Experimental procedure (pigments)
Start by gathering the materials required for the experiment. Record your findings in a table.
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Grind the flowers or leaves with a pestle and mortar then transfer the paste into a test tube.
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To the crushed material, add methanol or acetone. Cork the test tube and shake well. Filter the solution, and transfer the filtered solution into another test tube.
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Use a pencil to mark a line 3 cm from the end of the Whatman filter paper. This is the reference line.
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Using a finely drawn capillary, put one spot for the extract of leaves, and one spot for the extract of flowers, on the reference line with a gap in between the spots. Label these spots under the reference line and make a note of the labels in your lab book.
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Hang the filter paper in a jar containing 20 mL of one of the following solvent systems: a mixture with 19 mL of petroleum ether and 1 mL of chloroform (ratio 19:1), or a mixture with 18 mL of petroleum ether and 2 mL of acetone (ratio 9:2). The paper should hang so the solvent does not touch the reference line.
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Keep the jar as still as possible until the mobile phase has gone up two thirds the length of the paper.
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Remove the filter paper from the jar. Use a pencil to mark the solvent front and outline the spots. Allow the filter paper to dry.
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Measure the distance traveled by the solvent front and the center of the spots with respect to the reference line.
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Determine the number of pigments that are present in the extract of the leaves and the flowers.
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Calculate the Rf value of the different spots. Use the equation found here.
Figure 1 - Separation of pigments experiment. (A) The marked piece of filter paper, (B) The filter paper dipped in solvent, (C) Solvent moving up the paper, (D) The chromatogram.
References:
- NCERT Chromatography handbook