Lab Activity 3.3
Phycobilin 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


Phycobilin is an example of an accessory pigment found in Rhodophyta. It helps chlorophyll gather light energy for photosynthesis. It absorbs light of particular colors (wavelengths) and then passes the energy to chlorophyll in a flow of electrons. 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 in the next lab activity you will see how phycobilin expands a seaweed's ability to capture light.

Top  Instruction

Study the information, and photographs in the experiment that follows. Be sure to complete the table of wavelengths, colors and absorbances; produce a bar chart of wavelengths versus absorbances; 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 Phycobilin 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 phycobilin 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 phycobilin 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 scale. Then the absorbance of the phycobilin 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 Phycobilin

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

Recording the Absorption Spectrum of Phycobilin:
  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 Phycobilin

Create a graph of absorbance versus wavelength was created for the phycobilin absorption spectrum.

Graphing the Absorption Spectrum of Phycobilin:
  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 wavelenght 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 Phycobilin

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

Analyzing the Absorption Spectrum of Phycobillin:

  1. Use the table and chart of the wavelengths, colors, and absorbances to analyze the absorption spectrum of phycobilin.
  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 phycobilin absorb much red light?
  2. Relative to other colors did phycobilin absorb much blue light?
  3. Relative to other colors did phycobilin absorb much green light?
  4. At which wavelength did phycobilin absorb the most light? What color is this wavelength?
  5. At which wavelength did phycobilin absorb the least light? What color is this wavelength?
  6. What color is phycobilin? From what you've learned about its absorbance and transmittance being reciprocal can you explain why it has this color?

Lab Activity 3.4 Chlorophyll Absorption Spectrum