AP Biology Lab Four: Plant Pigments and Photosynthesis

The purpose of this lab is to separate and identify pigments and other molecules within plant
cells by a process called chromatography. We will also be measuring the rate of photosynthesis in
isolated chloroplasts. Beta carotene, the most abundant carotene in plants, is carried along near the
solvent front because it is very soluble in the solvent being used and because it forms no hydrogen bonds
with cellulose. Xanthophyll is found further from the solvent font because it is less soluble in the solvent
and has been slowed down by hydrogen bonding to the cellulose. Chlorophylls contain oxygen and
nitrogen and are bound more tightly to the paper than the other pigments. Chlorophyll a is the primary
photosynthetic pigment in plants. A molecule of chlorophyll a is located at the reaction center of the
photo systems. The pigments collect light energy and send it to the reaction center. Carotenoids also
protect the photosynthetic systems from damaging effects of ultraviolet light.
Procedure:
1) Obtain a 50 ml graduated cylinder which has about 1 cm of solvent at the bottom.
2) Cut a piece of filter paper which will be long enough to reach the solvent. Draw a line about
1.5 cm from the bottom of the paper.
3) Use a quarter to extract the pigments from spinach leaf cells. Place a small section of leaf on the
top of the pencil line. Use the ribbed edge of the coin to crush the leaf cells. Be sure the pigment
line is on top of the pencil line.
4) Place the chromatography paper in the cylinder.
5) Cover the cylinder. When the solvent is about 1 cm from the top of the paper, remove the paper
and immediately mark the location of the solvent front before it evaporates.
6) Mark the bottom of each pigment band. Measure the distance each pigment migrated from the
bottom of the pigment origin to the bottom of the separated pigment band. Record the distances.
7) Turn on the spectrophotometer to warm up the instrument and set the wavelength to 605 nm.
8) Set up an incubation area that includes a light, water flask, and test tube rack.
9) Your teacher will provide you with two beakers, one containing unboiled chloroplasts and the
other containing a solution of boiled chloroplasts. Be sure to keep these on ice at all times.
10) At the top rim, label the cuvettes 1,2,3,4, and 5, respectively. Using lens tissue, wipe the outside
walls of each cuvette. Using foil paper, cover the walls and bottom of cuvette 2. Light should not
be permitted inside cuvette 2 because it is a control for this experiment.
11) Refer to Table 4.3 to prepare each cuvette. Add 4 ml of distilled water to cuvette 1. To 2,3, and 4,
add 3 ml of distilled water and 1 ml of DPIP. To 5, add 3 ml plus 3 drops of distilled water and 1
ml of DPIP.
12) Bring the spectrophotometer to zero by adjusting the amplifier control knob until the meter reads
0% transmittance. Add 3 drops of unboiled chloroplasts and cover the top of cuvette 1 with
Parafilm and invert to mix. Insert cuvette 1 into the sample holder and adjust the instrument to
100% transmittance. For each reading, make sure that the cuvettes are inserted into the sample
holder so that they face the same way as in the previous reading.
13) Obtain the unboiled chloroplast suspension, stir to mix, and transfer 3 drops to cuvette 2.
Immediately cover and mix cuvette 2. Then remove it from the foil sleeve and insert it into the
spectrophotometer's sample holder, read the percentage transmittance, and record it as the time 0
reading in Table 4.4. Replace cuvette 2 into the foil sleeve, and place it into the incubation test
tube rack. Turn on the flood light. Take and record additional readings at 5,10,and 15 minutes.
Mix the cuvette's contents just prior to each readings. Remember to use cuvette 1 occasionally to
check and adjust the spectrophotometer to 100% transmittance.
14) Obtain the unboiled chloroplast suspension, mix, and transfer 3 drops to cuvette 3. Immediately
cover and mix cuvette 3. Insert it into the spectrophotometer's sample holder, read the percentage
transmittance, and record it in Table 4.4. Replace cuvette 3 into the incubation test tube rack.
 Take and record additional readings at 5, 10, and 15 minutes. Mix the cuvette's contents just prior
to each readings. Remember to use cuvtte 1 occasionally to check and adjust the
spectrophotometer to 100% transmittance.
15) Obtain the boiled chloroplast suspension, mix, and transfer 3 drops to cuvette 4. Immediately
cover and mix cuvette 4. Insert it into the spectrophotometer's sample holder, read the percentage
transmittance, and record it in Table 4.4. Replace cuvette 4 into the incubation test tube rack.
Take and record additional readings at 5, 10, and 15 minutes. Mix the cuvette's contents just prior
to each readings. Remember to use cuvtte 1 occasionally to check and adjust the
spectrophotometer to 100% transmittance.
16) Cover and mix the contents of cuvette 5. Insert it into the spectrophotometer's sample holder,
read the percentage transmittance, and record it in Table 4.4. Replace cuvette 5 into the
incubation test tube rack. Take and record additional readings at 5, 10, and 15 minutes. Mix the
cuvette's contents just prior to each readings. Remember to use cuvtte 1 occasionally to check
and adjust the spectrophotometer to 100% transmittance.

Results:
Table 4.1 Distance Moved by Pigment Band

Band Number Distance (mm) Band Color

1. 61 mm Dark Green

2. 54 mm Light green

3. 59 mm Faint Yellow

Distance Solvent Front Moved 66 mm.

Table 4.2 Rf Values

.89 = Rf for carotene (faint yellow to yellow-orange)

- = Rf for xanthophyll (yellow)
.81 = Rf for chlorophyll a (bright green to blue green)
.92 = Rf for chlorophyll b (yellow green to olive green)

Table 4.4 Transmittance (%)

 Cuvette 0 5 10 15

2 Unboiled/Dark 46.8 38.3 41.3 42

3 Unboiled/Light 42.6 46.4 24 22

4 Boiled/Light 56.5 84.8 8.5 7.3

5 No Chloroplasts 25.5 26 50.2 48.2

Analysis:

The solubility, size of particles, and their level of attraction to the paper are all involved in the separation
of pigments.The different solubilities of the pigments would change the Rf values. DPIP is the electron
acceptor in this experiment. DPIP substitutes for the NADP molecules. The electrons that reduce DPIP
come from the photolysis of water. The spectrophotometer measures the percentage of light transmit-
tance through the cuvette due to DPIP reduction. Darkness prevents the reduction of DPIP from occur-
ring. Boiling denatures the protein molecules and stops the reduction. In the dark cuvette, there was no
light energy available, so there was no flow of electrons and no photolysis of water, while in the lighted
cuvette these processes were allowed to continue.
Conclusion:
We discovered that the many pigments found in chloroplasts are all involved in gathering energy
from sunlight. The spectrum of color displayed on the filter paper showed the pigments and the solubil-
ity of each. In lab 4b, the spectrophotometer measured the light transmittance through the various cu-
vettes and the chloroplast solutions in each. This indicated whether photosynthesis was occurring and at
what rate. The cuvette with the unboiled chloroplasts that had been exposed to light showed the biggest
change in percent transmittance, which indicates that the amount of light available has a very big effect
on the rate at which the light reactions of photosynthesis occur. Errors could have occurred because of
damaged spinach, inaccurate reading, fingerprints on the cuvettes, or erroneous measurements.