Name: ____________________
Photosynthesis La
Learning Outcomes:
· Determine the abso
ance spectrum of the photosynthetic pigment chlorophyll
Introduction
Photosynthesis
The first Law of Thermodynamics states that energy can neither be created nor destroyed,
ut its form can be changed. In the process of photosynthesis, light energy is captured by
plant cells, converted to electrical energy, and then transformed into chemical energy. Light
energy is captured in Photosystems I and II and is converted into electrical energy in the form of
high energy electrons. The kinetic energy of the electrons is transformed into chemical energy in
the bonds of ATP by a form of active transport, the proton pump. The energy of ATP is then
used to make the glucose molecule (C6H12O6), a form of stored (potential) chemical energy. Thus, light energy from the sun is ultimately captured and stored in the chemical bonds of suga
molecules. The following chemical equation summarizes these processes:
sun + 6CO2 + 6H2O C6H12O6 + 6O2
The glucose molecules produced during the process of photosynthesis may be used by the
plant as a source of metabolic energy or as a building block in the synthesis of other organic
compounds, including complex ca
ohydrates, proteins, lipids and nucleic acids. In turn, othe
organisms may consume the plant tissues and utilize the various molecules for their own
growth, reproduction, or metabolic energy in a process called cell respiration. In this lab exercise, you will investigate the characteristics of chlorophyll, the green-pigmented
molecule that abso
s light energy for photosynthesis. You will extract chlorophyll
from plant leaves and then, using the spectrophotometer, determine the wavelengths
of light that chlorophyll can abso
and reflect. You will also investigate the relationship of light
intensity and the rate of photosynthesis using Elodea plants.
Exercise 1. CHARACTERISTICS OF PHOTOSYNTHETIC PIGMENTS
Chlorophyll Extraction from Spinach Leaf In order to study the abso
ance of various
wavelengths of light by the chlorophyll molecule, we must extract the pigment from the
leaf tissues. This can be easily accomplished by following the procedure below. This procedure
uses ethanol as a solvent to extract chlorophyll from the spinach leaf. ETHANOL IS FLAMMABLE. PLEASE USE CAUTION!!
1. Obtain a 50 ml beaker from your supply tray and a spinach leaf. Place the leaf in the beaker carefully. Try not to crush the leaf because that will cause the cell wall to
eak and organelles to leak out.
2. Add ethanol to the beaker to the 20 ml line (do not use extra ethanol).
3. Place the beaker on a hot plate to heat the beaker. DO NOT LET THE SOLUTION COME
TO A BOIL! The solution will start to turn green as the chlorophyll is leached out of the leaf. Once the solution is a
ight green, turn off the heat.
4. Fill a cuvette approximately 2/3 full with the ethanol/chlorophyll solution (chlorophyll extract).
Exercise 2. Light Abso
ance of Chlorophyll
At this point you need to determine if your chlorophyll extract has an adequate amount
of chlorophyll. If the chlorophyll extract is too concentrated or too weak, it will be difficult to get co
ect absorption values. Therefore, the transmittance (percent of light that can
pass through) of the solution needs to be determined. Follow the procedure below to
ensure the solution concentration is adequate:
5. Set the spectrophotometer to measure %T and set the wavelength to 550 nm (the
wavelength of green light) and cali
ate with a cuvette of pure ethanol at 100% of T. This is the only time during this experiment that you will use the transmittance scale.
6. Measure the % transmittance of your chlorophyll extract. If you obtain a reading between 65% transmittance and 85% transmittance, your solution contains enough chlorophyll to provide
adequate readings. If your solution falls out of the 65-85% transmittance range, you will need to
adjust your solution before moving on to Step 7.
7. Now, find the abso
ance of your chlorophyll extract at the wavelengths given in Table 1. Remember to switch the spectrophotometer to “Abso
ance” for your readings. Also remember to recali
ate the spectrophotometer at each new wavelength using pure ethanol as a “blank.
The numbers have been provided for you.
8. In order to more clearly visualize the actual abso
ance characteristics of your chlorophyll extract, you will plot the data from the Table 1. Remember to label the axes appropriately
(x = wavelength and y = abso
ance) and be sure that your units of measurement properly fit
the graph.
TABLE 1. Light absorption characteristics of chlorophyll. You will plot the data below (wavelength vs. abso
ance).
WAVELENGTH (nm) (X)
COLOR OF LIGHT
AT THIS WAVELENGTH
ABSORBANCE
(Y)
400
Violet
0.8
425
Blue/Violet
1.09
450
Blue
0.98
475
Blue
0.53
500
Blue/Green
0.1
525
Green
0.08
550
Green/Yellow
0.12
575
Yellow
0.18
600
Yellow/Orange
0.24
625
Orange
0.6
650
Red
0.08
Graph the data from Table 1 by hand or feel free to copy and paste a chart using your spreadsheet of choice (Excel, Google Docs, etc).
Answer the questions below.
1. Identify the colors of light and their co
esponding wavelengths that showed the highest amount of absorption by the chlorophyll extract. (Hint: look at the two peaks on your graph)
2. Would the colors and co
esponding wavelengths you identified in question #1 be capable or incapable in stimulating photosynthesis? Explain why.
3. What would you expect to happen to the rate of photosynthesis if you exposed a plant to an environment in which only green light was present? Explain your answer.
4. Write the general equation for photosynthesis:
6 _______ + 6 _________ → 1 ____________ + 6 __________
5. Reactants light Products
a. What reactant in photosynthesis is split to produce oxygen? ___________________
. What wavelength(s) of light energy are BEST abso
ed by chlorophyll? _____________
c. Light energy abso
ed by chlorophyll is transfe
ed to what subatomic particle? _____________________
d. What reactant in photosynthesis loses electrons? ______________________
e. Energized electrons and CO2 are used to form what product of photosynthesis? ___________