Paper Chromatography: Separation of Plant Pigments
PAPER CHROMATOGRAPHY
Chromatography is probably the most useful method of separating organic compounds for
identification. There are many different types of chromatography, but most work on the concept of
absorbance. The two important components of chromatography are the absorbent and the eluent.
A good absorbent is usually a solid material that will attract and absorb the materials to be
separated. Paper is often used as the absorbent. The eluent is the solvent which carries the
materials to be separated through the absorbent.
Chromatography works on the concept that the compounds to be separated are slightly
soluble in the eluent and will spend some of the time in the eluent (or solvent) and some of the time
on the absorbent. When the components of a mixture have varying solubility in the eluent, they can
then be separated from one another. The polarity of the molecules to be separated and the polarity
of the eluent are very important. Changing the polarity of the eluent will only slightly change the
solubility of the molecules but will greatly change the degree to which they are held by the
absorbent. This affinity for the eluent versus the absorbent is what separates the molecules.
In paper chromatography the absorbent is the paper itself, while the eluent can be any
number of solvents. The polarity of the eluent is very important in paper chromatography since a
small change in polarity can dramatically increase or decrease the solubility of some organic
molecules. Many times, a mixture of a non-polar solvent and a polar solvent is used to achieve an
optimum polarity. When placed in a chromatography chamber, the eluent moves up the strip of
paper, being drawn by capillary action. The organic molecules, which were “spotted” onto the
paper chromatography strip, separate as they are carried with the eluent up the strip at different
rates. Those molecules that have a polarity closest to the polarity of the eluent will be the most
soluble and will move up the strip the fastest.
The choice of the eluent or solvent is the most difficult task. Choosing the right polarity is
critical because this determines the level of separation that will be achieved. Common solvents
used in chromatography, in order of increasing polarity, are: petroleum ether or hexanes,
cyclohexane, toluene, chloroform, ethyl ether, acetone, ethanol, methanol, and water. Sometimes
mixtures of solvents are used to achieve the desired degree of polarity. A general rule of thumb is:
if the substances to be separated are polar, the developing solvent should be slightly less polar.
Likewise, non-polar substances would require slightly polar solvents.
PLANT PIGMENTS
The major pigments of photosynthetic organisms are the chlorophylls. Chlorophylls are responsible
for the green coloring of most plants, as these pigments absorb light strongly in the red and blueviolet regions of the visible spectrum and transmit or reflect most light in the green region. There
are two types of chlorophyll found in higher plants, chlorophyll a and chlorophyll b. Besides
chlorophylls, plants also contain other pigments used to collect light energy. Sometimes known as
accessory pigments, these molecules include carotenes, xanthophylls, and phycobilins. Normally,
the abundant chlorophylls mask the colors of these other relatively scarce pigments. However, in
autumn, as chlorophylls begin to break down and lose their color, it is these accessory pigments
(which are still active) that give autumn leaves their brilliant red, yellow, and orange colors.
Paper Chromatography – Separating Plant Pigments
PURPOSE:
To separate the various pigments in spinach leaves
MATERIALS:
Chromatography solvent, chromatography paper, spinach leaves, 250-mL Erlenmeyer flask,
Parafilm, wooden splint, stapler
SAFETY: Wear goggles. Work in a well-ventilated area.
PROCEDURE:
1. Place torn up leaves, a little quartz sand, and 10 mL of acetone into a mortar.
2. Vigorously grind the mixture for 5 minutes.
3. Cover the bottom of a 250-mL Erlenmeyer flask with chromatography solvent . Cover with
Parafilm.
4. Prepare the chromatography paper strip as shown.
5. Use a capillary tube to transfer pigment solution to the paper.
6. Remove the Parafilm and slowly lower the chromatography strip into the Erlenmeyer flask.
The green line containing the plant pigments should remain above the solvent.
7. Carefully replace the Parafilm to cover the mouth of the flask.
8. When the solvent front is within 0.5-1.0 cm of the top of the chromatography strip, remove
the strip from the flask.
9. Allow the strip to dry. Mark the location of each of the separated bands on the strip with the
appropriate color. (The brightness of the colors of the chromatogram will fade with time.)
Paper Chromatography – Separating Plant Pigments
ANALYSIS AND QUESTIONS:
1. Draw a representation of the chromatography strip including the pigment colors and
locations as well as the starting line and final solvent front location.
Identify the different pigments according to the following information.
Chlorophyll a – blue-green
Chlorophyll b – olive-green
Violaxanthin – yellow
Lutein – gray-yellow; hard to see
Carotenes – yellow-orange
3. Which band is the most prominent band and what pigment does it represent?
4. Which pigment has the greatest affinity for the eluent?
5. Why does a plant have different pigments?
Paper Chromatography – Separating Plant Pigments