In order to demonstrate the process of photosynthesis, we will utilize a “virtual” lab. Please read instructions carefully. This activity is analogous to
AP Lab 4. Please be sure to not only read the info here, but on the website too! You will be held accountable for all bolded terms, laboratory
concepts and be able to apply data and questions given to you in a post-lab assignment once we do the real labs!!
Introduction- In this laboratory you will separate plant pigments using chromatography. You will also measure the rate of photosynthesis in isolated
chloroplasts. The measurement technique involves the reduction of the dye DPIP. The transfer of electrons during the light-dependent reactions of
photosynthesis reduces DPIP, changing it from blue to colorless.
Exercise 4A: Plant Pigment Chromatography
Paper chromatography is a useful technique for separating and identifying pigment and other molecules from cell extracts that contain a complex
mixture of molecules. The solvent moves up the paper by capillary action, which occurs as a result of the attraction of solvent molecules to the
paper and the attraction of the solvent molecules to one another. As the solvent moves up the paper, it carries along any substances dissolved in it.
The pigments are carried along at different rates because they are not equally soluble in the solvent and because they are attracted, to
different degrees, to the fibers of the paper through the formation of intermolecular bonds, such as hydrogen bonds.
Beta carotene, the most abundant type of carotenoids in plants, is carried along near the solvent front because it is very soluble in the solvent being
used and because it forms no hydrogen bonds with cellulose. Another pigment, Xanthophyll differs from carotene in that it contains oxygen.
Xanthophyll is found further from the solvent font because it is less soluble in the solvent and has been slowed down by hydrogen bonding to the
cellulose. Chlorophyll's contain oxygen and nitrogen and are bound more tightly to the paper than the other pigments. Chlorophyll a is the primary
photosynthetic pigment in plants. A molecule of chlorophyll a is located at the reaction center of the photo systems. The pigments collect light energy
and send it to the reaction center. Carotenoids also protect the photosynthetic systems from damaging effects of ultraviolet light.
Exercise 4B: Plant components and photosynthesis
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 were once called the dark reactions of photosynthesis. Today they are referred to as the
Calvin Cycle.
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 DPIP) 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 DPIP 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 Colorimeter or Spectrometer to measure color changes due to photosynthesis.
• Study the effect of light on photosynthesis.
• Study the effect that the boiling of plant cells has on photosynthesis. And compare the rates of photosynthesis for plants in different light conditions.
1.
2.
Please go to this website: http://www.phschool.com/science/biology_place/labbench/lab4/intro.html
Click on Lab 4 “Photosynthesis and Pigments” if you are not taken there directly!
You will be “performing” both 4A and 4B. As you read through each page, pay close attention to each underlined term and when ready click on
“next” to advance. If there is an option to “take closer look” please do that!
Once you have completed 4A, take the self quiz. ANY you missed should be recorded. Keep trying until you get a 100% and KNOW WHY all the
answers that are correct are correct.
Once you have gotten all the answers correct, click “next”.
Play the movie, but before moving on identify all of these items and their jobs:
A. Large green oval
B. Small light green ovals on the big oval
C. Purple ovals
D. Yellow squiggly arrow
E. NADP
F. ADP
Once you have watched the video, briefly explain what the red outlined, spastic star is. What is it used to actually do?
Before you move on, A) re-state where the H comes from to form NADPH. And B) What heterotrophic molecule is analogue to NADP? C) What is
different about it than the heterotrophic version? Click “next concept”
8. The idea is to confirm that both light and pigments and the structures in the chloroplast’s membrane are needed for photosynthesis to occur. To do
this we need a measurable way to see that light oxidizes pigments that can supply energy to make something. Watch the movie again and explain
what DPiP is and how and why it is used to support I idea.
9. What is a spectrophotometer? How will it be used to measure whether or not light reactions have run in photosynthesis?
10. Go through the experiment and make sure you understand what is happening!
11. Please draw and complete the graphs (3) as instructed. You will attach these to the actual lab questions you will be doing for credit.
12. Again, perform the quiz until you get a 100% and you know what and why all the correct answers are correct!
13. Name 3 specific things that can directly affect the rate of photosynthesis.
Lastly, please watch this video regarding transpiration video: https://www.youtube.com/watch?v=U4rzLhz4HHk
It’s imperative you understand this concept prior to lab we do on this. Be able to define:
Transpiration
Guttation
Review water potential
Osmosis
Diffusion
Root pressure
Review cohesion, adhesion
Xylem
Phloem
Epidermis
Once done please read through the following summation:
Some Key Review Concepts
In photosynthesis, plant cells convert light energy into chemical energy that is stored in sugars and other organic compounds. Critical to the process
is chlorophyll, the primary photosynthetic pigment in chloroplasts.
4A Chromatography - Paper chromatography is a technique used to separate a mixture into its component molecules. The molecules migrate, or
move up the paper, at different rates because of differences in solubility, molecular mass, and hydrogen bonding with the paper. In paper
chromatography the pigments are dissolved in a solvent that carries them up the paper. To separate the pigments of the chloroplasts, an organic
solvent must be used. In this experiment a drop of leaf extract is placed near the bottom of chromatography paper. This paper is then placed in a
solvent, which moves up the paper by capillary action
4B Photosynthesis -In the light reactions of photosynthesis, light energy excites electrons in plant pigments such as chlorophyll, and boosts them to
a higher energy level. These high-energy electrons reduce compounds (electron acceptors) in the thylakoid membrane, and the energy is eventually
captured in the chemical bonds of NADPH and ATP. The rate of photosynthesis in chloroplasts in measured using a dye called DPIP. DPIP
changes color when it accepts electrons that would normally be accepted by NADP, the electron acceptor of the light-dependent reaction of
photosynthesis. AS DPIP accepts electrons, it changes from blue to clear. Then a spectrophotometer is used to measure the color change, which
gives an indication of the rate of the light reactions of photosynthesis under various conditions.