Photosynthesis Lab Report: Pigment Chromatography and Photosynthesis

LAB TITLE: Pigment Chromatography and Photosynthesis / AP LAB 4

        Please Review Before the Lab

        Paper chromatography is a useful technique for separating and identifying pigments and other molecules from cell extracts that contain a complex mixture of molecules. The solvent moves up the paper by capillary action, which occurs as a result of the attraction of solvent molecules to the paper and the attraction of solvent molecules to one another. As the solvent moves up the paper, it carries along any substances dissolved in it. The pigments are carried along at different rates because they are not equally soluble in the solvent and because they are attracted, to different degrees, to the fibers in the paper through the formation of intermolecular bonds, such as hydrogen bonds.

        Beta carotene, the most abundant carotene in plants, is carried along near the solvent front because it is very soluble in the solvent being used and because it forms no hydrogen bonds with cellulose.  Another pigment, xanthophyll, differs from carotene in that it contains oxygen.  Xanthophyll is found further from the solvent from because it is less soluble in the solvent and it has been slowed down by the hydrogen bonds with cellulose.  Chlorophylls contain oxygen and nitrogen and are bound more tightly to the paper then the other pigments.

                Photosynthesis may be studied in a number of ways.  For this experiment, a dye-reduction technique will be used.  The dye-reduction experiment tests the hypothesis that light and chloroplasts are required for the light reactions to occur.  In place of the electron acceptor, NADP, the compound DPIP will be substituted.  When the light strikes the chloroplasts, electrons boosted to high energy levels will reduce DPIP.  It will change from blue to colorless.  We will know how much photosynthesis has occurred by how much color change we see with the DPIP.  Keep in mind that light reactions are just the first part of photosynthesis.  We are assuming that carbon fixation will occur but are not testing that here.

PROTOCOL / SET UP 

Part 1 – Pigment Chromatography

→ Please wear goggles during this procedure as the solvent fumes can irritate the eyes.

1. Obtain a chromatography chamber that has solvent in the bottom. The cylinder
is tightly sealed because this solvent is volatile, and you should be careful to keep the
lid on as much as possible.

2. Cut a piece of chromotography paper that will be long enough to reach the solvent. Cut one end of this paper into a point. Draw a pencil line 1.5 cm above the point.