Recent studies have shown that the chromatographic separation of mixtures of saccharides may be improved by making use of activated charcoal, a promising low cost material for the separation of sugars, including fructooligosaccharides. In this work, the development of a methodology to separate fructooligosaccharides from glucose, fructose and sucrose, using a fixed bed column packed with activated charcoal is proposed. The influence of temperature, eluant concentration and step gradients were evaluated to increase the separation efficiency and fructooligosaccharide purity. The final degree of fructooligosaccharide purification and separation efficiency were about 94% and 3.03 respectively, using ethanol gradient concentration ranging from 3.5% to 15% (v/v) at 40°C. The fixed bed column packed with the activated charcoal was shown to be a promising alternative for sugar separation, mainly those rich in fructooligosaccharides, leading to solutions of acceptable degrees of purification. © 2014.313237241Gibson, G.R., Roberfroid, M.B., Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics (1995) J Nutr, 125, pp. 1401-1412Sangeetha, P.T., Ramesh, M.N., Prapulla, S.G., Maximization of fructooligosaccharide production by two stage continuous process and its scale up (2005) J Food Eng, 68, pp. 57-64Kuhn, R.C., Maugeri, F., Separation of fructooligosaccharides using zeolite fixed bed columns (2010) J Chromatogr B, 878, pp. 2023-2028Vankova, K., Polakovic, M., Optimization of single-column chromatographic separation of fructooligosaccharides (2010) Process Biochem, 45, pp. 1325-1329Nobre, C., Suvarov, P., de Weireld, G., Evaluation of commercial resins for fructo-oligosaccharides separation (2014) New Biotech, 31, pp. 55-63Kuhn, R.C., Maugeri, F., Purification of fructooligosaccharides in an activated charcoal fixed bed (2010) New Biotech, 27, pp. 862-869Nobre, C., Teixeira, J.A., Rodrigues, L.R., Fructo-oligosaccharides purification from a fermentative broth using an activated charcoal column (2012) New Biotech, 29, pp. 395-401Hernandez, O., Ruiz-Matute, A.I., Olano, A., Moreno, J.J., Sanz, M.L., Comparison of fractionation techniques to obtain prebiotic galactooligosaccharides (2009) Int Dairy J, 19, pp. 531-536Kuhn, R.C., Maugeri, F., Silva, V., Palacio, L., Hernández, A., Prádanos, P., Mass transfer and transport during purification of fructooligosaccharides by nanofiltration (2010) J Membr Sci, 365, pp. 356-365Gramblicka, M., Polakovic, M., Adsorption equilibria of glucose, fructose, sucrose, and fructooligosaccharides on cation exchange resins (2007) J Chem Eng Data, 52, pp. 345-350Boon, M.A., Vant Riet, K., Janssen, A.E.M., Enzymatic synthesis of oligosaccharides: product removal during a kinetically controlled reaction (2000) Biotech Bioeng, 70, pp. 411-420Hidaka, H., Hirayama, M., Sumi, N., A fructo-oligosaccharide producing enzyme from Aspergillus niger ATCC 20611 (1988) Agric Biol Chem, 52, pp. 1181-1187Maugeri, F., Hernalteens, S., Screening of yeast strains for transfructosylating activity (2007) J Mol Catal B: Enzym, 49, pp. 43-49Maugeri, F., Hernalteens, S., Barboza, G.N., EO Aguiar Processo de obtenção de frutooligossacarídeos em biorreator utilizando enzima frutosiltransferase. BR Patent 0705359-2Lu, T.S., Lee, T.Y., Separation of diethylbenzene isomers by column adsorption and desorption (1987) Ind Eng Chem Res, 26, pp. 2024-2028Morales, V., Sanz, M.L., Olano, A., Corzo, N., Rapid separation on activated charcoal of high oligosaccharides in honey (2006) Chromatographia, 64, pp. 233-238Toshiaki, E., Hidaka, H., Toshiaki, K., (1981) Method of producing high purity maltose. USP4294623Ahmedna, M., Marshall, W.E., Rao, R.M., Surface properties of granular active carbons from agricultural by-products and their effects on raw sugar decolorization (2000) Bioresour Technol, 71, pp. 103-112Kaplan, H., Hutkins, R.W., Fermentation of fructooligosaccharides by lactic acid bacteria and bifidobacteria (2000) Appl Environ Microb, 66, pp. 2682-2684Abe, I., Hayashi, K., Kitagawa, M., Adsorption of saccharides from aqueous solution onto activated carbon (1983) Carbon, 21, pp. 189-191