Detailed molecular characterization and fractionation were carried out for a polystyrene-block-poly(methylmethacrylate) (PS-b-PMMA) diblock copolymer prepared by linking PS-alkyne and PMMA-azide through a click reaction. PS and PMMA block precursors were prepared by atom transfer radical polymerization and anionic polymerization, and end-functionalized to have alkyne and azide group, respectively. As-synthesized block copolymer was characterized by solvent gradient reversed-phase liquid chromatography (RPLC) with multiple-detection for both separation and identification of byproducts. As-synthesized PS-b-PMMA contains various byproducts including unreacted precursor homo-polymers as well as byproducts formed during the synthesis of precursor polymers. To fractionate pure PS-b-PMMA out, two-step HPLC fractionation was employed. In the first step, PMMA homo-polymer was removed from the as-synthesized polymer using a C18 bonded silica column. In the second step, PS homo-polymer was removed to obtain pure PS-b-PMMA using a bare silica column. In both steps, multiple-injection method was used to facilitate the large scale fractionation procedure. A twodimensional liquid chromatography method, solvent gradient RPLC for the first dimension and SEC for the second dimension was also conducted to map the molecular characteristics of the block copolymer and its byproducts. (C) 2015 Elsevier Ltd. All rights reserved.