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Post-Lab Report

Experiment 5: Column and Paper Chromatography: Separation of Plant Pigments

Danielle Harrisson Tiffany Maurice February 23 2012 March 8 2012

Objective: the purpose of this lab is to compare separation of components of a mixture by two different techniques, and to demonstrate the effect of bromination on plant pigments of tomato juice. Precautions: Work with chemicals in fume hood. Dispose of hydrocarbons in appropriate waste container. Chemicals and Equipment: Melting point capillaries 25-mL buret Glass wool Whatman no. 1 filter papers (10x20cm) Hot plate Tomato Paste Alumina (Aluminium Oxide) Hexane 95% ethanol Toluene Acetone 0.5% B-Carotene in Pet Ether Saturated bromine water

Experimental Procedure: In the first part of this lab a sheet of Whatman no. 1 filter paper was obtained and cut to size. This paper was then marked with five lanes as to plan the spotting of the samples (refer to page 48 of lab manual for paper dimensions). The pigments of tomato paste were extracted in two steps. 10g of tomato paste was weighed into a 50mL beaker. 15mL of 95% ethanol was then added to the paste and stirred vigorously. A small amount of glass wool was placed inside a filter, and the tomato-ethanol mixture was filtered (as to separate the aqueous component from the mixture, leaving only residual paste). The residue and the glass wool were then added to a 50-mL beaker and 10mL of hexane was added. This new mixture was left to stir for two minutes as to extract the pigments. The aqueous component was filtered into a new 50-mL beaker. The aqueous solutions were then put on a hot plate in the fume hood and evaporated to about 1 mL volume. The next part of the experiment involved placing the chromatographic paper on a clean area, as to not contaminate it. The paper was spotted with the various tomato paste extracts and the Bcarotene solution. Once the spots were dry the paper was then curved into a cylinder and stapled. 20 mL of the eluting solvent was placed in a 600-mL beaker. The cylinder was then placed within the beaker, and covered with aluminum foil and left to develop. The second part of this experiment was performed by taking a 25-mL buret with a piece of glass wool near the stopcock (within the buret, in order to not let the stationary phase (alumina) to escape out the bottom when separating the pigments. The column was packed with alumina (20cm) as the stationary phase, and hexane for the mobile phase. The extract was then pipetted carefully into the column and allowed to elute.

Experiment 5 Column and Paper Chromatography: Separation of Plant Pigments Danielle Harrisson Tiffany Maurice REPORT SHEET Paper chromatography Sample B-Carotene: Lane 1 Lane 5 Tomato Extract: Lane 2: a) b) c) d) Lane 3: a) b) c) Distance from origin to solvent front Distance from origin to center Rf Color March 8 2012

d) Lane 4: a) b) c) d) Column Chromatography Number of bands 1 2 3 Describe the colors observed in the tomato juice rainbow experiment, starting from the bottom of the cylinder. 1 2 3 4 5 6 Distance Migrated from top of column Color

Experiment 5 Column and Paper Chromatography: Separation of Plant Pigments Danielle Harrisson Tiffany Maurice POST-LAB QUESTIONS 1. The tomato paste does in fact contain lycopene, as it is attracted to the alumina more strongly than the B-carotene and related carotenes. This has to do with the 13 double bonds, versus the carotenes only having 11 to 12 double bonds. This means that the yellow carotene band moves down the column faster than the orange-red lycopene band. The yellow pigments trail behind the lycopene because they contain a POLAR hydroxyl group (which are strongly attracted to Alumina). 2. March 8 2012

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