Kishore K. R. Tetala 1 Bo Chen 1,2 Gerben M. Visser 1 Teris A. van Beek 1 1 Laboratory of Organic Chemistry, Wageningen University, Wageningen, The Netherlands 2 Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research, Ministry of Education, Hunan Normal University, Changsha, P. R. China Original Paper Single step synthesis of carbohydrate monolithic capillary columns for affinity chromatography of lectins Carbohydrate monolithic beds were synthesized in a single step in capillary col- umns to study affinity chromatography of lectins. In this method, carbohydrates (b- galactose, b-glucose, and a-mannose) with an easy to synthesize alkene terminated tetraethylene glycol spacer were used as functional monomers along the monomer 2-hydroxyethyl methacrylate (HEMA). As crosslinkers (+)-N,N9-diallyltartardiamide (DATD) and piperazine diacrylamide (PDA, 1,4-bisacryloyl-piperazine) were used. SEM showed the successful formation of monolithic beds in the capillary columns. The permeability of the columns was high. The specific interaction of the lectins Con A, Lens culinaris (LCA) and Arachis hypogaea (PNA) with the carbohydrate station- ary phase was studied by frontal affinity chromatography (FAC). Con A and LCA were successfully eluted from the column using 0.1 M methyl-a-mannopyranoside and PNA with 0.1 M b-galactose. Dissociation constants (K d ) for carbohydrate – lectin interactions were determined and compared with literature. Keywords: Affinity monolithic bed / Dissociation constant / Frontal affinity chromatography / Lectin elution and scanning electron microscopy / Received: July 30, 2007; revised: September 19, 2007; accepted: September 19, 2007 DOI 10.1002/jssc.200700356 1 Introduction The increase in demand for the fast separation of biomo- lecules in separation science initiated the search for new solid stationary phases, which finally resulted in the invention of “monoliths (continuous beds)” in the year 1989 by HjertØn et al. [1]. Out of the two major different types of monolithic phases, organic stationary phases (polymethacrylate, polystyrene, etc.) and inorganic sta- tionary phases (silica), organic phases are preferred due to their efficiency in the fast separation of large biomole- cules [2, 3]. The use of different combinations of mono- mers, crosslinkers, and porogens makes monoliths flexi- ble to work with. Moreover, the distinct advantages such as (i) easy to introduce in microchannels (capillary and chip), (ii) high mass transport and permeability, (iii) high capacity, and (iv) high mechanical, chemical, and ther- mal stability are of great added value [4–6]. Monolithic materials are extensively used for enzyme or protein immobilization and in the separation of bio- molecules [7–16]. In recent years, various research groups explored the advantages of these materials in other fields, such as incorporating catalysts to perform organic synthesis and the separation of chiral compounds [17– 20]. However, one field receiving little attention so far is “the study of carbohydrate–lectin interactions” by utiliz- ing carbohydrate monolithic columns. Lectins, widely known as carbohydrate recognizing proteins, occur both in plants and animals and are used in understanding the fundamental aspects of “carbohy- drate–protein binding.” These interactions are reversible and play a pivotal role in cell interactions in the immune system, cancer metastasis and cell agglutination [21–25]. A better diagnosis or even contributions to treatments in such widely differing fields as oncology, autoimmune diseases, bacterial infections, and plant pathology can benefit from measuring the strength of carbohydrate– lectin interactions. For instance, it has been shown that binding constants can be enhanced several orders of magnitude by multivalency interactions [25]. So far only few publications appeared in the carbohydrate–lectin interaction field using monoliths as stationary phases [26, 27]. In these publications, the affinity monolithic bed was prepared in two or three steps. Recently, we Correspondence: Dr. Teris A. van Beek, Laboratory of Organic Chemistry, Dreijenplein 8, 6703 HB Wageningen, The Nether- lands E-mail: Teris.vanBeek@wur.nl Fax: +31-317-484914 Abbreviations: BB, binding buffer; DATD, (+)-N,N9-diallyltartar- diamide; FAC, frontal affinity chromatography; LCA, Lens culina- ris; PNA, Arachis hypogaea i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com 2828 K. K. R. Tetala et al. J. Sep. Sci. 2007, 30, 2828 – 2835