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Experimental procedure (pigments)

Start by gathering the materials required for the experiment. Record your findings in a table.

  1. Grind the flowers or leaves with a pestle and mortar then transfer the paste into a test tube.

  2. 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.

  3. Use a pencil to mark a line 3 cm from the end of the Whatman filter paper. This is the reference line.

  4. 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.

  5. 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.

  6. Keep the jar as still as possible until the mobile phase has gone up two thirds the length of the paper.

  7. 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.

  8. Measure the distance traveled by the solvent front and the center of the spots with respect to the reference line.

  9. Determine the number of pigments that are present in the extract of the leaves and the flowers.

  10. Calculate the Rf value of the different spots. Use the equation found here.

Step A. Draw a reference line 3 cm from the bottom with a pencil. Put a label under the reference line for samples a and b. Step B. Place the paper carefully into a jar of solvent. The paper is attached to a hook that is part of the lid. The reference line is above the solvent level. Step C. The solvent is running up the filter paper. The spots are visible. Step D. The chromatogram. There are 3 spots for sample a and 2 spots for sample b.

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

Referred from:

Article
How to note observations (pigments)