Biology 1012 K Lab Manual

Background:  

Have you ever wondered why leaves change colors in the fall but remain green during other seasons of the year? Leaves contain multiple types of pigments that are involved in many different functions in the plant, including photosynthesis, protection from UV radiation, and even attracting pollinators. Pigment molecules are stored inside of plastids, a class of cellular organelles that includes chloroplasts, the organelles responsible for photosynthesis. Found inside chloroplasts, chlorophyll is the most common pigment in a leaf. It comes in two varieties (chlorophyll a and chlorophyll b). During the spring and summer months, when day length is longest and sunlight is most direct, plants produce a large amount of chlorophyll, which is used to capture sunlight energy that drives photosynthesis.  

Figure 1: Photosynthesis Credit: [CC BY SA 3.0 (https://creativecommons.org/licenses/by-sa/3.0)]

Figure 2: Chemical Equation for Photosynthesis

Photosynthesis is the process by which plants use energy from the sun and water from the soil to convert atmospheric carbon dioxide into sugar (glucose) that can be used for energy production.  

All of the wavelengths of energy emitted by the sun are collectively called the electromagnetic spectrum. Waves at the low end of the spectrum, like radio waves and microwaves, emit less energy, while waves at the high end, like x-rays and gamma rays, emit higher energy. The very small section of the electromagnetic spectrum that humans can see is called the visible light spectrum. Plants can use these same wavelengths of light, from red at the low end to violet at the high end of the visible light spectrum, to power photosynthesis.  

Different pigments absorb different wavelengths of light, and therefore appear different colors to us. During the spring and summer months, when plants are most productive, they produce a huge amount of chlorophyll molecules. Chlorophyll a and chlorophyll b absorb mostly at the red-to-orange and blue-to-violet ends of the visible light spectrum; they reflect the rest of the wavelengths. The reflected wavelengths, mostly in the yellow-to-green range, are the wavelengths that are detected by the human eye. This is why plants appear green to us (Fig. 3). Because chlorophyll a and chlorophyll b are the primary pigments involved in photosynthesis, it is important to measure the concentrations of both chlorophyll a and b individually, total chlorophyll (a+b), and the ratio of chlorophyll a to chlorophyll b (a/b). These values help us to understand and compare the relative photosynthetic capacities of different plants within ecosystems.

Figure 3: Absorption Spectrum of Chlorophyll A and Chlorophyll B Credit: Chlorophyll_ab_spectra2.PNG: Daniele Pugliesiderivative work: M0tty [CC BY-SA 3.0 (https://creativecommons.org/licenses/by-sa/3.0)]

Another class of pigments found in plants is the carotenoids. Carotenoids include carotenes, which appear yellow-orange, and xanthophylls, which appear mostly yellow. These pigments are found in the plant year-round but are largely masked by the abundant chlorophyll molecules that are present during the spring and summer. Carotenoids play a minor role in photosynthesis, but they play a larger role in protecting the plant tissues from damage caused by UV radiation from the sun.  

As summer gives way to fall, day length gets shorter, temperatures decrease, and water may become less available. Plants stop producing new chlorophyll molecules, and the remaining chlorophyll molecules are broken down and reabsorbed to be used again in the following spring. Carotenoids break down more slowly, which is why we can see their yellow and orange colors showing through as chlorophyll is lost from the leaves. 

In this lab you will explore the amounts of photosynthetic pigments (chlorophyll a and chlorophyll b) produced by different plants. With your lab group, you will choose a question that you would like to investigate. You could compare two different species of plants that grow in similar conditions or habitats, species grown in full sun vs. shade, or woody vs. herbaceous plants. The previous examples are only suggestions; if you have another original question you would like to address in this lab, feel free to be creative! Just make sure to run your question by your lab instructor before you begin your experiments. 

RESEARCH QUESTION:     __________________________________________________________________________________________________________________________________________________________________________________________________________________________________________ 

Materials and Methods 

Part 1: Pigment Extraction 

1. Obtain leaves from your selected plant source. You will need approximately 5.0 grams of fresh leaf tissue, so obtain roughly a handful of leaves from each individual plant you are using in your experiment. Depending on your question, you may need more or fewer replicates. Determine the number of replicates you will perform for each group. Number of replicates: ______________________________________ 

2. Cut or tear leaves into small pieces, discarding the large midvein if there one is apparent. Use a balance scale to weigh out approximately 1-2 grams of leaf tissue into a weigh boat. (Make sure to use the TARE function to account for the weight of the weigh boat itself before adding leaves.) 

3. Place a small amount of clean quartz sand into a clean mortar, then add your leaf tissue. Use the pestle to grind the leaf tissue to a fine pulp. If your leaves are too dry, you can add 1 mL of 80% acetone to the mortar to help grind the leaf tissue into a paste-like consistency. 

4. Add 5mL 80% acetone to the pulp and continue to grind. If you need to add additional acetone, you may. RECORD the total volume of acetone used for EACH of your samples. 

5. When leaf tissue is thoroughly ground into a paste-like consistency, and the acetone appears a homogenous green color, cover the mortar with aluminum foil and allow the solution to rest for 1 minute.  

6. Carefully pour off the liquid portion of the mixture into a clean plastic screw top vial, leaving the pulpy remains in the mortar.  

7. Wrap each vial in aluminum foil to prevent the degradation of the chlorophyll molecules by exposure to light. 

Part 2: Centrifugation and Spectrophotometric Analysis 

1. Remove the foil and place the screw-top vials into the centrifuge. IMPORTANT: Make sure the centrifuge is balanced. You may need to fill extra vials with water to balance the load. **Check with instructor to ensure the centrifuge is properly loaded.** 

2. Spin samples for 10 minutes at 1500 rpm. When centrifuge stops, remove samples and rewrap them with aluminum foil.  

3. Using a clean transfer pipet, transfer 3mL of supernatant (the liquid portion of the sample) to a clean cuvette. 

4. Follow the directions given for the spectrophotometer to obtain an optical density (OD) value for each of your samples. **IMPORTANT: Set the wavelength on the spectrophotometer to 663 nm to obtain OD for chl a and 646 nm to obtain OD for chl b. You will need separate reading for each to calculate sample concentrations.** 

5. Record your OD readings for chl a and chl b for each sample, then use the following equations to calculate concentration of each pigment: 

Chl a = 12.21A663 - 2.81A646 

Chl b = 20.13A646 - 5.03A663 

where A is optical density (OD) at the respective wavelength.  

These equations will give you chlorophyll concentrations in micrograms per milliliter (ug/mL). To convert to for grams of fresh leaf tissue you started with, use the following equation: 

C = cVR/m x1000 

where: 

C = chlorophyll content (mg/g) 

c = chlorophyll content (μg/mL) 

V = total extract volume (mL) 

m = sample fresh leaf weight (g fr wt) 

1000 = convert factor for μg in mg 

Part 3: Analysis 

1.  What were the results of your experiment? 

2. Did you see what you expected to see? 

3. What challenges did you encounter, or what might you do differently if you were to repeat the experiment?