Separation of Lycopene and B-Carotene Via Column Chromatography

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Chapter # 9 Column Chromatography Purpose The purpose of this lab is to become familiar with the theory and process of column chromatography as it is used to separate two substances, in this case, Lycopene and β-Carotene. This is done by first extracting lycopene from tomato paste and β-Carotene from carrot puree. First, the each paste is treated with acetone to remove as much water as possible. Then, the extraction is performed with three 5-mL portions of dichloromethane. Each organic extract is then dried over anhydrous calcium chloride pellets and evaporated to dryness. The extract is then wet with a minimal amount of dichloromethane and a sample of each is obtained and mixed together with 200 mg of alumina and again evaporated to dryness. This mixture is then placed in the prepared chromatography column and eluted with hexanes until the yellow β-carotene band is collected. The solvent is then switched to a 90/10 mix of hexanes and acetone to speed up the elution of the more polar lycopene band, which is also collected in its own flask. Each of these samples is evaporated to dryness and rehydrated with a minimal amount of dichloromethane. Finally, a TLC plate is run comparing the crude lycopene and β-carotene samples with the purified lycopene and β-carotene samples. Theory The theory of column chromatography is much the same as other types of chromatography, such as running TLC plates. Additionally, column chromatography can be used to separate compounds of interest, where TLC plates cannot due to their microscale nature. In column chromatography, the stationary phase is alumina or silica powder layered in a thin, upright column. The mobile phase uses gravity to run through the column rather than capillary action as seen in TCL. Other than these differences, the compounds of interest are separated using the same principles as TLC, with the molecular

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