Lab Activity 3.4
Chlorophyll Absorption Spectrum

  Directions

Study the instructional material below. Be sure to click on each of the photographs for an enlarged view in a separate window. The larger version is necessary to complete the assignment. It opens in a separate window which can be resized by grabbing the bottom right corner and dragging it. It can also be moved by grabbing the top heading bar and dragging it. Be sure to close the extra window by using the X in IBM, or the close box in MAC when you are finished using them.

  Introduction


. This energy is then used to make sugar during the synthetic portions of the photosynthesis reactions. By comparing the results of this experiment with that of the preceeding lab activity you will see how phycobilin expands a seaweed's ability to capture light.

Top  Instruction

Study the information, photographs, and video clips in the experiment that follows. Be sure to complete the table of wavelengths, colors, and absorbances; produce a bar chart of wavelengths versus absorbances; compare the bar chart with the one you made for the preceeding lab activity; and answer the questions you're asked in your notebook.

Be sure to write about what you are learning in the lab section of your notebook. You will be expected to answer questions about the lab activity during the lab self test and lab quiz. It helps to have your text and coloring books open beside you for support.

 
Supporting Information
Refer to the Assigned Readings Below:
Marine Biology Textbook None
Marine Biology Coloring Book None
 
The Absorption Spectrum of the Chlorophyll Extract

An absorption spectrum is a measure of the light absorbing capacity of a substance across the entire range of wavelengths (colors). In this experiment we measured the absortion spectrum of the pigment chlorophyll extracted from a sample of coralline algae.

Using a Spectrophotometer to Measure Light Absorption

A spectrophotometer is a device that measures the relative amount of light absorbed by a sample dissolved in a liquid solvent.

Using a Spectrophotometer to Measure Light Absorption:
  1. A spectrophotometer was used to measure the light absorbed by a sample of phycobillin extract in a special tube placed in its sample port.
  2. The spectrophotometer had a Transmittance and Absorbance (T/A) Meter which had a scale that allowed us to read the amount of light that was transmitted by (passed through) the sample or its reciprocal, the amount of light absorbed by the sample.
  3. The spectrophotometer had a wavelength control that allowed us to set the wavelength (color) of the light that passed through the sample.
  4. The spectrophotometer also had 0% and 100% controls to set it up.

The Spectrophotometer
Observations:
  1. Observe the photograph of the spectrophotometer and familiarize yourself with its parts.
  2. Observe the photograph of the spectrophotometer Transmittance and Absorbance (T/A) Meter and familiarize yourself with how to read its scale.
  3. Note: You can read both the Transmittance (red upper scale) and Absorbance (blue lower scale) from the same meter.
  4. Note: the Transmittance and Absorbance scales run opposite to each other. As transmittance increases absorbance declines and vice versa. This is because the light that is transmitted by (passes through) a sample is not absorbed by it and vice versa.

The T/A Meter

Running the Spectrophotometer

The raw chlorophyll extract was diluted to a concentration that would absorb the proper amount of light to read within the limits of the spectrophotometer T/A meter scales. Then the absorbance of the chlorophyll sample was measured at a series of wavelengths (colors) across a range of visible light wavelengths from 400nm (violet) to 700nm (deep red).

Running the Spectrophotometer:

  1. We determined the absorbance spectrum of the pigment solution by obtaining absorbance readings at 25 nm intervals of wavelength starting with 400 nm and continuing to 700 nm.

Recording the Absorption Spectrum of Chlorophyll

Record the absorbance of the chlorophyll sample for wavelengths at 25nm intervals from 400nm to 700nm.

Recording the Absorption Spectrum of Chlorophyll:
  1. Produce a table of wavelengths, colors, and absorbances in your lab notebook like the one you see below or print it out and fill it in.
  2. Read the spectrophotometer meter for each wavelength of light by clicking the wavelengths in the first column of the table below and reading the lower absorbance scale of the T/A meter that appears.
  3. Record the absorbances in the last column of your table.
Wavelength Color Absorbance
400 nm Violet
425 nm Blue-Violet
450 nm Violet-Blue
475 nm Blue
500 nm Blue-Green 
525 nm Green 
550 nm  Green-Yellow
575 nm Yellow
600 nm Yellow-Orange 
625 nm Orange 
650 nm Orange-Red
675 nm Red 
700 nm Deep Red 

 

Graphing the Absorption Spectrum of Chlorophyll

Create a graph of absorbance versus wavelength for the chlorophyll absorption spectrum.

Graphing the Absorption Spectrum of Chlorophyll:
  1. Produce a bar chart of wavelengths versus absorbances in your lab notebook.
  2. You can configure it like the chart below or you can print it out and complete it by filling in the cells next to each wavelength to create bars.
A b s o r b a n c e
W

a

v

e

l

e

n

g

t

h

   0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
400 nm                    
425 nm                    
450 nm                    
475 nm                    
500 nm                    
525 nm                    
550 nm                    
575 nm                    
600 nm                    
625 nm                    
650 nm                    
675 nm                    
700 nm                    

 

Analyzing the Absorption Spectrum of Chlorophyll

Examine the absorption spectrum data for the chlorophyll sample to determine the relationship between absorbance and wavelength.

Analyzing the Absorption Spectrum of Chlorophyll:

  1. Use the table and chart of the wavelengths, colors, and absorbances to analyze the absorption spectrum of chlorophyll.
  2. Examine your table and chart and determine the answers to the questions that follow. Write the answers into your lab noterbook.

Observations:

  1. Relative to other colors did chlorophyll absorb much red light?
  2. Relative to other colors did chlorophyll absorb much blue light?
  3. Relative to other colors did chlorophyll absorb much green light?
  4. At which wavelength did chlorophyll absorb the most light? What color is this wavelength?
  5. At which wavelength did chlorophyll absorb the least light? What color is this wavelength?
  6. What color is chlorophyll? From what you've learned about its absorbance and transmittance being reciprocal can you explain why it has this color?

Comparing the Absorption Spectra of Chlorophyll and Phycobilin

Examine the absorption spectra data for the chlorophyll and phycobilin samples to determine how they differ.

Comparing the Absorption Spectra of Chlorophyll and Phycobilin:

  1. Use the tables and charts of the wavelengths, colors, and absorbances to compare the absorption spectrum of chlorophyll to that of phycobilin.
  2. Examine your tables and charts and determine the answers to the questions that follow. Write the answers into your lab noterbook.

Observations:

  1. Relative to other colors how did chlorophyll compare to phycobilin in the absorption of red light?
  2. Relative to other colors how did chlorophyll compare to phycobilin in the absorption of blue light?
  3. Relative to other colors how did chlorophyll compare to phycobilin in the absorption of green light?
  4. At which wavelength did chlorophyll absorb more light than phycobilin? What colors are these wavelengths?
  5. At which wavelength did phycobilin absorb more light than chlorophyll? What colors are these wavelengths?
  6. What light absorbing advantage would seaweeds containing phycobilin and chlorophyll have over those that have chlorophyll only? Under what environmental circumstances would this advantage give these seaweeds a competitive edge?