In this experiment the carotenoid bixin is isolated from annatto ("Bixa orellana") seeds using column chromatography. The experiment has several key advantages over previous pigment separation experiments. First, unlike other experiments significant quantities of the carotenoid (typically 20 to 25 mg) can be isolated from small quantities of plant material. Second, in this experiment students learn how to choose the appropriate solvent for a chromatographic separation by analyzing several potential solvents using thin-layer chromatography. After isolation, the purified bixin is analyzed by UV-vis and IR spectroscopy. (Contains 2 figures and 1 note.)