Plant Traveling Lab. TTU/HHMI at CISER. 2010
Plant Pigment Chromatography
Students will isolate and identify photosynthetic pigments in spinach leaves.
Students will calculate Rf values of photosynthetic pigments and graph the
absorption spectrum for each pigment.
Introduction
As primary producers in the food chain with some bacteria and algae, plants
produce their own food by using the sun’s energy to transform carbon dioxide and
water into glucose. In this process of photosynthesis, plants convert the sun’s
energy into chemical energy that is stored in the bonds of the glucose molecule.
This energy fuels the metabolic processes of cells and is essential for life on earth.
Glucose is a simple carbohydrate that provides immediate fuel to cells but it is also
a building block for more complex carbohydrates stored by living organisms for
future use.
For photosynthesis to transform light energy from the sun into chemical energy
(bond energy) in plants, the pigment molecules absorb light to power the chemical
reactions. Plant pigments are macromolecules produced by the plant, and these
pigments absorb specified wavelengths of visible light to provide the energy
required for photosynthesis. (Appendix A) Chlorophyll is necessary for
photosynthesis, but accessory pigments collect and transfer energy to chlorophyll.
Although pigments absorb light, the wavelengths of light that are not absorbed by
the plant pigments are reflected back to the eye. The reflected wavelengths are
the colors we see in observing the plant. (Example: green pigments reflect green
light) Plants contain different pigments, and some of the pigments observed
include:
chlorophylls (greens)
carotenoids (yellow, orange red)
anthocyanins (red to blue, depending on pH)
betalains (red or yellow)
The process of chromatography separates molecules because of the different
solubilities of the molecules in a selected solvent. In paper chromatography,
paper marked with an unknown, such as plant extract, is placed in a developing
chamber with a specified solvent. The solvent carries the dissolved pigments as it
moves up the paper. The pigments are carried at different rates because they are
not equally soluble. A pigment that is the most soluble will travel the greatest
distance and a pigment that is less soluble will move a shorter distance.
The distance the pigment travels is unique for that pigment in set conditions and is
used to identify the pigment. The ratio is the Rf (retention factor) value. Standards
are available for comparison. (Appendix B)
distance pigment travels (cm)
Rf = distance solvent travels (cm)