IB Biology
Determining the absorption spectrum of photosynthetic pigments using a colorimeter
Introduction:
In this experiment you will observe the ability of differing pigments in vegetables to absorb and transmit different wavelengths of light. For this experiment, spinach leaves and carrots have been soaked in acetone to extract the pigments from each.
Recall that the colors that we perceive with our eyes are the wavelengths of the visible spectrum that are transmitted rather than absorbed (example: a blue shirt is transmitting blue, but absorbing other colors of the spectrum). You will once again use the colorimeter to measure the ability of each pigment extract to absorb or transmit different wavelengths of light.
You will now observe the ability of spinach extract and carrot extract to absorb or transmit light of different wavelengths (400 – 700 nm). The spinach extract in this experiment is green in color, while the carrot extract is orange in color.
Additionally, figure 1 denotes the approximate range of wavelengths for each color of the visible light spectrum.
Color Wavelength
Violet 380-450 nm
Blue 450-500 nm
Green 500-570 nm
Yellow 570-590 nm
Orange 590-620 nm
Red 620-750 nm
Figure 1. Colors of the visible light spectrum with their approximate corresponding wavelength range.
Use the information given in figure 1 to write hypotheses about the wavelengths that you think spinach extract and carrot extract will absorb and transmit:
Based on what I know about transmittance and absorbance of the visible light spectrum, spinach extract will
______
Based on what I know about transmittance and absorbance of the visible light spectrum, carrot extract will
______
Photosynthetic Pigment Absorption Spectrum Experiment
Method:
1. Turn on the colorimeter using the power switch/zero control knob and allow it to warm up for five minutes.
2. With the black sample tube in the holder and the lid closed, turn the power switch/zero transmittance control knob until the meter read 0% transmittance.
3. Adjust the wavelength control knob until the display reads 525 nm.
4. You have been provided with three test tubes: one labeled B, which contains your blank (in this case the blank is acetone), one labeled S, which contains spinach extract (the extract was made by placing spinach leaves in acetone),
and one labeled C, which contains carrot extract (the extract was made by placing carrot shavings in acetone).
5. Insert the blank into the sample holder and close the lid. Using the 100% transmittance control knob, adjust the meter reading to 100% transmittance.
REMEMBER that as you go through this experiment you will be
changing the wavelength. EVERYTIME you change the wavelength, you
MUST again set the blank to 100% transmittance. Failing to do this may
drastically affect your results.
6. Remove the blank and insert the spinach extract tube. Measure the % transmittance, if it reads 90% transmittance record the
THE COLORIMETER DOES NOT READ 90% TRANSMITTANCE FOR THIS SAMPLE, dilute the sample with acetone until it yields a reading of close to 90% transmittance.
7. Press the mode button to obtain the absorbance of the spinach extract and record it in figure 3.
8. Place the tube containing carrot extract into the colorimeter and repeat steps 6 and 7.
9. After you have successfully obtained the transmittance and absorbance for both spinach and carrot extract at 525 nm, move the wavelength in 25 nm integrals both up and down the spectrum. REMEMBER to reset the blank to 100% transmittance at every step.
Results:
Wavelength (nm)
T%