AP Lab 4 (Addendum): Photosynthesis Pigments
Name ________________________________
AP Biology
Hour ___ Date ________________________
Paula Donham, Olathe East High School pdonhamoe@olatheschools.com
Adapted from J. Gehring AP Consultant, Beulah, MI
OBJECTIVES:
In this introduction to the spectrophotometer you will:
1.
extract pigments from a leaf of a known species and
2. using a spectrophotometer, develop an absorbance spectrum for the leaf’s pigments.
SAFETY:
PROCEDURE:
Goggles should be used due to the use of alcohol and glass with a boiling water bath.
1.
Get a small test tube rack with 1 small test tube and 1 medium test tube ½ full of 95% ethanol.
Using a Sharpie, put a small vertical mark on the lip of the test tube (this will help you align the
test tube consistently in the spectrophotometer).
2. Go outside and collect one green leaf from a plant you are familiar with (this means you at least
know the common name for the plant). If you get grass, get multiple blades of grass. The
different lab groups should strive to get different species of plants.
3. Using scissors, chop your leaf into the alcohol in your medium sized test tube.
4. Put the test tube with the leaf in alcohol into a hot water bath. When it boils (which it will do
quickly – remember, it’s alcohol) remove it with test tube tongs.
5. Dilute to a light chartreuse color with additional alcohol (too dark a solution will give you a flat
curve). Decant into the small test tube.
6. Hold the “blank” test tube (filled with just the 95% alcohol) near the top and using a Kim Wipe
remove fingerprints from the rest of the test tube.
7. You will now use the “blank” to “zero” the spectrophotometer. Place the blank in the cuvette
well taking care to align the mark on the lip of the test tube with the raised line on the opening.
Make sure the spec is set to measure absorbance and the wavelength is set at 400nm. Close
the cap on the well and adjust the % T/A knob on the front of the machine until the machine
reads 0.
8. You will re-zero using the blank each time you change the wavelength before you measure
your samples. [Note: if you were measuring transmittance, which you will be doing later, you
would need to set the machine to measure transmittance and you would then make sure the
machine read 100% with the blank in the machine.]
9. Now, hold your sample test tube near the top and using a Kim Wipe remove fingerprints from
the rest of the test tube.
10. You will now place your sample in the spectrophotometer, and read and record the percent
absorbance. Then the other lab groups should measure their samples at 400 nm.
11. Increase the wavelength by 25 nm, re-zero the machine using the blank and have all groups
measure the absorbance at that wavelength. All lab groups will now record % absorbance at
every 25 nm from 400 to 700 nm.
ANALYSIS OF
RESULTS:
Record your data in Table 4.1 below:
Table 4.1: Measurement of % Light Absorbance at Wavelengths from 400 nm to 700 nm for
______________________ Pigments
leaf species name
Wavelength
(nm)
%
Absorbance
400
425
450
475
500
525
550
575
600
625
650
675
ANALYSIS OF
RESULTS:
1. Graph the data points on the graph below and connect them with a smooth curve.
Graph 4.1: (Descriptive) Title: ___________________________________________
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700
2. Describe and explain the relationship between wavelength and absorbance:
_________________________________________________________________
_________________________________________________________________
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3. What color(s) light is your leaf absorbing? _________________________________
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4. What color(s) light is your leaf probably reflecting? __________________________
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