CHROMATOGRAPHY OF PHOTOSYNTHETIC PIGMENTS
PURPOSE: To separate various photosynthetic pigments in a leaf using paper chromatography.
MATERIALS:
Spinach leaves
Sand
80% acetone in water
Petroleum ether-acetone (9:1)
10 ml graduated cylinder
Mortar and pestle
Chromatography tube with cap
Chromatography paper
Capillary tube
Scissors
Ruler
Pencil
Tape
PROCEDURE:
1. Place a spinach leaf into a mortar and add 5 ml of acetone and a pinch of sand. Grind up
the leaf with a pestle until the solution is a very dark green colour. This process will
release the plant pigments.
2. Obtain a strip of chromatography paper. Handle the paper by its edges so that oil from
your fingers does not contaminate the paper. Cut the bottom of the chromatography strip
into a point.
3. Make a faint pencil line across the paper approximately 2 cm from the tip of the paper.
Use a capillary tube to apply a dot of plant extract in the middle of the drawn line. Blowdry the dot and repeat the application at least 10 times.
4. Place the square end of the chromatography paper into the cap of the chromatography
tube. The tip of the chromatography paper should be just off the bottom of the flask.
5. Put a few millilitres of petroleum ether-acetone solvent in the chromatography tube and
hang the chromatography paper (pointed end down) into the tube. There should be
enough solution to touch the bottom of the paper but not enough to touch the green dot.
Tape the cap onto the tube.
6. Remove the chromatography paper when the petroleum ether-acetone is within 1 cm
from the top of the chromatography paper and immediately mark the highest level the
solvent reached with a pencil. Let the paper dry and place the solvent and the green
acetone solution in the special waste container.
7. Mark with a pencil, the darkest, most concentrated, part of each of the different coloured
lines produced. Record the colours present and their positions, and calculate the Rf value
of each.
Rf = rate of flow = distance the coloured line moved from the original spot
distance the solvent moved from the original spot
OBSERVATIONS:
Pigment Colour
Distance Moved
Distance Solvent
Moved
Rf Value
QUESTIONS:
1. Why did several different lines form along the paper? Explain in detail.
2. Which pigment was responsible for each line?
3. What does a small Rf value indicate about the characteristics of the moving molecules?
4. Is it possible to have an Rf value greater than 1? Why or why not?
5. If yellow xanthophylls were present in the extract, why did the extract appear green?
6. Would the results be the same if you used a red leaf taken from an autumnal maple tree
instead of spinach leaves? Explain.
7. How could the chromatography of photosynthetic pigments be useful for further studies
on photosynthesis?