PAPER CHROMATOGRAHY OF PLANT PIGMENTS

EXP NO
DATE

Aim Perform paper chromatography for separation of various plant pigments.

Theory Components of the mixture with a stronger attraction to the paper (stationary phase)
than to the solvent will move more slowly that the components with a strong attraction to the
solvent (mobile phase). The difference in the rates with which the components travel along the
paper, over time, leads to their separation. Particular mixtures will have chromatographic
patterns that are consistent and reproducible as long as the paper, solvent, and time are constant.
This makes paper chromatography a qualitative method for identifying some of the components
in a mixture.
All living organisms require energy for their metabolic (chemical) processes. The ultimate
source of this energy is the sun. Photosynthetic organisms, including plants, protists (single-
celled 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.

Requirements - What man filter, Ethanol (95 %), acetone, petroleum ether, spinach, glass
chamber, beaker, glass vial, Test Tube.

Procedure -
1. The chloroplast pigment extract pictured at left was obtained by boiling fresh leaves of
spinach in 95% ethanol for several minutes filtering it using gravity filtration.
2. Pigments are then "painted" onto strips of chromatography paper with V-shaped tips using a
small, hollow glass tube or a small paintbrush.
3. 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).
4. The chromatography solvent is used to separate the mixture of pigments painted on the paper.
5. The solvent used was comprised of nine parts petroleum ether and one part acetone.
6. A small amount of this solvent is added to a large test tube and capped with a rubber stopper.
7. The V-shaped 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.
8. The paper is allowed to remain in the solvent until the uppermost pigment band nears the top
of the paper.

Note The chromatography solvent is highly volatile and flammable.

Observation
Four different Bands are seen on the chromatograph paper
1. The Topmost Band is of ___________ pigment.
2. The Second topmost Band is of ___________ pigment.
3. The Third Topmost Band is of ___________ pigment.
4. The last band is of ___________ pigment.


Result The chromatograph paper clearly shows ____ number of pigments, hence it can be
inferred that the pigments of the given sample could be easily separated.