Lab 6: Using spec20's to measure the Hill Reaction

Here is this week's lab:

The process of photosynthesis involves the use of light energy to convert carbon dioxide and water into sugar, oxygen, and other organic compounds. This process is often summarized by the following reaction:

6 H2O + 6 CO2 + light energy --> C6H12O6 + 6 O2

This process is an extremely complex one, occurring in two stages. The first stage, called the light reactions of photosynthesis, requires light energy. The products of the light reactions are then used to produce glucose from carbon dioxide and water. Because the reactions in the second stage do not require the direct use of light energy, they are called the dark reactions of photosynthesis. In the light reactions, electrons derived from water are “excited” (raised to higher energy levels) in several steps, called photosystems I and II. In both steps, chlorophyll absorbs light energy that is used to excite the electrons. Normally, these electrons are passed to a cytochrome-containing electron transport chain. In the first photosystem, these electrons are used to generate ATP. In the second photosystem, excited electrons are used to produce the reduced coenzyme nicotinamide

adenine dinucleotide phosphate (NADPH). Both ATP and NADPH are then used in the dark reactions to produce glucose. In this experiment, a blue dye (2,6-dichlorophenol-indophenol, or DCIP) will be used to replace NADPH in the light reactions. When the dye is oxidized, it is blue. When reduced, however, it turns colorless. Since DCIP replaces NADPH in the light reactions, it will turn from blue to colorless when reduced during photosynthesis.

OBJECTIVES

In this experiment, you will

• Use a Spec20 to measure color changes due to photosynthesis.

• Study the effect of light on photosynthesis (in a QUANTITATIVE way)

MATERIALS

Phosphate buffer

3 cuvettes

Isolated chloroplasts

3 disposable pipets

Aluminum foil

DCIP

Lamp

Large Erlenmeyer Flask, filled with water

Ice

Watch/clock

PROCEDURE

IT IS REALLY IMPORTANT THAT YOU ARE MINDFUL OF ANY LIGHT WHEN DOING THIS EXPERIMENT. IF CHLOROPLASTS ARE EXPOSED TO LIGHT, THEY WILL START TO REACT.

1. Hold the cuvette above the white circle only. If you touch the glass below the circle, make sure to wipe the glass clean with a Kim wipe.

2. Create a blank. This is what you will use to zero the spec20. (You will be told the amount of Phosphate Buffer and DCIP to add in lab).

3. Follow your instructor’s directions for zeroing the Spec 20. These pieces of equipment cost $1500 each. BE GENTLE WITH THEM. The Spec20’s will be on the dark side of the room
   

4. Obtain a piece of aluminum foil large enough to wrap around the cuvette to block out any light. You will use this in step 6.

5. While on the dark side of the room, create a cuvette containing ___ drops of chloroplast solution (again you will be told how much in lab), ____mL DCIP and ____mL of Phosphate Buffer. Put this in the Spec20 and write down the transference and absorbance in your data table.

6. After completing the test wrap the cuvette in foil.

7. Each member of the group should create an experimental sample using ___drops isolated chloroplast solution, ___mL of Phosphate Buffer and ____mL of DCIP.

8. Test the initial absorbance and transference of each sample and record in the data table.

9. Bring the EXPERIMENTAL test tubes to the light side of the room. Do not turn on the light until instructed to do so by your teacher.

10. Set up the cuvettes as instructed by your teacher.

11. Every ten minutes, turn off the light and take one sample at a time over to the dark side to test in the Spec 20. Record your data. Return the sample and obtain another one. Continue to record data every ten minutes for each sample until you are instructed to stop.

12. Conduct a final Spec 20 test on the sample that was left in the dark.

13. Clean up your materials thoroughly. Place the contents of the cuvettes in a waste beaker at the teacher station.

PreLab Questions

1. What is the control in this experiment?

2. What is being tested?

3. How does DCIP work?

4. What does a color change in DCIP represent?