A one step supercritical fluid chromatography process has been developed using a semi-preparative supercritical fluid chromatography scale plant designed and built in-house, for isolation and purification of polyunsaturated fatty acid ethyl esters from different origins (fish and algae). A wide range of experimental conditions (pressure, temperature, sample load, co-solvent rate and CO2 flow rate); columns (stationary phase and particle size); and UV/vis detectors have been investigated to optimise the isolation and purification of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DMA) from an algae oil rich in EPA, and a fish oil rich in EPA and DMA. The particle size, followed by the stationary phase, sample loading, temperature/pressure and feed oil composition, were found to be the most important parameters for achieving separation. Under optimised conditions, EPA at greater than 95% purity from both oils and DHA at greater than 80% purity from fish oil was obtained in a single pass and without co-solvent. (C) 2012 Elsevier B.V. All rights reserved.