Photosynthesis Lab

Introduction

To survive, all organisms need energy. Plants get most of their energy from photosynthesis. Plants are autotrophs, that generate their own chemical energy from the product of the sun through photosynthesis. The chemical energy that plants get from the sun is used to produce glucose that can be burned in mitochondria to make ATP, which is then used to drive all the energy-requiring processes in the plant.

Photosynthesis occurs in plants in the chloroplast, an organelle specific to plant cells. In prokaryotes that perform photosynthesis, there are no chloroplasts; instead, photosynthesis occurs in association with the plasma membrane or infoldings of the membranes. Chloroplasts are found mainly in the cells of mesophyll, the green tissue in the interior of the leaf.

In the lab we replaced the electron-accepting coenzyme, NADP, with the blue DPIP dye. When DPIP was reduced, it had changed its color from blue to colorless. The DPIP reduction led to the decrease in light absorbance measured by the colorimeter.

Procedures

Plant Pigment Chromatography

1. Obtain and wear safety goggles and a lab apron.

2. Obtain a chromatography bottle with 5mL of solvent.

3. Cut the filter paper so that the bottom will touch the bottom of the bottle and still stick out of the top.

4. The normal length is about 15 cm.

5. Cut the end going into the solvent into a point

6. Draw a pencil line about 2 cm above the pointed end of the paper.

7. Use a coin to extract the pigments from the spinach leaf.

8. Place a small section of the leaf on top of the pencil line.

9. Use the ribbed edge of the coin to push the plant cells into the chromatography paper.

10. The more times you do this, the better.

11. Place the pointed end of the chromatography paper into the solvent and make sure the pigment is not in the solvent.

12. Place the lid on the chromatography bottle and let the paper sit until the solvent is 3 or 4cm from the top of the paper. Trace the pigment fronts and solvent fronts.

13. Allow the paper to dry. Measure the distance each pigment moved from the starting line to the top of the pigment band. Record the distance each of the pigments and the solvent moved, in millimeters.

14. Leave the solvent in the chromatography bottle.

Photosynthesis

1. Obtain and wear safety goggles and a lab apron.

2. Obtain two plastic Beral pipettes, seven cuvettes with lids, one 5cm x 5cm of aluminum foil, red cellophane, green cellophane, and blue cellophane, and cuvette lids.

3. Make a jacket for the dark, red, green, and blue cuvettes. One layer only and a small piece of the tape. Keep finger oils off as much as possible. Prepare a master solution of 12mL buffer, 18mL dH2O, and 12mL DPIP. Add 3.5mL of this solution to six cuvettes. In the seventh cuvette, add 2.5mL dH2O and 1mL buffer. Do not add the chloroplasts until the experiment starts.

4. Locate the unboiled and boiled chloroplast suspension prepared by your instructor. Draw up about 1mL of unboiled chloroplast and add 3 drop to every cuvette.

5. Set the lamp 45 cm from your cuvettes. Turn the lamp on but don't put the cuvettes in the light yet.

6. Take absorbance readings for each cuvette every 5 mintues or less. Record the exact times and note the change in minutes. Invert each cuvette two times to resuspend the chloroplast

7. Take 4 measurements of each cuvette, the experiment will usually end in 15 minutes. Be ready