📄

Understanding Paper Chromatography Techniques

Oct 7, 2024

Paper Chromatography Lecture Notes

Overview of Chromatography

  • Chromatography is a technique used to separate molecules based on:
    • Size
    • Shape
    • Mass
    • Charge
    • Solubility
    • Absorption properties

Paper Chromatography Process

  • Procedure involves:
    • Spotting a mixture on paper
    • Drying the spot
    • Allowing solvent to flow through the paper by capillary attraction
  • As the solvent moves, different compounds separate into colored spots.
  • Final step involves drying the paper and visualizing the separated compounds.

Materials Required

  1. Fresh spinach leaves
  2. Filter paper strips
  3. Chromatography chamber
  4. Mortar and pestle
  5. Ether acetone solvent
  6. Measuring cylinder (5 ml of acetone)
  7. Capillary tube
  8. Thread
  9. Stapler
  10. Scissors
  11. Spatula
  12. Watch glass
  13. Scale
  14. Pencil

Steps in the Procedure

  1. Cut fresh spinach leaves into small pieces.
  2. Grind spinach in a mortar with 5 ml of acetone.
  3. Transfer extract to a watch glass using a spatula.
  4. Prepare filter paper:
    • Draw a horizontal line 2-3 cm from the notch tip.
    • Use capillary tube to place drops of pigment extract on the line (4-5 drops).
  5. Prepare chromatographic chamber:
    • Pour ether acetone solvent into the chamber.
    • Staple one end of the filter paper and hang it in the chamber (loading spot 1 cm above solvent).
  6. Allow the solvent to rise through the paper.
  7. Remove the strip when the solvent has traveled about 3/4 of the way up.
  8. Let the paper dry.

Observations

  • The dried chromatographic paper shows four distinct pigment bands.

Calculations

  • Calculate RF value for each pigment:
    • RF = (Distance traveled by compound) / (Distance traveled by solvent)
  • Measure distances and record in a table.

Results

  • Identified pigments from top to bottom:
    1. Carotene - Orange yellow band (topmost)
    2. Xanthophylls - Yellowish band
    3. Chlorophyll a - Dark green band
    4. Chlorophyll b - Yellowish green band (bottom)

Precautions

  • Use fresh and green spinach leaves.
  • Ensure loading spot is 2-3 cm from the notch and maintain a height of 1 cm above solvent level in the chromatography chamber.

Full transcript