Describe and use chromatography to separate and identify chloroplast pigments (reference should be made to Rf values in identification of chloroplast pigments)

Sourav PanOctober 31, 2024

Answered

To separate and identify the major chloroplast pigments (Chlorophyll a, Chlorophyll b, Carotene, and Xanthophyll) using paper chromatography, and to utilize Rf (Retention Factor) values for identification.

Materials:

• Chloroplast extract (e.g., from spinach leaves)
• Paper chromatography paper (e.g., Whatman No. 1)
• Solvent mixture (e.g., Petroleum Ether : Acetone : Methanol = 10:4:3)
• Ruler
• Pencil
• UV light source (optional)

Procedure:

1. Prepare the Chloroplast Extract:
   • Grind spinach leaves in a mortar with a small amount of sand and solvent mixture.
   • Filter the mixture to obtain the chloroplast extract.
2. Spot the Extract on the Chromatography Paper:
   • Apply a small drop of the extract to the paper, about 1.5 cm from the bottom edge.
   • Allow the spot to dry completely.
3. Develop the Chromatogram:
   • Place the paper in a chromatography chamber with the solvent mixture, ensuring the spot is above the solvent level.
   • Allow the solvent to ascend the paper ( approx. 30-40 minutes).
4. Observe and Record the Separated Pigments:
   • Under white light:
     • Observe the separated bands and note their colors.
     • Measure the distance each pigment traveled (Distance Traveled by Pigment).
     • Measure the distance from the origin to the solvent front (Total Distance).
   • Under UV light (if available):
     • Observe any fluorescent bands.
5. Calculate Rf Values:
   • Rf Value = (Distance Traveled by Pigment) / (Total Distance)
   • Record the Rf values for each pigment.

Identification of Chloroplast Pigments using Rf Values:

Example Chromatogram and Rf Value Calculation:

• Chromatogram:
  • Band 1 (Blue-green, 6 cm from origin)
  • Band 2 (Yellow-green, 3 cm from origin)
  • Band 3 (Yellow, 8 cm from origin)
  • Solvent front: 10 cm from origin
• Rf Value Calculations:
  • Band 1 (Chlorophyll a): Rf = 6 cm / 10 cm = 0.6
  • Band 2 (Chlorophyll b): Rf = 3 cm / 10 cm = 0.3
  • Band 3 (Carotene): Rf = 8 cm / 10 cm = 0.8

Identification:
Based on the Rf values and colors, the separated pigments are identified as:

1. Band 1: Chlorophyll a (Rf ≈ 0.6, close to the expected range of 0.7-0.9, but may indicate some variation in solvent or pigment interaction)
2. Band 2: Chlorophyll b (Rf ≈ 0.3, slightly lower than the expected range, potentially due to solvent or experimental conditions)
3. Band 3: Carotene (Rf ≈ 0.8, within the expected range of 0.9-1.0)