Lab Section:




Textbook reading:


Overview of photosynthesis- p. 172

Photosynthetic pigments- pp. 173-174



Photosynthesis is a series of chemical reactions that convert radiant energy in the form of light into chemical energy in the form of sugar. Why is this important? Most plants are autotrophic; they synthesize their own food. Heterotrophs, organisms that cannot produce their own food, rely on plants to provide them with the food they need for energy. Through the process of photosynthesis, plants provide heterotrophs with oxygen and nutrients necessary for life. In addition to being a food source, plants help remove carbon dioxide from the atmosphere and provide oxygen in return. Photosynthesis can be summarized in the following chemical equation:


6 CO2 + 12 H2O light_+___> C6H12O6 + 6 H2O + 6 O2

Carbon water chlorophyll Sugar Water Oxygen



With light and chlorophyll present, plants are able to take in carbon dioxide and water and convert them to sugar, which is used immediately for growth or stored as starch; they then release water and oxygen into the environment.

In todayís lab you will identify the pigments involved in photosynthesis using paper chromatography, a spectroscope and fluorescent observations. In addition, you will observe the uptake of carbon dioxide during photosynthesis as well as starch production.




Part I. Photosynthetic Pigments

Plants have a variety of pigments that are necessary for photosynthesis to occur. These pigments, chlorophyll a and b, and accessory pigments such as carotene and xanthophyll, absorb light. The accessory pigments ultimately transmit the light energy they absorb to chlorophyll a.

We can easily separate these pigments by using a technique called paper chromatography. Pigment is applied as a spot to one end of the paper; that end is put into the solvent (just below the spot). The solvent moves up the paper, taking the pigment particles with it. The lighter (in weight), the more polar, and the more soluble the pigment particles are, the further they travel, along with the solvent, up the paper.

We can identify each pigment by determining its Rf (ratio of fronts) value. The Rf value describes the relationship between the distance moved by the pigment and the distance moved by the solvent and is calculated as follows:


Rf = __________Distance moved by pigment________

Distance from pigment origin to solvent front


Obtain a piece of chromatography paper from your lab instructor. Make a faint pencil (not pen) line across the paper about 2 cm from the tip of the paper. Using a glass capillary tube, apply a line of plant extract over the pencil mark. Donít use too much extract or it will smear. Blow on the strip until it is dry and repeat several times, making sure the extract is dry before reapplying. The idea is to make a concentrated line of extract.


Working under the hood, carefully pour about 2 ml of chromatography solvent into a large jar. This is a powerful solvent, so use in a well ventilated area. Place your chromatography paper into the jar with the pigment at the bottom. Be sure to allow the tip of your paper to touch the solvent, but donít cover the pigment with the solvent.


You may take the jar to your bench, but be sure to keep the lid on. Watch the edge of the solvent move up the paper; this is the solvent front. Remove the paper when the solvent front is about 2 cm from the top of the paper. Immediately mark where the solvent front is with a pencil and then allow the paper to dry.


How many different colors do you see? Describe them. _4; 2 shades of green, one yellow and one orange


Draw your results below:


Solvent front







Pigment extract





Now measure the distance between the pigment extract line and the solvent front line:____________ cm. Calculate the Rf values for each of the four pigments and record them in the table below. Use these values to identify each of the pigments (ask your instructor for the known Rf values).














What does a small Rf value tell you about the characteristic of the moving molecules?


______A small Rf value tells you that the pigment particles are less soluble in the solvent and tend to adhere more readily to the paper.








Which is more soluble in the chromatography solvent, xanthophylls or chlorophyll a ?






Why did the plant extract appear green if yellow xanthophylls were present?


___The green light reflected by the chlorophyll is a more dominant color that masks the color of the other pigments



Based on your observations during this experiment, what is responsible for the change in leaf color during the fall?


________During the fall, trees retract the chlorophyll from the leaves and store it in their roots. The other pigments are left behind and produce the oranges, yellows, and browns that we commonly see.





Next, you will be using an instrument called a spectroscope. A spectroscope can separate white light into its individual colors.


Look through the spectroscope and see the light spectrum from violet to red. Now insert a sample of plant extract between the light and the spectroscope.


How does the spectrum change? Some colors are missing while others are not as bright as before. _____________________________________________






Are there very faint colors or colors that are missing? _____________________________








Complete the graph below by estimating the relative absorbance of each color (whether the extract absorbed each color or let it pass through) by placing an X above the color name (located on the x-axis) at the correct absorbance along the y-axis. Connect the Xs to generate our spectrum.




















Based on your graph, which color of light probably contributes the least to plant growth?


_Green____ __


Remember that the color we see is the light being reflected not absorbed. Thus, the light reflected from the plant extract is green. Not all of the light, however, is reflected; some is absorbed and reemitted at a longer wavelength. This wavelength is determined by the structure of the molecule. The process is called fluorescence.


Obtain a tube of plant extract. Place it in front of a bright light and view it from the side. Alternatively, place it under ultraviolet light if one is available.


What color is being fluoresced? __red


Would you expect a chlorophyll extract to fluoresce blue light? Why or why not? ___No, the wavelength is of fairly high energy.






Part 2- Uptake of Carbon Dioxide During Photosynthesis

Recall that plants require CO2 to photosynthesize during the light dependent reaction. To detect the uptake of CO2 by a plant, you will use an indicator. An indicator is a solution that changes color depending on pH. Today you will be using phenol red (phenol sulfonphthalein). Phenol red turns yellow in acidic solutions (pH<7.0) and is red in neutral to basic solutions (pH>7.0).


In order to see this reaction take place, your breath will provide the CO2 to a solution of phenol red. This results in the following chemical reaction:



H2O + CO2 <-------> H2CO3 <-------> H+ + HCO3-

water carbon carbonic hydrogen bicarbonate

dioxide acid ion ion



By adding a plant to the solution, it can "fix" the carbon dioxide which will cause the pH to rise (become more alkaline). Your solution should then turn red.


Mark two test tubes with your initials. Fill them halfway with a solution of phenol red. Using a straw, gently blow into one of the test tubes, being careful to avoid splashing. Stop blowing as soon as your solution turns yellow; otherwise, the experiment will take longer because of the additional carbonic acid formed.


Add about 10 cm of Elodea (it can be in pieces) to the other test tube. Pour off any excess phenol red solution so that the solution just covers the Elodea.


Place both of the test tubes approximately 0.5 m in front of a bright light bulb for 30-60 minutes. Observe every 10 minutes.


NOTE: While waiting for this reaction to occur, read ahead. If time permits, you can begin the next experiment.


Has the color in either test tube changed? If so, why? _The tube with the elodea plant has turned back to red. This took place due to the fact that the plant was photosynthesizing and using up the CO2 that created the carbonic acid in the first place.








As mentioned above, CO2 is "fixed" by the plant. Using p. 186 in your textbook, explain what this means.















Part 3- Starch Production


The cell membrane of cyanobacteria, and the photosynthetic membranes called thylakoids in plants and algae, are the sight of photosynthesis during the light-dependent reaction. The thylakoid membranes are located within an organelle called a chloroplast. Thylakoids are stacked in columns called grana. The grana, in turn, are connected by lamellae. A fluid called the stroma surrounds the thylakoid membrane and provides the enzymes that catalyze the light-independent reactions.


When sugars are produced during photosynthesis, they are often stored as starch. Light energy is needed to split water molecules, providing the hydrogen ions and electrons needed for the rest of the reaction to take place. Chlorophyll is also required for photosynthesis to occur since this is where light energy is captured during the light- dependent step. However, it is during the light-independent step that the hydrogen ions and electrons gained during the light reaction are used to convert 5-carbon sugars into glucose and ultimately starch.


The following exercises will demonstrate the need for light and chlorophyll by detecting starch using iodine.


Obtain a Geranium leaf that has been half or completely covered with metal foil for several days. Sketch or describe its color in the table on the next page. Immerse the leaf in boiling water for about one minute (Wear goggles!). Carefully remove the leaf from the boiling water and place it in boiling ethanol for 3 minutes. Make sure the ethanol is not directly on the heat source.


Carefully remove the leaf from the ethanol and place it in a petri dish. Add five to eight drops of iodine and observe any change in color. Sketch or describe the color in the table below.


Now obtain a leaf of a variegated Coleus plant (these are the ones with white patches on the leaves). Sketch or describe its color pattern in the table below. Extract the pigments in the same way you did the Geranium , describing the color changes in the table.


Repeat the procedure using a Coleus with red leaves.



What does boiling the leaf in water do? In alcohol? ______________________________







Was there starch formation in the leaf that was covered? If so, was this what you expected?


_______Yes there was. I would not have expected that since the covered part of the plant was unable to photosynthesize and ultimately produce starch to be stored there. Maybe the starch was produced before the leaf was covered.













Before Treatment

Stained with Iodine


(leaf covered)






















(red leaves)
















Why do plants store starch? ____Starch is stored food that can be utilized later in respiration._____________________________________________







How would you use the information you learned in lab to describe to other people (maybe non-biologists) how plants function?