The objective of this study was to purify stearidonic acid (SDA, 18:4 x-3) from modified soybean oil containing a mixture of over 20 fatty acids (23% SDA). Interest in obtaining purified fractions of SDA arises from reported health benefits associated with polyunsaturated fatty acids (PUFA), such as cardiovascular disease prevention. In addition, SDA may also provide improved stability characteristics since its unsaturation index is less than longer PUFA, such as eicosapentaenoic acid (EPA, 20:5 x-3). First, a chemical ethanolysis of modified soybean oil was performed to transform the triacylglycerols into fatty acid ethyl esters (FAEE). Then, the FAEE were fractionated and SDA-EE was purified by argentation silica gel (10% AgNO 3 ) open column chromatography, which allows selectivity based on degree of unsaturation. Different FAEE sample loads and mobile phases were explored until the best purification of SDA-EE was achieved. The solvents used were hexane and hexane:acetone mixtures (99 and 95%). Under the optimal conditions, a fraction with high SDA-EE purity (96%) was obtained with 77% yield. Besides, it was possible to obtain another fraction enriched in a-linolenic acid-EE (37% purity and 68% yield) and c-linolenic acid-EE (22% purity and 61% yield). A scaled-up process resulted in 840 mg of final product composed of 97% SDA-EE with 71% yield.