Spectral Analysis of Photosynthetic Pigments

Chlorophyll and accessory pigments are used by leaves to collect energy from light and transduce it to chemical energy be used to synthesize sugar. These pigments can be separated by chromatography in which a solvent system travels through paper by capillary action, carrying the pigments with it. Because each pigment has a different solubility and/or affinity for paper, they move at different rates, and separate along the paper. Pigments separated in the way in a previous lab have varying absorption spectra which may be demonstrated using the technique for determining the absorption spectra.

Alternatively, simple analysis of the composition of plants may be performed by drying, extracting with various organic solvents, and determining the absorbtion spectra of each extract. In this experiment, we will determine percent water, and relative pigment concentrations in a variety of plant materials, notably salad greens.


It is required that you know how to use a spectrophotometer and correctly plot data on a graph to complete this lab. Ask the instructor for previous protocols if you lack this knowledge.

Which solvent is most effective extracting chlorophyll?
How does the chlorophyll content compare among salad greens?
What differences do you note when comparing spectra of various greens?
How does the absorption spectrum of plant extract compare with known dyes?
Might the concentration of photosynthetic pigments correlate with the nutritional quality of these salad greens?


  • Variety of salad greens, fresh and unfaded
  • Organic solvents:
    • 95% ethyl alcohol
    • petroleum ether
    • acetone, etc.
  • Whatman #1 filter paper
  • Diluted standard microbiological stains:
  • Hucker’s: 1.0 uL into 10 mL dH2O
  • Safranin O: 3.0 uL into 3 mL dH2O
  • Methylene Blue: 2.0 uL into 3 mL dH2O


  • Large strainers with feet
  • Drying oven, 80°C
  • Balance
  • Mortar and pestle
  • 16 x 150 mm test tubes with corks
  • Filter funnel
  • Spectrophotometer
  • 5 cuvettes in plastic rack
  • Lens paper

Continued from the extraction protocol…

8. Dilute filtered extract 1:10: 5.0 mL EtOH + 0.555 mL extract (we are ignoring effect of diluting alternate solvents into EtOH.).

9. Prepare cuvettes: Rinse five cuvettes with 95% EtOH. Fill each with 3.00 mL EtOH, polish outside, read A350. If the difference is >than 0.005, clean and polish again. Mark the cuvette with the lowest A350 as “B” (blank), the others S1, S2, etc (S = sample).
10. Read absorbencies at 350 nm
11. Dilute the extract to adjust absorbency so that A350 ~ 0.800 to 1.000

12. Read absorption spectra for all group samples every 25 nm from 350-800 nm. (Read all samples at a given wavelength, then re zero and reblank for the next wavelength and read all samples at that wavelength, etc). Rotate reading and recording roles. Enter into the computer as instructed.

13. Graph the data: Graph all data on the same graph, plotting wavelength (on the X axis) versus absorbance (on the Y axis), noting maxima for each of the samples. Follow graphing protocol previously distributed. Note maxima for each of the solutions tested. Which of the original questions can you answer? What conclusions do you draw?

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