Purification of Lycopene by Reverse Phase Chromatography

This work investigates the potential of various hydrophobic matrices for the separation and purification of lycopene from microbial biomass obtained from fermentation using Blakeslea trispora. The lycopene purification method was developed in two steps. In the first step, carotenoids were extracted from the biomass of B. trispora with petroleum ether/acetone (1:1). In the second step, this partially purified carotenoid extract was further purified by reverse phase chromatography technique. Various binding conditions were studied to achieve maximum amount of lycopene bound onto the chromatographic matrices. Column studies on the elution conditions of lycopene using linear and step gradient method were investigated. Purification of lycopene using packed bed column by reverse phase chromatography matrix HP20 was carried out using step gradient of 55% isopropyl alcohol in acetone followed by 65% isopropyl alcohol in acetone. The purity and recovery of lycopene was checked using Knauer high-performance liquid chromatography (HPLC) system. A 89% recovery of lycopene of HPLC purity 95% was achieved with substantial success. Proton magnetic resonance of the purified sample showed close resemblance with the chemical shifts (delta values) of the standard lycopene.