Chromatography with leaf colours

Use chromatography to separate out leaf colours - the yellows, greens and reds. Find out that yellow is always in the leaf, but usually hidden by the green (until the fall).
Science content
Biology: Features, Adaptations of Living Things (K, 1, 3, 7)
Chemistry: Physical Changes, Solutions, Mixtures and Separating (2, 4, 5, 6)
Earth/Space: Weather, Seasons, Climate Change (K, 1, 4, 7)
  • white coffee filter papers
  • scissors
  • spinach leaf, or other green leaves that are easily smashed. try maple leaves of various colours
  • tape (clear)
  • coin
  • tall tube/jar and rack
  • toothpick
  • rubbing alcohol (95% ethanol; 70% isopropyl alchohol works but is much slower)

Please note that in a class of students it is likely that one of them is at least partially colourblind (1 in 12 males are colourblind). As this is an activity distinguishing colours, these students will not be able to tell some colours apart and perceive some colours differently, although the activity will be no less interesting for them. The common red/green colour blindness means reds and greens (or colours containing reds and greens such as browns) look similar. More information at and

One of the fall leaf colours is in leaves all year but is hidden until the fall. Do an experiment to find out which fall colour is hidden in green leaves.

Cut a strip of filter paper about 1cm wide and about as tall as the tube (I make strips 12cm long for my 50ml "Falcon tube" pictured).
Cut a strip of spinach leaf, 1-2mm wide, and tape across the filter paper strip near one end (about 2cm in).
Use a coin to roll over the spinach leaf to transfer the colour from the leaf to the filter paper. (Leave the tape on when you are done, as it might rip the paper if you try and tear it off.)
Fold/roll the end of the filter paper over/around the toothpick, and hang it in the empty tube. Adjust the height so that the bottom of the filter paper hangs about 1cm above the bottom of the tube, and the green pigment is about 3cm from the bottom of the tube. Remove the filter paper and toothpick again.
Add rubbing alcohol to the tube to a height of about 2cm, then hang the filter paper back in the tube, making sure that the green pigment is not submerged, but the end of the filter paper strip is submerged. If it looks like the colour will dip into the liquid, fold/roll the filter paper a little higher. Also make sure that the filter paper is not touching the sides of the tube.

Allow the chromatogram to run. Almost immediately the colours will start to move up the filter paper, but the further up it moves, the more dramatic the separation of the colours will be. I suggest a minimum running time of 10 minutes; 20 minutes or more is ideal, as in the photo.
Note that in a class of students the results will be very variable, depending on how tidy their chromatogram assembly is; however almost all students should see some yellow separating from the green. (Using 70% isopropyl alcohol, the colours move more slowly and are not separated as well with 95% ethanol, so in this case a minimum would be 30 minutes. I have not tried 99% isopropyl alcohol but would guess that it is more like the 95% ethanol.)
To see the colours more clearly while the chromatogram dries, lay on a white paper towel or other white paper.

The leaf pigment molecules are soluble in alcohol and are carried along by it, but they also get caught up on the surface of the filter paper, before moving along again. Each molecule interacts with the filter paper and the alcohol a little differently (as the molecules have different shapes and charges), so they move at slightly different rates up the filter paper. The molecules become separated from each other more and more clearly as the chromatography proceeds.

In spinach leaves, a bright yellow colour separates out from the green.
The yellow is a pigment molecule (called xanthophyll) which moves ahead of the green chlorophyll molecules.
If you try other leaf colours, you will see their colours separate out too. Green leaves only have green and yellow pigment in them. Red leaves may also have yellow pigment in them. Yellow leaves only have yellow pigment in them. Therefore yellow pigment can be masked by the green or red pigments, so is only visible when neither are present.

Relate to fall leaf colours:
The yellow pigment molecules are always in leaves, and help with photosynthesis, but their colour is usually masked by the green chlorophyll. In the fall, the green chlorophyll molecules break down as the leaves die in preparation for winter. As the green molecules disappear, the yellow molecules become visible.
Red and brown pigments are not in leaves before the fall, but made as leaves break apart sugar molecules in preparation for winter. The red molecules appear around the same time that the green molecules disappear.

Attached documents

New York Hall of Science and JEMZ used the original technique (in the booklet attached). This new technique takes longer but is way better for seeing the yellow.

Idea for additional activity while waiting for leaf colours to separate: use food colouring how green covers up yellow but not red (mix green and yellow, green and red, yellow and red).

Grades taught
Gr K
Gr 1
Gr 2
Gr 3
Gr 4
Gr 5
Gr 6
Gr 7