Separation of Pigments by Paper
Chromatography
All living organisms require energy for their metabolic (chemical) processes. The ultimate
source of this energy is the sun. Photosynthetic organisms, including plants, protists (singlecelled organisms), and blue-green algae (cyanobacteria), convert light energy into the chemical
energy of sugars, which can be used to power metabolism. During photosynthesis, molecules
referred to as pigments (due to the wavelength, thus color, they reflect) are used to capture light
energy. Four primary pigments of green plants can easily be separated and identified using a
technique called paper chromatography. These pigments include two greenish pigments called
chlorophylls and two yellowish pigments called carotenoids. Pigments are separated according to
differences in their relative solubilities.
In order to extract these pigments from the thylakoid membranes of the chloroplasts, the
organelles in which photosynthesis occurs, fresh, ground or torn leaves (preferably spinach) may
be soaked in acetone or concentrated alcohol. The chloroplast pigment extract pictured at left
was obtained by boiling fresh leaves of spinach in 95% ethanol for several minutes and then
filtering using gravity filtration. Pigments are then "painted" onto strips of chromatography paper
with V-shaped tips using a small, hollow glass tube or a small paintbrush. For best results, allow
the line of pigments to dry, then repeat the process until a dark green line of pigments is evident
(about six times is sufficient to achieve a dark pigment line). See photo at left for technique.
Next, chromatography solvent is used to separate the mixture of pigments painted on the paper.
In the experiment pictured at left, the solvent used was comprised of nine parts petroleum ether
and one part acetone. A small amount of this solvent is added to a large test tube and capped with
a rubber stopper. Note that chromatography solvent is highly volatile and flammable. The Vshaped tip of the paper is placed in the chromatography solvent and acts as a wick to draw the
solvent up the paper, separating pigments according to their relative solubility and molecular
weights. The paper is allowed to remain in the solvent until the uppermost pigment band nears
the top of the paper.
This photograph shows the four main pigments separated from green plants using paper
chromatography. The primary pigments in green plants are chlorophylls, represented by
chlorophyll a and b, which appear green. Visible light, or white light, is made up of the colors of
the rainbow. Some of these colors are absorbed ("used") by pigments and others are reflected.
Pigments appear the color of the reflected light, so the chlorophyll pigments do not use the green
portion of the spectrum. The other two pigments are types of carotenoids, which appear yellow,
orange, or brown. The top band of pigments in the separation are carotenoids called carotenes,
most likely beta-carotene, and appear yellowish-orange. The second type of carotenoid separated
in the experiment are xanthophylls, which appear bright yellowish and are most likely lutein. The
"loading line" is the location of the original pigment line painted on the paper.