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Analyzing the absorption spectrum of leaves

Learn how to use the solution spectra page by performing an absorption experiment.

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 you wear a dark T-shirt on a sunny day (which is why doing so will make you much warmer than if you wear a light-colored shirt).

As the seasons change, some plants' leaves change from green to red, orange, yellow, and brown. This occurs 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 best support the photosynthetic process.

Required materials

  • Spectrometer
  • Cuvettes (SE-8739)
  • 4× cuvettes and lids
  • 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
  • Mortar and pestle

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 one of the samples into 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 as homogenous 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 Steps 2 through 5 for the other leaf samples. Store all samples on ice until you are ready to begin testing them.

Connect and calibrate the spectrometer

Before the experiment, your chosen spectrometer needs to be properly calibrated to both the absence of light and a reference solution. To do this:

  1. Prepare a reference solution by filling a cuvette ¾ full with 95% ethanol. Handle this cuvette only by the lined sides and wipe the smooth sides clean with a lint-free cloth.
  2. Start Chemvue, then connect the spectrometer to the program. The Spectrometry Module should automatically open to the solution spectra page .
  3. Cover the spectrometer's sample chamber to block out ambient light, then select Calibrate Dark at the bottom of the screen. The spectrometer will turn off light sources to perform a calibration. The icon will change to a new appearance when the calibration is complete.
  4. Place the cuvette containing the reference solution into the spectrometer so that one of the clear sides is facing towards the white light source icon.
  5. Select Calibrate Reference to begin the reference calibration. The icon will change to a new appearance when the calibration is complete.

Collect data

  1. Using a lint-free cloth, clean the smooth sides of the cuvette containing the first leaf extract. Place the cuvette into the spectrometer's sample chamber and select Start . The absorbance spectrum for the extract will appear.

    Note

    If the data plateaus at any point in the scan, remove the sample, dilute it with 95% ethanol, and replace the cuvette in the chamber. Make sure to dilute all other samples with the same amount of 95% ethanol to preserve accurate readings.

  2. In the Tools panel, adjust the Smoothing and Number of Scans to Average sliders as desired.

  3. When you are satisfied with the result, select Stop to end data collection.
  4. Select Annotate Graph to create a note indicating which sample or species was analyzed in that run of data.
  5. Repeat Steps 1 through 4 with the remaining leaf extract samples.

Analyze data

For each of the samples for which data was collected in the previous section:

  1. Select the Coordinates tool and drag the coordinate data target to locate the peaks for the sample.
  2. Select Snapshot with the coordinates active, or record the values for each sample in a data table.
  3. The table below 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 from a sample 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?