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PSU CHEMISTRY 36H - Extraction and Thin Layer Chromatography

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Extraction and Thin-Layer Chromatography of Chlorophyll Aand B from SpinachAdapted by R. McLaughlin and K. Masters from Griffin, G. William; Quach, Hao T.; Steeper, Robert L. J.Chem. Ed. 2004, 81, 385-387; revised 12/6/04IntroductionExperiments that focus on extracting pigments from plants have long beenpopular in student chemical laboratory courses. These types of experiments are useful forteaching students about the techniques involved in thin-layer chromatography (TLC)because they provide a number of colorful pigments that students can easily observe andstudy. In this experiment, we will learn TLC techniques by analyzing pigments extractedfrom spinach.Prelaboratory Exercise1. What is the purpose of the MgSO4 in the ground spinach mixture?2. Why should you not mark TLC plates with ink?3. Answer PreLab questions in chapter 6 of the Lab Guide.CautionsThis experiment does not involve any chemicals that are significantly hazardous.However, remember that substances used in the laboratory are never to be eaten. Be sureto properly handle and dispose of all solvents and reaction mixtures.ProcedureThin-Layer Chromatography (TLC) Solvent PreparationPrepare a solution of 6 mL petroleum ether, 1.6 mL cyclohexane, 1 mL ethylacetate, 1 mL acetone, and 0.4 mL methanol. Place just enough of this solution in a TLCjar to cover the bottom of the jar. Tightly cap the jar while it is not in use. Obtain twoTLC plates, and in pencil, mark a straight line across the plate 1 cm from the edge.Sample PreparationWeigh out 0.5 g of fresh spinach. Combine this with 0.5 g of anhydrousmagnesium sulfate and 1.0 g of sand. Using a mortar and pestle, grind the mixture until itbecomes a fine, light green powder. Transfer this solid into a test tube and add 2 mL ofacetone. Stir the solution using a stir bar for 2 minutes. Allow the mixture to sit for 10minutes. The solid should settle to the bottom, leaving a green liquid layer on top.Transfer this green layer to another test tube using a pipet. Seal the test tube containingthe spinach extract with parafilm when you are not using it.TLC of the ExtractSpot the extract on the line you marked on one of the TLC plates using a capillarytube. For this particular experiment, you will spot across the entire pencil line youmarked on the TLC plate. This will make the various components of the extract easier tosee on the plate. Place the plate in the TLC jar with the pencil line right above thesolution. Cap the jar and monitor the movement of the solution up theplate. When the solvent front reaches about 1 cm from the top of theplate, remove it from the jar. Mark a line for the solvent front before itevaporates. The components of the spinach extract are visible withouta UV light; however, they decompose quickly. To slow thedecomposition, immediately place a piece of tape over the entireTLC, and tape it in your laboratory notebook. Mark the lines you seebefore they are no longer visible.Demetalation of the Spinach ExtractRemove 100 µL of the spinach extract using a manual pipettor with a disposablepipet tip, and place it in another test tube. Weigh out 100 mg of Dowex 50WX8 H+ resin,and place that in the same test tube. Allow the mixture to stand for three minutes.TLC of the Demetalated ExtractUsing a capillary tube, spot the liquid mixture on a TLC plate. Becareful not to get any of the Dowex resin on the TLC plate. Repeat theprocedure you used for the TLC of the spinach extract. Again, thecomponents decompose quickly so be sure to tape the TLC plate in yournotebook immediately and mark the visible lines.Clean-upThe ground spinach solid in the first test tube can be left to dry and then thrownaway in the trash. The acetone extract solution can be diluted with water and poureddown the drain. The Dowex resin solution can also be dried and then thrown away in thetrash.AnalysisThe TLC plate of the spinach extract should reveal four pigment lines. From thebottom of the plate up, there should be a yellow line for xanthophyll, a green line forchlorophyll b, a brighter green line for chlorophyll a, and a yellow line for β-carotene.You can obtain Rf values for each of your pigment lines using the equation in Figure 1.The literature reports the following Rf values for each component: Rf = 0.16 forxanthophyll, Rf = 0.32 for chlorophyll b, Rf = 0.44 for chlorophyll a, and Rf = 0.95 for β-carotene.The TLC plate of the demetalated extract should reveal five pigment lines. Thelines for xanthophyll, chlorophyll b, and β-carotene should remain. In addition, a darkgreen line for pheophytin b should appear after the chlorophyll b, and an even darkergreen line for pheophytin a should appear after that. According to the literature, the Rf =0.49 for pheophytin b; the Rf = 0.60 for pheophytin a.Final ReportDiscuss what happened when you demetalated chlorophyll a.1. Several solvent systems were tried before the five solvent system used in thisreport. Below are the TLC plates for those systems as well as the one used inthis experiment. Why was the five solvent system we used chosen? Why wasthe largest component ofthe five solvent systempetroleum ether ratherthan cyclohexane? Whatwould happen if you usedFigure 1 Equation for determining Rf valuesRf = Distance between the starting point and pigment line Distance between the starting point and solvent fronta solvent system that consisted mostly of cyclohexane instead?2. Rank the components of the spinach extract from most to least polar.3. Rank the components of the demetalated extract from most to least polar.4. Answer PostLab question #3 in Lab

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