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The results of copolymer characterization by coupling of chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques and subsequent calculation of copolymer composition using a novel software tool MassChrom2D are presented. For high-resolution mass analysis copolymer samples were fractionated by means of liquid adsorption chromatography (LAC). These fractions were investigated off-line by MALDI-TOF MS. Various mono-n-butyl ethers of polyethylene oxide-polypropylene oxide copolymers (PEO-co-PPO) were investigated. As well as the copolymer composition presented in two-dimensional plots, the applied approach can give additional hints on specific structure-dependent separation conditions in chromatography.