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LAB 6: PHOTOSYNTHESIS Introduction Some organisms are autotrophic or self-feeders. The largest group of the autotrophs are the photosynthetic organisms. Photosynthesis is made possible through the unique characteristics of pigment molecules like the chlorophylls. In today’s lab we are going to study the properties of the photosynthetic pigments as well as examining the gas flux during photosynthesis. A simplified overall reaction of photosynthesis looks like this:
6 CO2 + 12 H2O + sunlight [solar energy] à C6H12O6 + 6 H2O + 6 O2
Photosynthesis involves two sequences of reactions. The first (the “photo” part) harnesses light energy from the sun to excite and “bump” electrons from special pigments in complex arrays of molecules called photosystems. The second portion of photosynthesis (the “synthesis” part) involves a circular sequence or cycle of reactions. This takes the ATP and reduced nucleotide coenzymes from the first part and “fixes” or secures CO2 to produce carbohydrates (sugars) like glucose. Pre-Lab Questions 1. How do heterotrophs differ from autotrophs?
2. What is Paper Chromotography?
3. What is an Absorption Spectrum?
4. What is Elodea?
5. What is fluorescence and what does it have to do with photosynthetic pigments? Procedures: A. Where do plants conduct photosynthesis? In this simple exercise you will locate and observe the chloroplast organelles in plant cells.
1. Remove a single leaf from the growing end of an Elodea water plant and place it on a microscope slide. Add a drop or two of distilled water and place a cover slip over the leaf.
2. Observe the leaf under low power (10X objective). Note the color, number, and size of the chloroplasts. You may also notice that the chloroplasts are moving around a large central water vacuole in the cell. This movement is the result of cytoplasmic streaming.
3. Look for details of chloroplasts, such as the grana of thylakoid membranes. B. What photosynthetic pigments do plants use? Chloroplasts contain several photosynthetic pigments. The major ones are chlorophyll a, chlorophyll b, and the carotenoid pigments. In this simple exercise you will extract and separate these pigments to observe them on a sheet of chromatography paper.
1. Select any of the leaves provided. Record the name of the plant species (if known).
2. Fold a piece of chromatography paper (8 cm x 15 cm) the long way and then open the fold.
3. Cut your leaf into thin strips and, using the handle of the scissors, rub the leaf pigments into the paper about 1.5 cm from the bottom of the paper on each side of the fold (but only on one side of the paper). Rub the leaf into the paper at least five times for each side of the fold.
4. Print your name in pencil on the top edge of the paper and stand the folded paper in the chromatography chamber. The chromatography solvent (usually a 9:1 mixture of petroleum ether and acetone) is toxic, so do not breathe it and keep the chamber covered at all times.
5. Leave your paper in the chamber until the solvent approaches the top of the paper but does not go over it. This should be about twenty to thirty minutes, but may vary—watch closely.
6. Examine the chromatogram to distinguish the pigments. Carotenes and xanthophylls (both carotenoid pigments) are yellow-orange and pale yellow, respectively. Chlorophyll a is a dark bluish-green, whereas chlorophyll b is a lighter yellow-green.
7. Using colored pencils, sketch your chromatogram. Label the photopigments present.
C. What wavelengths of light work best for photosynthesis? In this exercise you will prepare an extract of photosynthetic pigments and determine which wavelengths of light these pigments absorb and which they reflect.
1. Obtain some leaves (spinach or other available leaves) and cut them into small pieces.
2. Crush the leaves in a mortar and pestle with a little sand and 10ml of 80% acetone.
3. Vacuum filter the resulting solution through filter paper with a vacuum apparatus. Your resulting extract should have photopigments suspended in acetone (with no sand, of course).
4. Using a spectrophotometer, measure the absorbance of your extract at 25 nm intervals starting at 350 nm and ending at 700 nm. Record all absorption data!
5. Prepare a plot of your spectrophotometry data to create an absorption spectrum, with wavelength on the x axis and absorbance on the y axis. If using a computer program, you will get a clear graph by making a semi log plot.
IMPORTANT: Do not discard your pigment extract!! You will use it in the next exercise.
D. How do photosynthetic pigments capture light energy? Photopigments are excited by light energy, which is what drives all of photosynthesis. In this exercise you will see a simple demonstration of how the pigment molecules capture light energy.
1 Hold your tube of photopigment extract (from exercise C) under an ultraviolet (“black”) light. Notice that the fluid appears wine-colored. What you observe is excitation of pigment electrons, but since these pigments are no longer on membranes there is no place to pass this energy (excited electrons), so the energy is given off as visible (red) light. This is called fluorescence.
2. Take your chromatogram (from exercise A) to a UV light and look at the various pigments. Do any show fluorescence in this separated state? How do you know? Do you get the same red color as with the tube of pigment extract?
Materials:
Elodea Distilled water Slide/cover slip Compound Scope Coleus, geranium, zebrina Chromatography paper cut into strips 2 inches wide 6 inches long Chromatography solvent (petroleum ether:acetone 9:1 soln) Chromatography chambers (1000 mL beakers with watch glass) Pencils Colored Pencils Spinach Leaves Mortars/pestles Sand 80% acetone Graduated cylinders 25 mL Vacuum apparatus for sink with Erlenmeyers, vacuum adapters, hosing Spectrophotometer Spec tubes Kimwipes Small plastic funnels Small scissors Test tube rack Filter paper UV light Power lab systems with oxygen and carbon dioxide sensors
REFERENCES/SOURCES:
Helms, D. R., C. W. Helms, R. J. Kosinski, and J. R. Cummings. 1998. Biology in the Laboratory, 3e. New York: W. H. Freeman.
Kull, R. C., Jr. 1992. Revised Laboratory Manual to Accompany The Nature of Life (Postlethwait & Hopson), 2e. McGraw-Hill.
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