Biology 203

Spring 08

Photosynthesis Introduction Photosynthesis is the process in which light energy is captured by plants and is used to build carbohydrates from water and carbon dioxide. Photosynthesis involves many reactions. These reactions can be grouped into a two-step process. •

Certain specific wavelengths of light are captured by pigments and converted to chemical energy in the pigments. The captured energy in the pigments is used in the light reaction to make two unstable energy-rich compounds, ATP and NADPH. In this process water is broken down and oxygen is released.

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The chemical energy in the ATP and NADPH is used in the Calvin Cycle to make stable energy-rich sugar using carbon dioxide and water.

Sunlight contains all the colors (wavelengths) of light, but plants can only make use of those colors absorbed by the pigments in the plant. In today’s lab we will investigate pigments involved in photosynthesis. During the lab you will extract pigments from spinach leaves and then separate the pigments using paper chromatography. You will then determine what wavelengths of light each pigment can absorb by making an absorption spectrum for each pigment. You will also run an experiment to determine the rate of photosynthesis of spinach leaf discs by indirectly measuring oxygen evolution.

Objectives: 1. Learn how to use differential extraction and paper chromatography 2. Learn how to determine an absorption spectrum of a pigment 3. Learn one simple way to measure the rate of photosynthesis 4. Know the difference between net and gross photosynthesis

Introduction to Spectrophotometry Every molecule has its own absorption spectra, i.e., it absorbs different quantities of different colors (wavelengths) of light. A spectrophotometer is often used to determine a molecule’s absorption (or transmittance) spectra. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector. If the sample absorbs some of the light less light will reach the detector. By only passing one wavelength (color) of light at a time through the sample it is possible to determine which wavelengths the sample absorbs. The absorption spectrum for that sample can then be drawn.

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Before beginning this lab your instructor will briefly explain more on how spectrophotometers work and explain their proper use.

Separation and Characterization of Pigments in Spinach Leaves Extraction of Pigments 1.

Tear up 3-4 leaves (discard petioles). Using about 15mls of acetone, grind gently for about 30 seconds. Discard the extract and keep the “pulp” (This gets rid of most of the water.) 2. Regrind the pulp vigorously with about 5-10 mls of acetone for one minute. Allow this to stand for several minutes and then filter this through cheesecloth. This is the extract that you will use for the paper chromatography.

Paper Chromatography of the Pigments 1. Obtain a piece of paper chromatography paper and draw a pencil line ¾ of an inch from the edge of the paper. 2. Using the extract that you obtained from the spinach leaves apply extract to the pencil line. Do NOT go right to the edge of the paper but avoid by ½-1 inch (if you get the extract out near the edge of the paper the extract will smear instead of running cleanly). Apply the extract with a small pasteur pipette by capillarity. After applying one thin line allow the extract to dry completely. 3. Repeat applying thin lines of extract about 20 times, ALLOW THE EXTRACT TO DRY ON THE PAPER EACH TIME BEFORE APPLYING A NEW LINE.

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4. After allowing the paper to dry completely insert it into the solvent with the EXTRACT LINE TOWARDS THE BOTTOM BUT WITH THE LINE ABOVE THE SOLVENT (pet ether: acetone, 9:1). Cover. 5. Watch the solvent front run up the paper. Do NOT let the solvent run off the paper. This should take anywhere from 10-20 minutes. When the solvent has run up the paper you should be able to see several distinct bands. The top band, at the solvent front is carotene (yellow), next are two yellow bands, each is a mixture of several different carotenes and carotenoids. Underneath those bands come two green bands—the top is chlorophyll a and underneath should be chlorophyll b. 6. Please draw and label the results from your paper chromatography on the question sheet at the end of this lab. Please label the bands. (Keep the chromatography paper out of the light to prevent photo-oxidation of the pigments.)

Absorption Spectrum Each table will make an absorption spectrum for one pigment (or group of pigments); chlorophyll a, chlorophyll b or the carotenes. To get enough pigment each table should make two paper chromatograms. 1. Turn on your Spec 20 and allow it to warm up as you obtain your pigment 2. Cut out the pigment band from your chromatogram. Cut it into small enough pieces to fit into a test tube and add 5-6 mls of acetone. Agitate until most of the pigment comes off the paper and goes into solution in the acetone. If the color of the solution is very faint, obtain the same band from another group, cut it into pieces and add it to the acetone. 3. Pour the pigment into a cuvette. You will need at least 4 mls of solution to get proper readings. 4. Read the instructions for the proper use of the spectrophotometer below. Use those instructions to finish 5 and 6. 5. Before doing the entire absorbance spectrum you will want to know if your pigment solution is too concentrated or dilute for this machine to read accurately. The absorbance value should not be above 0.8 or below 0.3. If the reading it too high, dilute the solute with more acetone. If the reading is too low you will need to get another group’s pigment band and elute it into your solution. To check for correct concentration read the absorbance at the wavelength listed: Chlorophyll a 430 nm Chlorophyll b 450 nm Carotenoids 440 nm 6.

Measure and record the absorbance at 25 nm intervals between 400 and 700 nm. Remember to BLANK THE MACHINE TO ZERO ABSORBANCE AT EVERY WAVELENGTH.

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Instructions for the Spectrophotometers: 1) Allow 15 minutes for the machine to warm up 2) Using the % transmittance knob (the power knob) set transmittance to zero. You should not need to reset this again for the rest of the lab. 3) Set the correct wavelength. This is the knob on the top of the spec. 4) If your spectrophotomer has a filter that you need to adjust (bottom left side) make sure this is set to the correct wavelength. 5) Insert the blank. This is a cuvette that includes the same solvent as the sample. Using the 100% T/ 0A knob, set the absorbance reading to zero. This is called “blanking” the machine. Blanking subtracts the amount of light that is absorbed or transmitted by the solvent. 6) Insert sample. Read and record the results. For this lab please record results in absorbance. 7) You must blank the spectrophotometer every time you change the wavelength. Each group must make an absorption spectrum and attach it to one of the group members questions sheet. Wavelength will be on the X-axis and absorbance on the Y-axis.

Measurement of Photosynthesis Using a Leaf Disc Assay Discs made from spinach or bean leaves will float in a water solution because of the air (approximately 80% N2) spaces in the leaf. If the air is removed they will sink. If those air spaces are refilled with oxygen they float. In this experiment you will investigate photosynthesis by measuring how quickly the leaves re-float (i.e., how much oxygen has been evolved) after being vacuum infiltrated with a solution. There is a complication. Oxygen is constantly being consumed as a part of cellular respiration. Measuring the rate that the leaves float therefore does NOT show the rate of photosynthesis, it shows the rate of photosynthesis in excess of respiration, i.e., the amount of oxygen evolved from photosynthesis that is not being consumed by respiration or photorespiration. This is referred to as net photosynthesis. To determine the rate of gross photosynthesis the rate of respiration must be determined. The rate of net photosynthesis is described by the equation below. Rate of Net Photosynthesis = Rate of Gross Photosynthesis - Rate of Respiration

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Measurements 1. Obtain three 10 ml syringes and label them. Remove the plungers and wrap the needle ends with parafilm. Fill two of the syringes with 10 mls of sodium bicarbonate solution (a source of CO2). These will be syringes 1 +2. Fill the third syringe with water. 2. Obtain one or two turgid spinach or bean leaves. Using a straw, punch out 24 leaf disks. Avoid cutting through areas with large veins. Put 8 disks into each syringe. Put the plunger back in the syringe and vacuum infiltrate the discs. Your instructor will show you how. Do this with all three syringes. 3. Wrap syringe #1 in aluminum foil. 4. Stand syringes #2 and 3 in the test tube rack in front of the lights. 7. At two-minute intervals for 30 minutes (or until all the discs float) count the number of discs floating in each syringe. 8. After 9 discs have floated in syringe #2 wrap that syringe in aluminum foil. Check for how many discs are floating every two minutes. (Try to avoid letting too much light fall on them). 9. Make a table and record your results. Determine the rate of net photosynthesis for the three treatments. To do this, calculate the number of minutes required for half the discs to float. This is an arbitrary number but can be used to compare treatments.

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Please answer the following questions.

Name______________________

Please draw in and label the results from your paper chromatography.

1) When making an absorption spectrum, why must you re-blank at every wavelength?)

2) To determine which color of light land plants can use you should check the absorption spectrum of each of the different pigments. Why?

3) If you did an absorption spectrum for all the pigments in the red algae, do you think it would be the same as the combined absorption spectrum for all the land plants? Why or why not.

4) When performing the leaf disc experiment, how do you know that there was photosynthesis in excess of respiration?

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5) Even if discs did not float does this mean that there was NO photosynthesis? Explain.

6) Using the floating disc technique, how do you think you can determine the rate of respiration?

Each person must hand in their own lab questions, but only one absorption spectrum per group needs to be turned in. Please attach the absorption spectrum your group made to one of the pages with the questions and put all group members’ names on the front of the absorption spectrum.

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