Required Practical 7: Chromatography of Leaf Pigments

Overview of Practical

In this example experiment, I will be comparing the pigments present in 3 different salad leaves: Spinach, Dark Baby Red Leaf and Red Chard.Please be aware that this may not be the practical that you have done/will be doing, but it is an example. In the exam, the chromatography question may have a completely different experiment, but the fundamental principles will be the same.

Equipment

3 salad leaves, 1 of each: Spinach, Dark Baby Red Leaf, Red Chard 3 boiling tubes solvent: 9:1 petroleum ether and propanone bungs with paper clips attached test tube rack marker pen 3 strips of chromatography paper pencil ruler cork borer white tile glass rod small glass measuring cylinder

Risk Assessment

Solvent: extremely flammable and can cause irritation if touched or inhaled wear goggles at all times replace the bung in the tube as soon as possible clean up spills as soon as possible avoid skin contact keep away from flames

Method

Add 3cm^3 of solvent to each of the 3 boiling tubes and put a bung in the top before storing in the rack. Label the tubes 'S' (Spinach), 'D' (Dark Baby Red Leaf) and 'R' Red Chard using the marker pen. Label the strips of paper 'S' (Spinach), 'D' (Dark Baby Red Leaf) and 'R' Red Chard using a pencil. Using a ruler and the pencil, draw a line 2cm from the bottom of each strip. This is the origin line. Cut a disk of Spinach using the borer, avoiding the veins as much as possible. Put the disk on the centre of the origin line on the paper marked 'S'. Place the paper onto the white tile and crush the disk using the glass rod to leave a stain. Attach the paper to the paper clip of the bung of the 'S' tube and replace the bung in that tube. Wait for the solvent to travel up the paper until it almost reaches the top. Remove the paper and immediately draw a pencil line to mark where the solvent reached. Remember to replace the bung in the tube. This paper is your chromatogram. Wait for the paper to dry and then draw a ring in pencil around each coloured spot to be seen. Repeat steps 4-9 for Dark Baby Red Leaf and Red Chard. Calculate the Rf value for each pigment spot on the chromatogram using the formula below. Compare the Rf values with an appropriate reference to estimate and identity of the pigments. Compare results with other groups. Rf value =          distance between spot and origin                               distance between solvent line and origin

Application to Biology

different plants have different types of photosynthetic pigment each type of pigment has a different optimum wavelength where maximum absorption of light energy occurs plants will have adapted to their habitat so that their photosynthetic pigment proportions match the wavelengths of light may also have adapted to contain other non-photosynthetic pigments to protect/help them (see below) Shade-tolerant plants  have chloroplasts adapted to low light conditions but are really sensitive to higher light levels often contain anthocyanins, a purple pigment that is thought to protect the chloroplasts when exposed to higher levels of light.