Photosynthetic Pigments and Fall Foliage Color Changes

Transcription

1 Photosynthetic Pigments and Fall Foliage Color Changes Objectives: 1. To determine the pigments responsible for leaf color. 2. To investigate differences in leaf color within and between trees. I. BACKGROUND MATERIAL During autumn in Vermont, tree leaves "paint the landscape" with awe-inspiring colors. While some plants exhibit a single shade of color in the fall, such as birch and aspen that have yellow leaves and sumacs that have deep red leaves, other species can have multiple color signatures, such as maples that dazzle with red, orange, and gold, and ashes that show maroon and yellow. The colors of maples and ash, among others, can vary considerably from one locality to another, or even from one leaf to another, depending on the combination of pigments present in the fall leaves. In this experiment, you will separate out the pigments in leaves by paper chromatography, and then measure the quantity present by spectrophotometry. Through chromatography, individual pigments are isolated from the many other substances found in living tissues. Once separated, the amount of pigments present can be determined with spectrophotometry, which measures the light absorbed by a given substance. Figure 1: The green pigments of leafy material are underlain by yellows and reds.

2 A. PHOTOSYNTHESIS Photosynthesis n: A process by which green plants and other organisms produce simple carbohydrates from carbon dioxide and hydrogen, using energy that chlorophyll or other organic cellular pigments absorb from radiant sources. Photosynthesis is the most important series of chemical reactions on earth. Without photosynthesis, life as we know it would not exist. It is a complex chemical process that converts radiant energy (light) to chemical energy (sugar): Light + 6 CO H 2 O C 6 H 12 O 6 (sugar) + 6 H 2 O + 6 O 2 B. PIGMENTS Pigment n: A natural substance in plant or animal tissue that absorbs light and gives the tissue its color) The chloroplasts (those photosynthesizing organelles) of mature leaves contain several groups of pigments: Chlorophylls chlorophyll a grass green chlorophyll b yellow-green Carotenoids carotenes orange/yellow xanthophylls pale yellow Each of these pigments plays a role in photosynthesis. Research suggests that the large amounts of chlorophylls and their intense green color usually hide the presence of the carotenes and xanthophylls. Most plants regularly destroy and re-synthesize their chlorophyll during their growing season, but as the fall progresses, the rate of chlorophyll synthesis lags behind that of its breakdown. The decreasing amounts of chlorophyll no longer mask the other pigments, and the fall color change begins. (Additional color may also occur due to an increase in production of two other common pigments, anthocyanin or betacyanin. These water-soluble pigments are non-photosynthetic and are not present in the chloroplasts, Figure 2: A fiddlehead, photosynthesizing early in the spring, shows through dead oak leaves that have lost their pigment.

3 but instead are localized in vacuoles, especially in epidermal cells.) Eventually the leaf cells break down and die, and the leaves eventually turn a shade of brown or tan. This browning is due to a reaction between leaf proteins and tannins stored in the cell vacuoles. (This is akin to the tanning of animal hides that produces leather, in which tannins react with proteins.) C. PAPER CHROMATOGRAPHY Chromatography n: A method for determining which chemical components a gaseous or liquid mixture contains. It involves passing it through or over a medium that absorbs the different components at different rates. Separation of a compound from others present in a given tissue can be done with a simple piece of paper! Pigments have an affinity for paper, and are also easily dissolved in solvents. This technique takes advantage of these two facts. Paper is made up of cellulose, which has many -OH groups present. These groups hydrogen bond to other hydrophilic groups, and as a result many substances (like chlorophyll) hydrogen bond to cellulose. Yet, pigments can be dislodged from their cozy hydrogen bonds if a solvent is present. If we apply a tissue extract of pigments to paper and then wet the paper gradually with a solvent, a pigment molecule can be displaced slightly from its original position. The pigment will migrate over the paper as the solvent flows over it. The other pigments present in the tissue extract also bind to the cellulose, but with different affinities since different types of pigment molecules have different chemical structures, sizes, polarities and solubilities. Therefore, the substances in the mixture separate: some are slightly soluble in solvent and don't migrate very far on the paper, while others are more soluble and migrate farther, separating from each other. II. Context for the Exercise Recall the components of the Scientific Process: The Observations behind this exercise include: Plant leaves are various shades of green while they are photosynthetic but often change to other colors as the autumn season progresses. This leads to two basic questions: Why are tree leaves green? What changes occur when leaves change color? You should now try to rephrase these questions as testable Hypotheses, which include an aspect of the mechanism of color production, using the background material discussed above. You will use the experimental techniques of paper chromatography and spectrophotometry to test these hypotheses as they relate to specific trees on campus. See methods, section III. Record your results in self-explanatory, legible tables and graphs. What inferences can you make from your own results or from compiled results?

4 What you will do in this experiment: Week #1: Selection of research tree, sampling and ESIQ. You and your partner will select a tree as your test specimen. Using the basic observation, question and hypothesis outlined above, plan a set of experiments to test your hypothesis and predictions concerning why your tree is green. In the first week you will sample leaves and learn the basics of ESIQ: Extraction, Separation, Identification and Quantification of the pigments responsible for most leaf color. Week #2: Measuring differences in leaf color. Plan a set of experiments that address the observation, question and hypothesis concerning why leaf color differs within your tree between seasons. III. METHODS 1. PIGMENTS Routine laboratory procedures including paper chromatography and spectrophotometry can be used to (E) extract, (S) separate, (I) identify, and (Q) quantify leaf pigments - ESIQ. Procedure for ESIQ: A. Chromatography 1. Each group of students will collect leaves to analyze for pigments. 2. Weigh approximately 5 grams of fresh leaves. Record the actual weight! Chop the leaves into small pieces, and place some in a chilled mortar. Add 10 ml of cold acetone, (a *small* pinch of sand may help the grinding process) and then grind the leaves with a pestle. After you have partially ground the first batch, add the remaining chopped leaf. As you grind you will need to add acetone in small amounts (5-10 ml each time) as the mixture in the mortar dries out. Ideally, you would extract all the pigments in the leaf, and leave the pulp colorless (Why?). This would be very difficult with your equipment, but do the best you can. The pulp will begin to look faded, and that s good enough. You can pour off extract as you work (see number 3. below), and add fresh acetone. You should create about 20 ml of extract. 3. Pour off the liquid extract from the mortar into a 50 ml graduated cylinder being careful to get as little pulp in the cylinder as possible. Rinse the pulp in the mortar with a small amount of acetone, and add this to the graduated cylinder. Place the extract in an ice bath allowing any solids to settle for 5 minutes. Be sure to record the total volume of liquid extract. 4. Draw a light pencil line across the chromatography paper 2 cm from the bottom. Remove 1.0 ml of extract from the graduated cylinder using a pipettor, and put it in one of the

5 vials provided. Using the glass pipette, apply the extract carefully and as evenly as possible along the pencil line. Make sure you don t tear the paper by dragging the pipette tip aggressively. Allow the paper to air-dry before applying each successive sample. The tighter and neater the line of pigment you can make on the paper, the better the chromatography will work! That s why you should practice using the glass pipette first. You should apply all of the 1.0 ml to the origin using as many partial applications as required. Record the volume applied. The line of green tissue extract is called the origin. 5. Roll the paper into a tube, with the origin on the bottom, hold edges 1/8" apart, and staple. Do not let the paper overlap! Open the chromatography tank as briefly as possible, and only in the hood. Place the tube in the chromatography tank. The tank contains 50 ml of 10% acetone/90% petroleum ether solvent, which is 1 cm deep. (Solvent must not cover the origin.) Cover tightly. 6. The solvent will travel upward, wetting the paper. This process is called irrigation. Allow the solvent to irrigate until the solvent front is 1-2 cm from the top of the paper (this should take about 15 minutes). Remove the chromatogram from the tank, mark the solvent front and let the paper dry in the hood. Cylinder of chromatography paper Front as solvent moves Pigments loaded on Pencil line Solvent Figure 3: Roll of chromatography paper in place, absorbing solvent. 7. Locate the chlorophyll a and b bands and the xanthophyll and carotene bands. (Note: In this procedure, you have extracted only non-polar photosynthetic pigments. For example, the water-soluble anthocyanin pigments were not extracted and cannot be measured. Fortunately, these usually represent a small percentage of most leaf pigments.)

6 Front Golden yellow carotene Pale yellow xanthophylls Grass green chlorophyll a Yellow-green chlorophyll b Figure 4: Chromatography paper, unrolled, showing the movement fronts of pigment. 8. Cut out each band of separated pigment as carefully as you can, and place each band in a vial with the correct labeled top to which 10 ml of acetone has been added. You need to include all the paper with any particular color on it in the vial since the color is the extracted pigment you are after. The acetone will dissolve the pigment in about 5 minutes. Note: Some species may contain more than four pigment bands. B. Spectrophotometer 1. Refresh yourself on the procedure for using the spectrophotometer they are delicate instruments! IN PARTICULAR DO NOT SPILL ACETONE ON THEM! Acetone attacks many types of plastics! 2. Carefully swirl each of the four vials of acetone with dissolved pigment. 3. Set the wavelength of the spectrophotometer to 400 nm and blank (set the absorbance to zero) with a cuvette of pure acetone. 4. Transfer aliquots of the four well-mixed acetone and pigment solutions to cuvettes and measure the absorbance of each sample at 25 nm intervals from 400 nm to 775 nm. Blank with acetone each time the wavelength is changed. You can measure all four pigment samples at each wavelength. 5. Record your data in a table and plot it to produce an absorption spectrum for each pigment. What is the peak absorbance for each pigment? You can hone in on the true peak by going back and testing the extracts in 10 nm intervals right near the previously found peak. Once you know the peak absorbance for each pigment, you need only measure the absorbance at this wavelength for successive trials in week For each pigment, calculate the number of grams that were in 1 gram of leaf material: a. g of pigment / g fresh weight of leaf material. See section C. below.

7 C. Calculations For the calculations you will need the following information: Pigment Molecular weight Molar extinction coefficient (E) chlorophyll a = 894 g/mol 89 / mm cm (milimole/l)(cm) chlorophyll b = 907 g/mol 56 / mm cm carotene = 536 g/mol 2500 / mm cm xanthophyll = 568 g/mol 2500 / mm cm NOTE: don t miss the division symbol in these values of molar extinction! Absorbance, A, = peak absorbance measured. Path length, l, = 1 cm. In order to calculate the amount of each pigment in the original sample of leaf you ground up, there are several steps detailed below. You will need to do this calculation for each different pigment you isolated. Pay attention to the units for all the variables that you use! 1. Start by solving the Beer-Lambert equation (A=ElC) for concentration, C, which is expressed as molarity (M), or moles/l. 2. Using the absorbance value you measured at the peak absorbance wavelength, calculate a value of C (concentration) for each pigment. Can you see how the units cancel in the equation? 3. Multiply this value (C) by the molecular weight for that pigment to convert the units of concentration from molarity, to grams/liter. Be sure to pay attention to units! This value is the concentration (g/l) of pigment in your cuvette, and thus, the vial you extracted the strip of chromatography paper in. 4. Now you need to calculate the amount (g) of pigment in that vial. Multiply the concentration of the solution in the vial by the total volume in the vial (g/l x L = g - Watch your units!) This represents the amount of pigment in your vial, which is only a fraction of the total amount of pigment extracted from the leaf. Where did this pigment in the vial come from? It was on the chromatography paper. 5. Now you know the amount (g) of pigment on the chromatography paper. Where did the pigment that was on the paper come from? You added a certain volume of your raw extract to the paper. If you divided the amount of pigment you found in step 4 by this volume, you calculate the concentration of the pigment in the original raw extract (g/ml = concentration)! 6. No you know the concentration of the pigment in your extract. How do you calculate the amount (g) of pigment in that extract? Finally, you can calculate the % pigment in the original mass of leaves you extracted.

8 7. Eventually you should get the number of grams of pigment in the sample of leaves used. This value needs to be adjusted to express your final value as grams of pigment/ gram fresh weight of leaf material. IV. THOUGHT QUESTIONS These questions can be used to gain perspective on this experiment. 1. Do your individual results support or fail to support your hypotheses? Do the compiled results from the class support or fail to support the hypotheses? 2. Do the amounts of pigment you calculated for each pigment make sense? For instance, did you calculate that the leaf is 50% pigment by weight? Does that sound reasonable to you? 3. Can you identify the biggest sources of error in your experimental protocol? 4. Can you suggest alternative methods that might reduce these sources of error? 5. Can you use the absorption spectra that you created for each pigment to describe why the plant you extracted appears green?

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