Purpose of Required Practical 7

To separate and identify the different pigments present in leaves using paper or thin-layer chromatography. This helps us understand how plants absorb a wide spectrum of light for photosynthesis using different pigments.

1. Extracting the pigments

• • Grind fresh leaf material (e.g. spinach) with propanone (solvent) using a pestle and mortar.

  • Filter to remove solid fragments, retaining the pigment solution.

2. Preparing the chromatography strip

• • Draw a pencil line near the bottom of the chromatography paper or Thin Layer Chromatography / TLC plate (made of silica gel).

  • Why pencil?
    Pencil is insoluble in the solvent, so it won’t run or mix with the pigments in the leaf.
    If you use ink, it could dissolve and mix with the pigments, distorting the chromatogram.

  • Use a capillary tube to spot a concentrated dot of pigment extract onto the line.

  • Allow it to dry and reapply for stronger results.

3. Running the chromatogram

• • Suspend the strip in a beaker with a small amount of solvent (e.g. propanone or petroleum ether).

  • Ensure the solvent is below the pencil line.

  • Why must the solvent be below the origin line?
    If the solvent is above the pigment spot, it would wash the pigments away into the solvent reservoir instead of pulling them up the paper.
    This step ensures that the pigments travel upwards with the solvent to allow for separation and identification.

  • Cover the beaker to reduce evaporation of the solvent.

  • Allow the solvent to travel up the paper, carrying pigments with it.

Why do pigments separate?

• Each pigment has a different solubility in the solvent. They also have different attractions to the paper or TLC plate (the stationary phase).

• More soluble pigments move faster/further; less soluble ones lag behind.

• This results in distinct pigment bands at different heights.

4. Analysis

• Once the solvent front nears the top, remove the strip and mark the solvent front.

• Why stop it early?
  If the solvent reaches the top, you cannot accurately mark the solvent front, making it impossible to calculate Rf values.
  Stopping it early preserves the solvent front, which is crucial for data analysis.

• Why mark the solvent front immediately?
  The solvent evaporates quickly, so you must mark how far it travelled before it disappears, otherwise your Rf calculations will be inaccurate.

• Allow it to dry and take a photo of your chromatogram.

• Why dry the chromatogram?
  Ensures that measurements are accurate, and that pigment movement has fully stopped.
  Wet pigment may continue to diffuse, blurring bands and reducing resolution.

• Calculate the Rf value for each pigment:

Rf = Distance moved by pigment / Distance moved by solvent front.

You can compare your Rf values to reference values in textbooks or data tables.

Expected Pigments and Their Colours

Why Use Chromatography in Biology?

Chromatography allows:

• Separation of compounds in a mixture.

• Identification based on solubility in the solvent used and interaction with the stationary phase (the chromatography paper or TLC plate).

• Calculation of Rf values to help identify pigments.