Chromatography and PhotosynthesisSamantha A. Price

AP BiologyLab # 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 extracts5) 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

1. Blank (no DPIP)

2. Un-boiled Chloroplast dark

3. Un-boiled chloroplast light

4. Boiled chloroplast light 5. NO Chloroplast light

Phosphate buffer 1 mL 1 mL 1 mL 1 mL 1 mLDistilled H2O 4 mL 3 mL 3 mL 3 mL 3 mL+3DropsDPIP None 1 mL 1 mL 1 mL 1 mLUn-boiled chloroplast 3 drops 3 Drops 3 Drops None NoneBoiled 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

BandDistance

(mm) Band Color Rf Pigment NameSolvent Front 45 None None None

1 22 Lemon yellow 0.48 Xanthophyll2 25 Pea green 0.55 Chlorophyll b

3 35 Mint green 0.77 Chlorophyll a4 42 Ash gray 0.93 Chlorophyll b5 43 Charcoal gray 0.95 Chlorophyll b

Blue-Green Algae

BandDistance

(mm) Band Color Rf Pigment NameSolvent Front 50 None None None

1 33 Tan 0.66 Carotene2 34.5 Olive green 0.69 Chlorophyll b3 35 Blue-green 0.7 Chlorophyll a4 39 Golden orange 0.78 Carotene5 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 39.5 423 un-boiled/ light 33 65 54 63

4 boiled/ light 44 45 45 496 no chloroplasts/ light 54 54 54 59

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 sheet2) Class discussions3) 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 pigmentb. Chlorophyll b – also catches lightc. Carotene – get energy from other wave lengthsd. Xanthophyll – get energy from other wave lengthse. Lycopene - get energy from other wave lengths

Photosynthesis:1) What is the function of DPIP in this experiment?

a. To accept electrons2) What molecule found in chloroplast does DPIP “replace” in this experiment?

a. NADP3) 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. 2i. 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?)