Chromatography and Photosynthesis

Samantha A. Price
AP Biology
Lab # 4
Introduction:

If chlorophyll is boiled or not exposed to light then no photosynthesis will be

preformed.
Pigments have different masses and also different wavelengths. The
chromatography aspect of this lab shows what pigments are in a sample of blue-green
algae and spinach. Factors that affect the outcome are the mass of each pigment, and its
affinity for the paper used, and how soluble the pigment is in the solvent.
Photosynthesis is a process in which light is used to make NADPH and ATP in plants.
Materials & Procedure:

Chromatography:
• Beakers, borosilicate, 50-ml, 2
• Blue-green algae extract, 0.5 mL
• Chromatography solvent, 4 mL
• Spinach extract, 0.5 mL
• TLC plate (sheet)
• Marker or wax pencil
• Pencil
• Pipet, Beral, graduated
• Pipets, Beral, thin-stem, 2
• Ruler
• Scissors
• Watch glasses, 2

1) Label two 50-mL beakers with your group name or number. Label one
beaker “spinach extract” and the other “blue-green algae extract”
2) Touching only the sides of the TLC sheet, use scissors to cut the TLC into
small plates approximately 6.5cm x 1.5cm. Be careful not to scrape any of
the silica gel of the plates-this will adversely affect results. Note: Some
silica gel may chip from the edges of plate-this is not a problem.
3) Using a pencil, draw a faint line 0.5cm from the bottom edge of each TLC
plate
4) Fill the stem of a thin-stemmed pipet with one of the extracts
5) Carefully dip one small drop of extract onto the center of the pencil line on
the TLC plate. Note: It is important to keep the spot centered and as small
as possible.
6) Blow gently on the spot to help the solvent evaporate.
7) Repeat steps 5 and 6 five to ten times in the same spot to create a dark spot
of extract on the TLC plate. Note: Too much extract on the initial spot will
cause the resulting pigment bands to broaden and tail. Too little extract
will create very faint bands that are difficult to analyze.
 8) Repeat steps 4 to 7 using the second extract and the second TLC plate.
9) Use a graduated pipet to transfer 2 mL of chromatography solvent into the
bottom of each 50-mL beaker.
10) Carefully place each TLC plate, sample end down, into the appropriate 50-
mL beaker. Note: The upper level of the solvent must not touch the sample
extract. If it appears that the sample will be within the solvent, take out the
sample before the TLC plate touches the solvent and remove some of the
chromatography solvent from the 50-mL beaker.
11) Carefully, place the watch glass onto the beakers. Note: Do not disturb the
TLC plate.
12) When the solvent front (top of the solvent) is 0.5-1cm from the top of the
TLC plate, remove it from the 50-mL beaker. Replace the watch glass on
top of the 50-mL beaker to prevent the solvent from evaporating.
13) Use a pencil to immediately mark the location of the solvent front before it
evaporates.
14) Mark the center of each pigment band.
15) Measure the distance each pigment migrated from the sample spot to the
center of each separated pigment band. Record the distance, in
millimeters, that each pigment and the solvent front moved on the
chromatography worksheet.
16) Calculate the Rf value for each pigment and record this value for each
pigment on the chromatography worksheet
17) Record the identity of each band on the chromatography worksheet.

Photosynthesis:

1) Teacher set up incubation site and spectrophotometer.

2) Get one beaker containing boiled and one containing un-boiled chloroplast,
being sure to keep on ice at all times.
3) At the top rim, label the cuvettes 1-5 respectively. Using lens tissue, wipe the
outside walls of each cuvette. Cover the walls and bottom of cuvette 2 with
 foil and make foil cap to cover top. Light should not be permitted inside this
cuvette because it is the control of the experiment.

Cuvettes
2. Un-boiled 3. Un-boiled 4. Boiled
1. Blank Chloroplast chloroplast chloroplast
(no DPIP) dark light light 5. NO Chloroplast light
Phosphate buffer 1 mL 1 mL 1 mL 1 mL 1 mL
Distilled H2O 4 mL 3 mL 3 mL 3 mL 3 mL+3Drops
DPIP None 1 mL 1 mL 1 mL 1 mL
Un-boiled
chloroplast 3 drops 3 Drops 3 Drops None None
Boiled Chloroplast None None None 3 Drops None

4) Be sure to follow direction on how to assemble cuvettes in above diagram.

Cover the tops with Parafilm and invert to mix.
5) Bring the spectrophotometer to zero by adjusting the amplifier control knob
until the meter reads 0% transmittance. Insert cuvette 1 into the sample holder
and adjust the instrument to 1--% transmittance by adjusting the light-control
knob. Cuvette 1 is the blank to be used to recalibrate the instrument between
readings. In other words, you will measure the light transmitted through each
of the other tubes as a % of the light transmitted though this tube. 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.
6) Remove cuvette 2 from its foil sleeve and insert it into the
spectrophotometer’s sample holder, read the % transmittance, and record it as
the time 0 reading on your data table. Put back into foil sleeve and place it in
the incubation test tube rack.
7) Do the same thing for the rest of the 5 cuvettes.
8) Put all the cuvettes in front of heat sink. And let sit until 5 min is up.
9) Record data for 0-15 min at 5 min intervals repeating steps 4-6 each time data
is recorded.

Results, Data Collection & Analysis:

Chromatography:
Spinach
Distance
Band (mm) Band Color Rf Pigment Name
Solvent Front 45 None None None
1 22 Lemon yellow 0.48 Xanthophyll
2 25 Pea green 0.55 Chlorophyll b
3 35 Mint green 0.77 Chlorophyll a
4 42 Ash gray 0.93 Chlorophyll b
5 43 Charcoal gray 0.95 Chlorophyll b

Blue-Green Algae
Distance
Band (mm) Band Color Rf Pigment Name
Solvent Front 50 None None None
1 33 Tan 0.66 Carotene
2 34.5 Olive green 0.69 Chlorophyll b
3 35 Blue-green 0.7 Chlorophyll a
4 39 Golden orange 0.78 Carotene
5 43 Light-yellow 0.86 Xanthophyll

Photosynthesis:

NOTE: Time is in minutes [#s 0-15 in 1st row]

Cuvette 0 5 10 15
2 un-boiled/ dark 358 37 39.5 42
3 un-boiled/ light 33 65 54 63
4 boiled/ light 44 45 45 49
6 no chloroplasts/ light 54 54 54 59
 Percent Photosynthesis Done

70

60

50
Transmittance %

40

30

20

10

0
1 2 3 4
Time (5 min intervals)

2 un-boiled/ dark 3 un-boiled/ light

4 boiled/ light 6 no chlloroplasts/ light

Linear (3 un-boiled/ light)

Discussion/ Conclusion:

The Hypothesis that we had was not confirmed or proven wrong. Our data was
not perfect from a few possible errors. The spectrometer may have been read incorrectly,
the cuvette with no chloroplast may have had some in it because it preformed some
photosynthesis, the boiled also was not boiled enough to denature all the enzymes
because that too did photosynthesis.

Literature Citation(s):

1) Lab sheet
2) Class discussions
3) Internet photos
Questions:

Chromatography:
1) What factors are involved in the separation of the pigments?
a. Solubility of the pigment in the solvent.
b. Density of the pigments (the more dense, the less movement).
c. Affinity for the paper (grater affinity, the less movement).
2) Would you expect the Rf value of a pigment to be the same if a different solvent
were used? Explain.
a. No, the solubility may be different and cause either more or less
movement in the pigments. It may also change density and that will
change results also.
3) Which pigment directly captures light energy? What are the roles of the other
pigments?
a. Chlorophyll a – main light capturing pigment
b. Chlorophyll b – also catches light
c. Carotene – get energy from other wave lengths
d. Xanthophyll – get energy from other wave lengths
e. Lycopene - get energy from other wave lengths

Photosynthesis:
1) What is the function of DPIP in this experiment?
a. To accept electrons
2) What molecule found in chloroplast does DPIP “replace” in this experiment?
a. NADP
3) What is the source of the electrons that will reduce DPIP?
a. Transmittance of light being shone through the sample. Directly
proportional to amount of photosynthesis being done.
4) What is the effect of darkness on the reduction of DPIP? Explain.
a. It decreases it because it needs light to excite the elections in order to
move them from the chlorophyll.
5) What is the effect of boiling the chloroplasts on the subsequent reduction of
DPIP? Explain.
a. It denatures all the enzymes that make the reactions possible stopping the
reduction of DPIP.
6) What reasons can you give for the difference in the percentage of transmittance
between the live chloroplasts that ere incubated in the light and those that were
kept in the dark?
a. Light accepts electrons so they can leave chlorophyll a and be bicked up
by DPIP.
7) Identify the function of each of the cuvettes.
a. 1
i. Act as a blank to compare all other cuvettes to (sets up the machine
to measure the color to DPIP).
b. 2
i. To see the effect of darkness on the light reaction in
photosynthesis.
c. 3
i. Effect of light.
d. 4
i. Control (Any reduction if chloroplast is denatured?)
e. 5
i. Control (Any reduction if no chloroplast?)