Laboratory Experiment 4: Plant Pigments and Photosynthesis for AP Biology
This experiment draws on material from photosynthesis. The general objective here is to use chromatography to separate plant pigments and to measure the rate of photosynthesis in chloroplasts.
For a quick review, refer to the following concepts:
- The Players in Photosynthesis for AP Biology
- The Reactions of Photosynthesis for AP Biology
- Types of Photosynthesis for AP Biology
Part 1: Separation by Chromatography
Before diving into this experiment, let's briefly review how paper chromatography works in relation to this lab. An extract from the leaves is dabbed onto a piece of paper, which is hung in such a way that its bottom tip is touching the chromatography solvent. As time passes, this solvent, by way of capillary action, runs up the paper, and like a public bus, carries any dissolved substances along for the ride. The rate with which a pigment migrates up the paper depends on two variables: how well it dissolves into the chromatography solvent and how well it can form bonds with the cellulose in the paper. Faster pigments dissolve more and bond less to the paper. Slower pigments dissolve less and bond more to the paper.
Of the plant pigments studied in this lab, the order of migration rate is as follows:
Beta carotene > xanthophyll > chlorophyll a > chlorophyll b
The last concept to take from part 1 of this experiment is Rf —a number that relates the relative rate at which one molecule migrates compared to the solvent of a paper chromatography. The faster a substance migrates, the larger its Rf will be. Beta carotene has the largest Rf of the four pigments listed above. Rf values change depending on the solvent used for the chromatography because different substances have different solubilities in different solvents.
Part 2: Photosynthetic Rate
The second portion of this experiment examines photosynthetic rates and tests the theory that photosynthesis requires light and chloroplasts to occur. If you are feeling shaky about your grasp of photosynthesis, it might be wise to review Chapter 8 before looking at the rest of this experiment.
The three products of the light reactions of photosynthesis are ATP, NADPH, and oxygen. NADPH is formed by the reduction of NADP+. In this experiment, the NADP+ is replaced by a compound, DPIP (2, 6-dichloroindopheno), which changes from a beautiful blue color to colorless when reduced. Thus, when the light reactions have occurred, one can tell by the color of the solution. A machine called a spectrophotometer is used to determine how much light can pass through the sample. This is useful because it will help us know exactly how much of the DPIP has changed from blue to colorless—and thus determine how much photosynthesis has occurred.
The experiment plays out as follows. Each student is given two beakers—one with boiled chloroplasts, the other with unboiled chloroplasts. An initial reading is taken on the spectrophotometer to determine how much light passes through the unboiled chloroplasts before any photosynthesis occurs. Since the experiment tests, whether or not photosynthesis, can occur in the absence of light, the first sample tested measures how much photosynthesis occurs as time passes while the sample is kept in the dark. The second sample tested measures how much photosynthesis occurs if light is permitted to strike the solution. A third sample tests the effects of boiling chloroplasts on the rate of photosynthesis.
Results from Part 2: (Photosynthetic Rates)
Much to the delight of the believers of the theory that photosynthesis requires light and chloroplasts to occur, this experiment proves just that. When the sample is left in the dark, the DPIP is not reduced. The electrons just cannot become excited in the absence of light. When the sample containing boiled chloroplasts is used, the heat denatures the organelle, stripping it of its photosynthetic capacity.
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