Abstract A preparative high-performance liquid chromatographic method, based on recycle chromatography, to separate vitamin D 2 (ergocalciferol) from previtamin D is described. The method provides efficient separation by means of a mixture of methanol, acetonitrile and hexane as eluent on a reversed-phase C 18 column. Scale-up to a 2-in. diameter column resulted in the collection of 100% pure fractions based on UV detection at 265 nm. The total throughput and the economics of the purification were also optimized.