An investigation into the separation of
photosynthetic pigments by means of thin layer
chromatography
Extracting the leaf pigments
Once you start, work quickly. Pigments can be broken down during the extraction
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Take a small handful of leaves from your selected plant and cut off the stem
Place your leaves in a pestle and mortar along with a few drops of propanone (acetone)
Grind the leaves into a paste
Using a muslin cloth and funnel, filter the liquid into a small beaker
Separation of the leaf pigments
1. Cut a strip of TLC paper that fits in the beaker you are using (you want to get the paper
as big as you can but can still fit inside a beaker with the lid on)
2. Using a pencil, mark a faint line across the bottom of the TLC plate, this line should be
about 1cm from the bottom
3. Take a drawn out capillary tube and dip the pointed end into the green solution, this
should draw up the liquid
4. Press the capillary tube on your pencil line and wait a few seconds for the dot to dry
5. Once the dot is relatively dry apply another dot, keep repeating this step until you have
dark green spot
*it is important you wait for the dot to dry as you only want a small circle*
6. Hold your TLC plate up to the beaker and note where your line would be on the beaker
7. Move the beaker into the fume cupboard
8. Carefully pour solvent into the beaker ensuring you don’t go over the pencil line
9. Place your TLC plate into the beaker and cover using a glass sheet
10. Watch the solvent move up the strip causing the leaf pigments to separate.
*Do not move the beaker once the solvent is running*
11. When the solvent reaches the top of the plate (should be about 1.5cm from the top)
remove the TLC strip and immediately draw a line to show the solvent front
This is what your TLC plate should look like:
12. Calculate the Rf value for each pigment using the following formula:
Rf = distance run by pigment (distance from pencil line to top of pigment)
distance run by solvent (distance between the two pencil lines)
13. Identify the principal pigments using the table of Rf values below.
Pigment colour
Pigment
Rf value
orange yellow
carotene
0.96
grey
a breakdown product 0.70
blue green
chlorophyll a
0.58
green
chlorophyll b
0.48
deep yellow
xanthophyll
0.44