AP Photosynthesis Lab http://www.phschool.com/science/biology_place/labbench/lab4/intro.html

Plant Pigment Chromatography Measuring the Rate of Photosynthesis

Paper chromatography :technique used to separate a mixture into its component molecules. The molecules migrate, or move up the paper, at different rates because of differences in solubility, molecular mass, and hydrogen bonding with the paper. EX – felt tip marker dot

ANALYSIS If you did a number of chromatographic separations, each for a different length of time, the pigments would migrate a different distance on each run. However, the migration of each pigment relative to the migration of the solvent would not change. This migration of pigment relative to migration of solvent is expressed as a constant, Rf (Reference front). It can be calculated by using the formula:

PHOTOSYNTHESIS In the light reactions of photosynthesis, light energy excites electrons in plant pigments such as chlorophyll, and boosts them to a higher energy level. These high-energy electrons reduce compounds (electron acceptors) in the thylakoid membrane, and the energy is eventually captured in the chemical bonds of NADPH and ATP.

In this activity you will measure the rate of electron excitation when light hits chlorophyll. You will use DPIP, a blue compound, as an electron acceptor. The thylakoid membranes of the chloroplasts are mechanically disrupted so that the natural electron transport chain no longer functions normally. When light strikes the chloroplasts, the DPIP is reduced by the excited electrons from chlorophyll, and it changes from its original blue color to colorless as it accepts the electrons. You will use a spectrophotometer to measure the color change, which gives an indication of the rate of the light reactions of photosynthesis under various conditions.

Spectrophotometer Instrument that can be adjusted to illuminate a sample with a specific wavelength of light. The spectrophotometer then measures the amount of light energy that is absorbed or transmitted by the sample. In this laboratory, as DPIP goes from blue to colorless, the amount of light of wavelength 605 nm transmitted through the sample will increase. Though you can note this color change visually, the spectrophotometer quantifies the change.

If DPIP is in an oxidized state, it will be blue, and the percentage of light transmitted will be low. If, on the other hand, chlorophyll's electrons have been excited and move on to reduce the DPIP, the sample will become progressively paler, allowing more light energy to pass through the sample. We can measure this change over time until the sample has been completely reduced, is almost colorless, and the percentage of transmittance is very high. For this experiment, one tube (the blank) will contain all the solutions used in the reaction except the DPIP. Since the blank contains chloroplasts, it will be green; you will use this tube to calibrate the machine. The other tubes will be experimental, and will contain either boiled or unboiled chloroplasts.