In this study, chromatographic purification of de-esterified marigold lutein was evaluated to obtain high purity free lutein. The quantification and identification of fatty acid in the de-esterified marigold lutein, which have not been reported before, were determined. The amount of fatty acid, mostly palmitic acid, was 49.12mg/g oleoresin. The separation of free lutein and fatty acid was then performed. The high purity and recovery of free lutein were observed to be 99.2% and 95.2%, respectively, for the suitable chromatographic separation condition, i.e., step-gradient of hexane-ethyl acetate mixture at 85:15v/v in the first 12min, followed by 70:30v/v.