Two routes aimed at the manufacture of unprecedented stereoisomeric combretastatin A-4 analogue were described: flash chromatography vs supercritical fluid chromatography. The latter has many advantages over liquid chromatography and was therefore chosen for the small scale separation of methyl 1-[(3-hydroxy-4-methoxyphenyl) (3,4,5-trimethoxyphenyl)methyl]-5-oxo-l-prolinate 5, with potential antitumoral activity. After a screening of six different polysaccharide based chiral stationary phases and four co-solvents, the percentage of co-solvent, the flow-rate and the outlet pressure were optimized through a design of experiments (DoE). The preparation of 50 mg of each stereoisomer was achieved successfully on a Chiralpak AD-H with isopropanol as a co-solvent. Productivity (kkd), solvent usage and environmental factor (E Factor) were calculated. Flash chromatography and supercritical fluid chromatography approaches were compared in terms of yield and purity of each stereoisomer manufactured.