Christmas Chromatography
Chromatography is a technique that is used to separate and analyze complex mixtures of chemical
compounds, such as plant pigments. You will use this technique to examine the pigments in a green leaf.
Purpose: to determine which pigments are present in poinsettia, spinach, and coleus.
Prediction: using the following table, predict which pigments you expect to find in each of the samples.
Pigment or Pigment Group
Anthocyanins
Chlorophyll a
Chlorophyll b
Carotenoids
Pheophytin
xanthophylls
Phycocyanin
Phycoerthrin
Colour
Purple
Bluish-green
Yellowish-green
Orange
Olive-green
Yellow
Blue
Red
Materials:
 Coleus leaves
 Spinach leaves
 Poinsettia leaves
 Isopropanol (rubbing alcohol)
 Chromatography paper
Poinsettia:
Spinach:
Coleus:
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3 Paper clips
Test tube rack
3 stoppers
3 medium test tubes
Mortar and pestle
Procedure:
Pigment Solution Preparation: repeat the following steps for each sample (3 total)
1. Using a mortar and pestle grind a leaf into small bits.
2. Add approximately 15-20 ml of rubbing alcohol to the mortar and pestle and
allow the mixture to sit while you prepare your chromatography apparatus
setup.
Chromatography Apparatus Setup:
1. Set up the cork stopper and the paper clip as per the example shwon. Be
sure to set up one chromatography apparatus for each leaf sample (3 total)
2. Measure a piece of chromatography paper so that it is long enough to hang
from the paper clip but not so long that it touches the bottom of the test tube.
Cut the paper to a point at one end and mark a straight line 2 cm above the
tip. This is your pigment line.
Chromatography: repeat the following steps for each sample (3 total)
1. Using a micropipette, pipette one drop of your pigment solution just above
the pigment line on your chromatography paper. Place the chromatography
paper into the drying oven for 5 minutes to evaporate the solvent (rubbing alcohol). Complete this
procedure 5 times to ensure you have a heavy pigment stain on the chromatography paper.
2. Hang your chromatography paper from the paper clip on your stopper and insert it into your medium
test tube.
3. Using a marker, mark the height of your pigment line on the side of your test tube.
4. Fill your test tube with solvent (rubbing alcohol) to just below the pigment line you marked on the side.
5. Place your stopper with hanging chromatography paper into the test tube so that the tip of the paper is
submerged in the solvent. ENSURE THE SOLVENT DOES NOT SUBMERGE ABOVE YOUR
PIGMENT LINE!
6. Allow the solvent to move up the chromatography paper until the solvent is approximately 1 cm from
the top. Remove the chromatography paper from your test tube apparatus and place it in the drying
oven for 5 minutes.
7. Once dry, remove the chromatography paper from the drying oven. This is your finished
CHROMATOGRAM.
Analysis:
1. Using the table above identify each of the pigments seen on your chromatogram.
This activity has been modified from “Using Chromatography to Separate Plant Pigments”, Biology 12, McGraw Hill Ryerson, 2011, p. 176