly N-isopropylacrylamide ; High performance size-exclusion chromatography 1. Introduction wx Since its introduction by Porath and Flodin 1, Ž . Size-Exclusion Chromatography SEC has become one of the most promising techniques for both analy- sis and purification of proteins. Moreover, the avail- ) Corresponding author. Fax: q33 1 4823 2801. ability of numerous stationary phases with excellent mechanical and chemical properties has improved the efficiency of this method. Proteins separation can be achieved by a SEC procedure based upon proteins diffusion in the pores of a support according to its wx hydrodynamic volume 2 . This method allows the elution of proteins in reverse order of their molecular size without any interaction. Unfortunately, non- 0304-4165r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. Ž . PII S0304-4165 97 00110-4