13.1: Instructor Guidelines

Purpose    

Students are introduced to photosynthesis during this lab. Students will extract plant pigments and separate these pigments using paper chromatography. The spectrophotometer will be used again in this lab to allow students to identify the plant pigments present in various plant leaves (e.g., Coleus). Students will also monitor the rate of photosynthesis through oxygen production, and observe and compare the quantity of stomata found on the upper versus lower parts of a leaf. 

Tasks    

Students will complete the following during this lab:

• Use acetone to extract plant pigments from spinach (or equivalent) and then separate these pigments based on polarity.
• Use paper chromatography and retention factor (Rf) calculations to identify the relative polarities of major plant pigments. 
• Use the spectrophotometer to identify common pigments present in plant leaves.
• Investigate the impact of CO2 availability on the rate of photosynthesis. 
• Identify and determine differences in stomata in the upper versus lower portions of a leaf. 

Lab instructors should provide students with an introduction photosynthesis and contrast photosynthesis with cellular respiration. The examination of the overall chemical equations for each is important for students to understand the relationship between humans (and other animals) and plants.

Students should wear gloves and goggles when working with acetone and the chromatography solvent. Remind students that these solutions require special waste containers that will be found in the biological safety cabinet. 

Students will need guidance on how to use the chromatography paper. Remind students to use the glass capillary tube to overlay the plant pigment on the pencil line several times, allowing the pigment to completely dry each time before applying the next later. Students should apply at least three layers, although five layers appears to generate the best results. Students can tape or staple the edges of the chromatography paper together to generate the tube that will be placed in the jars of solvent. Students should stand next to the solvent jars and check their papers frequently in order to ensure the solvent front does not to end of the chromatography paper.

Four bands of plant pigment should appear once the experiment has been completed:

• Lowest band (most polar): chlorophyll b (appears yellow-green)
• Chlorophyll a (appears grass green)
• Xanthophyll (appears light yellow)
• Highest band (least polar): carotene (appears dark/golden yellow)

In Exercise 2, students may need to dilute their plant pigment solution with more ethanol if distinct peaks do not appear in their absorbance spectrum. Using 1 gram of leaf tissue (as opposed to 4g as stated in the original manual) may be sufficient for dark-colored leaves. 

In Exercise 3, students may need to perform the vaccuum syringe step (see Exercise C Step 6) several times in order to remove the gas from the plant leaf disks. This step is critical to get optimal results for this experiment.

In Exercise 4, students should be able to view the stomata under 100x total magnification. We recommend having the instructor or student(s) take a picture of their samples (upper and lower leaf) through the microscope ocular lens in order to compare the two and discuss their findings with their peers.