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.
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
- Mortar and pestle
- 16 x 150 mm test tubes with corks
- Filter funnel
- 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?
The spectrophotometer is an instrument which measures the amount of light of a specified wavelength which passes through a medium. According to Beer’s law, the amount of light absorbed by a medium is proportional to the concentration of the absorbing material or solute present. Thus the concentration of a colored solute in a solution may be determined in the lab by measuring the absorbency of light at a given wavelength.
Wavelength (often abbreviated as lambda) is measured in nm. The spectrophotometer allows selection of a wavelength pass through the solution. Usually, the wavelength chosen which corresponds to the absorption maximum of the solute. Absorbency is indicated with a capital A.
To familiarize yourself with the spectrophotometer, illustrate and label the following features which are important to its proper use. You should know the function and/or significance of each of these features before you use the instrument.
At the spectrophotometer, you should have two cuvettes in a plastic rack. Solutions which are to be read are poured into cuvettes which are inserted into the machine. One should be marked “B”for the blank and one “S” for your sample. A wipette should be available to polish them before insertion into the cuvette chamber. Cuvettes are carefully manufactured for their optical uniformity and are quite expensive. They should be handled with care so that they do not get scratched, and stored separate from standard test tubes. Try not to touch them except at the top of the tube to prevent finger smudges which alter the reading. For experiments in which minor imprecision is acceptable, clean, unscratched 13 x 100 mm test tubes may be used.
Answers to the spectrophotometer dial readings:
10. Remove cuvette from machine, carefully wash and store spectrophotometer cuvettes keeping them separate from regular test tubes.
Return spectophotometer to its storage location.