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Lab 5 Column Chromatography: Isolation of Logotype from Tomato Paste Reading: subtract, pages 79-82, 127-130, 138-139, 141-143, and 235-240 Pre-lab: look up the structure of logotype. Introduction: Logotype is the red pigment in ripe tomatoes and, as an antioxidant, helps to fight certain cancers. In this lab you will isolate logotype from tomato paste. To do this you will first extract carotid pigments from the paste and then use column chromatography to isolate the logotype from the other pigments.
You will then use TTL to evaluate the column chromatography separation. Note: because logotype is light-sensitive, prevent any unnecessary exposure to light. Procedure: Weigh roughly 1. 0 g of tomato paste into a 15 ml centrifuge tube. Add 4 ml of a 50/50 (% volume) mixture of petroleum ether and acetone. Cap the centrifuge tube and shake until the solid becomes fluffy. Open the cap and crush the solid with a spatula. Close the tube and shake again. Repeat this crushing and shaking two more times.
Centrifuge the tube to separate the extract and residue. Remember to balance the centrifuge so that it does not walk (perhaps you and your neighboring lab group an share and balance each other’s tubes) and do not stop the centrifuge with your fingers! Transfer the extract (liquid) to a clean centrifuge tube. In the original centrifuge tube, add a new 4 ml of solvent and repeat the entire extraction procedure. Add the resulting extract to the first extract (in the second centrifuge tube).
Now wash (microscope) the combined extracts with saturated NCAA solution (ml), then with 10% aqueous potassium carbonate (ml), then with saturated NCAA solution (5 ml) again. Dry the organic layer with anhydrous sodium sulfate. Decant the organic layer into a small beaker and concentrate to roughly 0. ml by evaporation in the hood (do not apply heat! ). If the sample goes to dryness, re- dissolve in hexane (0. 2 ml). *Set aside a small amount of your crude extract for a TTL. Pack your chromatography column (a ml pipette). Use 1. 5 to 2. Goff neutral Broomcorn grade I alumina as your adsorbent. Gather your organic solution, 10 ml of hexane (the first element), 10 ml of 10:90 (% volume) acetone:hexane (the second element), a small Erlenmeyer flask,(for collecting the logotype fraction), and a beaker before you start running your column. Place the beaker under the column. Add the iris element, hexane, to the column until the liquid wets all of the alumina. Then add the logotype extract via Pasteur pipette to the top of the column (use a little of the hexane to rinse the extract vial and add this to the column as well.
As soon as the extract enters the alumina layer, fill the column almost all the way with hexane. Add hexane as necessary to keep the solvent level in your column relatively constant. When the first yellow band starts to drain out of the column, add your second element (10:90, % volume, acetone:hexane) to the top of the column and keep the element level constant as before. When the logotype layer (orange-red) begins to leave the column, collect the orange-red layer into the Erlenmeyer flask.
When the band is almost completely off the column, remove the sample vial and replace it with the waste beaker you used earlier. Take a TTL of your crude and purified products. Use 10:90 acetone:hexane as the developing solution. Results: Review the TTL lab to see what should be reported. Discussion: Use your TTL results to comment on the separation efficiency of the column chromatography separation. Waste Disposal: Solvents from this lab go into the non-halogenated waste container.