Absorbance Spectra of Photosynthetic Pigments

Prelab Exercise

  1. Fill the Color field in the table below
  2. Use plot.ly to create a line graph with the 3 samples below (A, B, C)
    • plot % Reflectance on the Y-axis and Wavelength (nm)  on the X-axis

% Reflectance

Color

nm

A

B

C

400

68

92

78

425

40

71

77

450

90

38

51

475

97

49

57

500

100

92

45

525

100

100

66

550

96

97

100

575

98

96

100

600

96

98

100

625

97

80

100

650

79

71

100

675

56

96

100

700

88

100

100

Stop and Think: Reflectance

A sign of plant health is viewed through the near infra-red. While we cannot see this spectrum of light with our eyes, we can use other sensors to detect this light. Compare the images of the Black & White with the Infra-red image. What differences can you see in the 2 images that will help you understand how this is a useful measure of plant health? How do you think this is corresponds to the table above?

English Garden in Color
The English Garden

Reflectance Slider shows an overlay of the next images

English Garden in Black and White
The English Garden (black & white)
English Garden in Infra-red
The English Garden (near infra-red)

Visible light wavelengths (between 400nm-700nm) are strongly absorbed by the pigments in leaves (Chlorophylls, Xanthophylls, Carotenoids). These pigments utilize the energy of these wavelengths to take part in the light reactions. The cellular structure of leaves do not absorb wavelengths longer than these wavelengths (>700nm in the infra-red range). By comparing the amount of visible light to the amount of near infra-red light that are reflected, one can gauge the relative health of leaves, forests or jungles . This is the rough description of the Normalized Differential Vegetation Index (NDVI) that scientists use in conjunction with satellite imagery to assess the health of vegetation.

The Role of Light in Carbohydrate Synthesis

  1. Pick a leaf from a geranium exposed to light and one kept in the dark for 48 hours.
    • keep the stem on the leaf grown in light
    • remove the stem from the leaf grown in the dark
  2. Hydrolyze the cell walls of the geranium leaves by boiling in a water bath for 5 minutes or until it looks like over-cooked vegetables)
  3. Bleach the leaves by  removing the pigments. Place the leaves in hot alcohol for 7 minutes or until they turn white.
    1. Save this green solution for Absorbance Spectrum exercise
  4. Remove the leaves and place it in a petri dish.
  5. Add iodine to the dish. If starch is present, the leaf will turn a deep bluish-black color.
  6. Photograph the leaf with your phone to document the effects of light on carbohydrate storage.

Measuring Absorbance

  1. Connect the Spectrovis to the LabQuest2
  2. Turn on the Labquest2 units
  3. Choose the Labquest app
  4. Select the icon that looks like X|Y
  5. Press the green Play button on the bottom left
  6. Press OK to calibrate
  7. Let the machine calibrate for 90 seconds
  8. Choose “Finish calibration”
  9. Insert the Geranium pigment from the bleaching reaction
    1. Do NOT use Acetone in these plastic cuvettes since it will frost over the plastic
  10. Press the Red Stop button
  11. Students should record the absorbance values at every 10 nm  from 380nm-700nm
  12. The professor will prepare Spirulina extract diluted in ethanol in a cuvette and obtain the continuous absorbance spectrum.
  13. Plot Relative Absorbance against wavelength using a line graph and compare the absorption spectrum of the extracts.
    1. Relative Absorbance sets the maximum value in each dataset as a denominator
    2. Every value is divided by this maximum value
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