Glucosinolates (GSLs) are a class of phytochemicals found in all calciferous plants (Brassicaceae) family as well as in the whole order of Brassicales (syn. Capparales). GSLs and their hydrolysis products (e.g. isothiocyanates) are known to play a defensive role by protecting the plant against exterior aggressions and may be potent antitumor, anticancer, antioxidant and antibiotic agents. In order to obtain pure GSLs standards for developing research, an efficient two-step method was developed to preparatively isolate and separate GSLs from papaya (Carica papaya), upland cress (Barbarea verna), cauliflower (Brassica oleracea L. var. botrytis L.), and broccoletti seeds (Brassica rapa ruvo). In this process, solvent extraction was followed by a strong anion-exchange centrifugal partition chromatography protocol. TLC, HPLC-PDA, ESI-MS and NMR analysis of the collected fractions, demonstrated that the GSLs from papaya and upland cress seeds (i.e. glucotropaeolin and gluconasturtiin, respectively) as well as all the GSLs from cauliflower and broccoletti seeds (i.e. sinigrin, glucoiberin, glucoiberverin, gluconapin, glucobrassicanapin and gluconasturtiin) can be purified in a single chromatographic step. Gram amounts of pure reference standards were obtained. These results will facilitate investigation of the biological activities and the isolation of such compounds in other crucifers. (C) 2011 Published by Elsevier B.V.