Objective: Use and design of chromatographic multi-column processes with internal recycling for separation of electrolytes and nonelectrolytes was investigated. Fractionation of concentrated acid lignocellulosic hydrolysates was used as a model system. Methods: Equilibrium theory of chromatography was used to develop a first sketch of the separation process, which was then refined with numerical simulations. The process was validated experimentally using both synthetic solutions and authentic softwood hydrolysate. Results: A four-column process scheme with four steps and closed loop recycling operation was developed. Good separation performance was achieved with both feed solutions. Recovery yield and purity were 98% and 94% for sulfuric acid, 85% and 95% for monosaccharides, and 88% and 93% for acetic acid. Due to implementation of internal recycling steps in the process, the eluent consumption was found to be 20 times lower than in a single-column batch chromatographic process with four parallel columns. (C) 2014 Elsevier B.V. All rights reserved.