Spectrophotometry: determination of absorption spectrum

MATERIALS FOR EXERCISE B:

• spectrophotometer

• Photosynthetic pigments

• 80% acetone

• 2 cuvettes

• Kimwipes®

1. Fill to 1/3 a spectrophotometer cuvette with 80% acetone. This will be your “blank” to use for standardizing the spectrophotometer.

2. Your instructor will give you a beaker containing your eluted pigment. Transfer the pigment solution from the beaker to a second spectrophotometer cuvette.

3. WipethesidesofcuvettewithaKimwipe©andavoidtouchingthesurfaces with your fingers.

A spectrophotometer directs a beam of white light through a grating (which separates the light into bands of specific wavelengths) to the specimen to be analyzed. At each specific wavelength, some of the light is absorbed by the specimen while some is reflected or passes through. The spectrophotometer records the percentage of light transmitted and the percentage of light absorbed (absorbance value). You will determine the absorbance of the pigment(s) at different wavelengths to generate the absorption spectrum for the different pigment solutions.

Students should work in groups of 3-4; there should be at least 5 groups. Each lab group will determine the absorption spectrum of a different solution (if there are 6 groups, one solution will be analyzed by two groups.) The five solutions are: (1) chlorophyll a, (2) chlorophyll b, (3) xanthophyll, (4) carotene, and (5) the original (whole) prepared spinach leaf extract. You will be asked to identify your solution (and those from the other groups) based on the absorption spectrum that you will plot from your data.

My questions:

What hypothesis is being tested in this experiment?

What is your production?

What is the independent variable in this experiment?

What is the dependent variable in this experiment?

What is the control?

What is the result?