Lab 4:Plant Pigments & Photosynthesis

Intro:

*Photosynthesis: when plant cells convert light energy to chemical energy which is stored in sugar and other organic compounds.

*Chlorophyll: primary photosynthetic pigment pigment in chloroplasts- crtitcal to photosynthesis.

*This lab contains 2 seperate activities: Plant Pigment Chromatography & Measuring the Rate of Photosynthesis

Plant Pigment Chromatography:

*Key Concepts:

-Paper chromatography: technique used to seperate a mixture into its component molecules. These molecules move up the paper at different rates because of differences in solubility, molecular mass, and hydrogen bonding with the paper.

• Example: draw a large circle in the center of filter paper with black water-soluble, felt-tip pen. Fold the paper into a cone and place the tip in a container of water. In a few minutes you will have tie-dyed filter paper.

The green, blue, red , and lavender colors that come from the black ink should help understand that what might appear to be a single color may be composed of different pigments.

*Design of the Experiment I:

-Pigments in paper chromatography are dissolved in a solvent that carries them up the paper. The solvent in the ink example is water. To seperate the pigments, you must use an organic solvent.

In this activity, you will seperate plant pigments using an organic solvent such as a mixture of ether and acetone. Make sure to keep the bottle tightly closed except for when using it because the solvent can be readily vaporized and produce fumes you shouldn't breathe.

*Depositing the Pigment:

-Deposit pigment by rolling a quarter over a spinach leaf about 15 times to make a heavy green line.

*Analysis of results I:

-If you would do many chromatographic seperations, each for a different length of time, the pigments would migrate a different distance. However, the migration of each pigment relative to the migration of the solvent would'nt change. This migration of pigment can be calculated by using this formula:

Measuring the Rate of Photosynthesis:

*Key Concepts:

-In the light reactions of photosynthesis, light energy excites electrons in plant pigments like chlorophyll and raising them to a higher energy level. These electrons reduce compounds in the thylakoid membrane, and the energy is eventually captured in the chemical bonds of NADPH and ATP.

-Using DPIP As an Electron Acceptor:

In this activity, you will measure the rate of the electron excitation when light hits chlorophyll. You will use DPIP, a blue compound, as an electron acceptor.

When light strikes the chloroplasts, the DPIP is then reduced by the "excited" electrons from chlorophyll and becomes colorless as it accepts the electrons. You wil use a spectrophotometer to measure the color change, which tells us the rate of the light reactions under various conditions.

-The Spectrophotometer: an instrument that can be adjusted to illuminate a sample with a specific wavelength of light. it measures the amount of light energy that has been absored or transmitted by the sample. As DPIP becomes colorless, the amount of light of wavelength 605 nm transmitted through the sample will increase. Even though you can see the color change, the spectrophotometer quantifies the change.

1. Close the lid on the empty sample chamber
2. Use the knob to select desired wavelength (605 nm)
3. Use th e"On-Off" knob to adjust the meter needle to 0% transmittance
4. Put the blank tube in the sample chamber and close the lid
5. Use the third knob to adjust tje meter to 100% transmittance
6. Put the sample in the sample chamber and close the lid
7. Without moving any knobs, read the % transmittance on the meter

*Design of the Experiment:

-After illuminating the reaction tubes, use the spectrophotometer to measure the percentage of transmittance at wavelength 605 nm.

-If DPIP is in an oxidized state it will appear blue and the percentage of the light transmitted will be low. If chlorophyll's electrons have been excited and reduce the DPIP, the sample will become paler allowing more light energy to pass through the sample. You can measure this change over time until the sample is almost colorless and the percentage of transmittance is high.

-In this experiment, one tbe will contain all solutions used except DPIP. since the tube contains chloroplasts, it wil be green. the other tubes will be experimental, containing either boiled or unboiled chloroplasts.

*Helpful Hints:

• Be sure to read the % transmittance IMMEDIATELY after adding the chloroplasts to each tube.
• Keep the test tubes clean!
• Mix each sample thoroughly before measuring
• Be sure to place all test tubes into the spectrophotometer in the same orientation each time so that variations in the glass do not mask your results.
• Maintain a constant distance from the light source to the sample for ALL tubes.

*Analysis of Results II:

-Print the graphs below and based on your understanding of light reactions, draw in the approximate shapes of the curves you predict.

-Expose the tubes to light and make an initail spectrophotometer reading at 5, 10, and 15 minutes.

*Some questions:

1. If a different solvent were used for the chlorophyll chromatography described earlier, what results would you expect? (activity I)

a)the traveling distances for each pigment will be different, but Rf values stay the same.

b)Relative position of bands will be different.

c)If time is held constant, results will be the same.

2.What is the role of DPIP in this activity II?

a)It does the same thing as chlorophyll by taking in light energy.

b)Its an elctron donor and stops the formation of NADPH.

c)Its an electron acceptor and is reduced by electrons from chlorophyll.