. Describe how the functional units for beta carotene, xanthophyll, chlorophyll A, and chlorophyll B are different.

Pre-Lab Questions

1. Describe how the functional units for beta carotene, xanthophyll, chlorophyll A, and chlorophyll B are different. Be sure to identify the subunits that adhere to paper during chromatography.

2. Describe a technique for measuring photosynthetic rate.

3. Many deciduous trees have leaves which turn yellow in the fall. What do you suppose is happening in the leaves at the cellular and molecular level?

4. Chloroplasts and mitochondria are both are unusual in that they have double membranes and contain their own set of DNA. Can you think of any explanations for this observation?

Experiment 1: Paper Chromatography

In this experiment, you will separate plant pigments using chromatography. You will also measure the rate of photosynthesis in isolated chloroplasts using the reduction of the dye 2,6-Dichloroindophenol (DPIP) as the measurement tool. The transfer of electrons during photosynthesis reduces DPIP, changing it from blue to clear.

Materials 10 mL 4.5% Acetic Acid (Vinegar), C2H4O2 10 mL Acetone (Nail Polish Remover), C3H6O 30 cm Aluminum Foil (4) 100 mL Beakers (1) 250 mL Beaker 20 cm Cheesecloth 3 mL 1% 2,6-Dichloroindophenol, DPIP (1) 12 x 12 cm Chromatography Paper Piece (1) 100 mL Graduated Cylinder 10 mL Mineral Oil 3 mL 0.1 M Phosphate Buffer, P 8 Pipettes (1) Resealable Plastic Bag Rubber Band (Large; contain latex, handle with gloves on if allergic) Ruler Wooden Stir Stick	100 mL 0.5 M Sucrose Solution (cold!), C12H22O11 3 Test Tubes (Glass) Test Tube Rack *Cutting Board *Kitchen Knife *Light Source *Pencil *Quarter *Scissors **Spinach Leaves (Fresh) *Tape (masking or Scotch®) *Water *You Must Provide *Keep these leaves if performing Experiment 2 within a few days of Experiment 1.

Procedure

Part 1: Paper Chromatography

1. Place the bottle containing 100 mL of sucrose solution in the refrigerator. Allow the solution to rest here for the remainder of Part 1; it will be required in Part 2.

2. Use the permanent market to label each 100 mL beaker as 1, 2, 3, or and 4.

3. Add 10 mL water to Beaker 1, 10 mL acetone to Beaker 2, 10 mL mineral oil to Beaker 3, and 10 mL acetic acid to Beaker 4. Cover the beakers with aluminum foil, and set them aside.

4. Use a pencil to draw a line approximately one cm from the bottom (across the bottom edge) of each piece of chromatography paper. Then, carefully cut each piece in half so that you have four, pieces of identical size (each piece should have the pencil line going across the bottom edge of the paper).

5. Use a quarter to extract pigment from a fresh spinach leaf by placing the leaf in between the filter paper and the coin. Firmly rub the edge of the quarter back and forth over the pencil line drawn in Step 2. Use a fresh section of the leaf for each rubbing (Figure 5).

6. Place one piece of filter paper in Beaker 1, with the pigment-side towards the bottom of the beaker. Tape the filter paper to the beaker so that the bottom of the filter paper is submerged in the solvent, but the pigment line is not. DO NOT ALLOW THE PIGMENT TO TOUCH THE SOLVENT!

7. Monitor the set-up as the solvent travels up the paper. Remove the paper when the solvent is about 1 cm from the top of the paper. Immediately mark the solvent front and the location of each analyte band.

8. Measure the distance from the original pencil line to the solvent front, and the original pencil line to each of the pigments. Record the data in Table 1.

9. Calculate the Rf using the following equation:

10. Repeat Steps 6 – 9 for Beakers 2, 3, and 4. Be sure to use a new piece of filter paper for each beaker.

Rf =	distance pigment migrated (mm)
	distance solvent migrated (mm)

Part 2: Chloroplast Isolation

11. Obtain a knife and cutting board. Carefully cut across a few spinach leaves to isolate chloroplasts from the leaves.

12. Place the leaves in a resealable bag. Measure and pour 100 mL of the cold sucrose solution (prepared in Part 1: Step 1) into the resealable bag with the spinach leaves.

13. Remove any air from the plastic bag and seal completely. Mash the solution for 2 minutes.

14. Lay a piece of cheesecloth over a 250 mL beaker, and push the cheesecloth into the beaker approximately four to five cm with your finger. Secure with a rubber band.

15. Slowly pour the spinach/sucrose solution into the cheesecloth. Allow the cheesecloth to filter the solution for several minutes; or, until all of the liquid has passed through the cheesecloth.

16. Once the liquid has drained, discard the collected solids. You may wish to squeeze the cheesecloth to extract any remaining liquid before throwing it away if liquid appears to be caught.

Part 3: Photosynthesis

17. Use the permanent marker to label three test tubes as 1, 2, and 3. Place the tubes in the test tube rack.

18. Add 3 mL water, 1 mL phosphate buffer, and 1 mL DPIP to each of the three test tubes.

Figure 6: A heat-sink for test tubes placed in front of a light source.

19. Use the wooden stir stick to swirl the chloroplast solution (prepared in Part 2). Then, add two drops of the solution to Tubes 2 and 3.

20. Immediately cover Tube 2 with aluminum foil.

21. Place Tube 1 and Tube 3 in a sunny location or under a strong light. If you place the tubes in front of a light, fill a beaker or clear glass with water and position in between the light source and the test tubes as shown in Figure 6.

22. Monitor the test tubes for several hours, and record how long it takes each tube to turn from blue to clear. After the tubes exposed to light turn clear, remove the aluminum foil from Tube 2 and immediately note the color. Record your data in Table 2.

Table 1: Part 1: Chromatography Data
Solvent	Distance from Original Line to Solvent Front	Number of Bands	Rf Factor
Acetic Acid
Acetone
Mineral Oil
Water

Table 2: Part 3 – Photosynthesis Data
Test Tube	Time Required to Change Color
1
2
3

Post-Lab Questions

1. What did the different colored bands signify in each solvent for Part 1? What pigments can you associate them with?

2. What is the osmolarity fluid used in Part 2? Why is this important? Why is it essential to keep it cool?

3. How could you modify this experiment to show the effects of different wavelengths of light on the photosynthetic rate?

4. Some plants (grasses) tend to contain a greater concentration of chlorophyll than others (pines). Can you develop a hypothesis to explain this? Would it be testable?

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