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Analyzing the absorption spectrum of leaves

Learn how to use the Analyze Solution page by performing an experiment on absorption.

Plants use light energy to synthesize energy-rich chemical compounds like sugars and starches from carbon dioxide (CO2) and water (H2O). What colors of light or wavelengths do chloroplasts actually need for this process? The leaves of many plants are green, which indicates that most of the green light falling on leaves is being reflected or transmitted, since you can see it! Other colors are absorbed by the leaf. This is similar to the energy absorption that occurs when wearing a dark T-shirt on a sunny day (which is why doing so will make you much warmer than wearing a light-colored shirt). As the seasons change, some leaves change from green to red, orange, yellow, and brown. This happens when leaves stop producing chlorophyll, causing other pigments such as carotenoids or anthocyanins to become visible. Analyzing the absorption spectrum (what wavelengths are absorbed versus transmitted) can tell us what pigments are present in a leaf and what wavelengths of light will support the photosynthetic process.

Gather the materials

  • Spectrometer
  • Cuvettes and lids (4)
  • 3 disposable pipettes
  • Plastic or glass funnel
  • Lint free cloth or wipes
  • 3 leaf samples
  • 3 test tubes
  • 80 mL of 95% ethanol
  • Filter paper
  • Test tube rack
  • 20-mL graduated cylinder
  • Scissors
  • Distilled water

Prepare the samples

  1. Using a balance, measure 0.50 g of each leaf sample. If possible, avoid veins and stems by cutting out small leaf tissue sections with scissors.
  2. Place the sample in the clean mortar and add 20 mL of 95% ethanol.
  3. Grind the mixture with the pestle for 2-3 minutes until the mixture is homogenized as much as possible.
  4. Filter the solution into a labeled test tube using the filter paper and funnel.
  5. Fill a cuvette ¾ full with the filtered leaf extract.
  6. Repeat the steps with additional samples. Store all samples on ice until ready for testing.

Calibrate the spectrometer

Before the experiment, the spectrometer needs to be properly calibrated to both the absence of light and a reference solution. To do this, follow the instructions under Two point calibration.

Collect data

  1. Using a lint-free cloth, clean the smooth sides of the cuvette with the first leaf extract. Place the cuvette into the sample well and click Record in the lower left of the screen. The absorbance spectrum for the extract will appear.

    Note

    If the data plateaus at any point in the scan, remove the sample and dilute with 95% ethanol. Dilute additional samples with the same amount of 95% ethanol.

  2. In the tools panel, adjust the smoothing and number of scans to average. Click Stop when you are satisfied with the result.

  3. Click the run title and then Rename to rename the run, or click Annotation Tool to make a note indicating which sample or species was analyzed.
  4. Repeat Steps 1 through 3 with the remaining leaf extract samples.

Analyze data

  1. Click Coordinates Tool and use the Coordinate tool to locate the peak(s) for each sample.
  2. Take a Snapshot of each run with the coordinates active, or record the values for each sample in a data table.
  3. Table 1 shows the absorption peaks for the most common plant pigments. Based on your data, which pigments are visible in your leaf extract samples?
Pigment Peak Absorption Wavelength (nm)
Chlorophyll A 430 and 662
Chlorophyll B 453 and 642
Carotenoids 460-550
Anthocyanins 520

Note

The absorption wavelength of anthocyanins is pH dependent. The value shown is at a pH of 4.5. At lower pH values, the pigments undergo a structural change and do not absorb visible light wavelengths.

Questions to consider

Will the visible pigments in a leaf from the same plant species change throughout the year?