How can photosynthetic pigments be separated by chromatography?

Sourav PanNovember 8, 2024

Answered

To separate photosynthetic pigments using chromatography, a systematic approach can be employed. Here’s a detailed experimental procedure, along with the principles behind the technique:

Overview of Chromatography

Chromatography is a method used to separate mixtures based on the differential affinities of components for a stationary phase and a mobile phase. In the context of photosynthetic pigments, this technique allows for the identification and quantification of various pigments such as chlorophyll a, chlorophyll b, carotenoids, and xanthophylls.

Materials Needed

• Plant Material: Fresh leaves (e.g., spinach, kale).
• Solvent: A mixture of solvents, commonly a 3:1:1 ratio of petroleum ether, acetone, and ethanol or hexane.
• Chromatography Paper: Filter paper or specialized chromatography paper.
• Mortar and Pestle: For grinding the plant material.
• Capillary Tubes: For spotting the pigment extract on the chromatography paper.
• Ruler: To measure distances traveled by pigments.
• Chromatography Chamber: A container to hold the solvent and paper during development.

Experimental Procedure

Step 1: Prepare Pigment Extract

1. Grinding: Use a mortar and pestle to grind a small amount of fresh leaves with a few milliliters of solvent (e.g., acetone or ethanol) to extract the chlorophyll and other pigments.
2. Filtration: Filter the mixture through cheesecloth or filter paper to obtain a clear pigment solution.

Step 2: Setting Up Chromatography

1. Prepare Chromatography Paper:
   • Cut a strip of chromatography paper about 10 cm long and mark a horizontal line approximately 2 cm from one end using a pencil (this is where you will spot the pigment).
2. Spotting the Pigment:
   • Using a capillary tube, spot a small amount of the pigment extract onto the pencil line. Allow it to dry before proceeding.

Step 3: Developing the Chromatogram

1. Prepare Solvent:
   • Pour the chosen solvent into the chromatography chamber so that it covers the bottom by about 1 cm.
2. Run the Chromatography:
   • Suspend the chromatography paper in the chamber so that the bottom edge is immersed in the solvent but does not touch the pigment spot.
   • Cover the chamber to minimize evaporation and allow the solvent to rise up the paper by capillary action until it reaches about 1 cm from the top.

Step 4: Analyzing Results

1. Remove and Dry:
   • Once the solvent front has reached its desired height, remove the paper from the chamber and mark the solvent front with a pencil immediately.
2. Identify Pigments:
   • Measure and record how far each pigment spot has traveled from the original pencil line (the starting point).
3. Calculate Rf Values:
   • Calculate the retention factor (Rf) for each pigment using the formula:
     Rf=Distance traveled by pigment/Distance traveled by solvent front​
   • Each pigment will have a unique Rf value that can be compared to known standards for identification.