Department of Zoology
Durham, North Carolina 27708-0325
Alexander Motten is an Assistant Professor of the Practice of Biology at
Duke University. He received a bachelor's degree in Botany from the University
of North Carolina at Chapel Hill (1970) and a bachelor's degree in Zoology
from the University of Washington (1976). In 1982 he received a Ph.D. in
Zoology from Duke University. Since 1984 he has taught in the introductory
biology program at Duke, where he is currently the director of the introductory
courses. He also teaches in the university writing program and conducts workshops
on ways to teach effective writing. His research interests are in evolutionary
ecology, especially pollination systems and plant reproductive biology.
|Reprinted from: Motten, A. F. 1995. Diversity
of photosynthetic pigments. Pages 81-98, in Tested studies for laboaratory
teaching, Volume 16 (C. A. Goldman, Editor). Proceedings of the 16th
Workshop/Conference of the Association for Biology Laboratory Education
(ABLE), 273 pages.
Although the laboratory exercises in ABLE proceedings volumes have been tested and due consideration has been given to safety, individuals performing these exercises must assume all responsibilities for risk. The Association for Biology Laboratory Education (ABLE) disclaims any liability with regards to safety in connection with the use of the exercises in its proceedings volumes.
Photosynthetic organisms (plants, algae, and some bacteria) rely on a variety of pigments to capture light energy from the sun. These colored compounds are particularly diverse in the algae, and their prominence in the biology of algae is indicated by the very names of many algal divisions. Indeed, photosynthetic pigments reveal much about the physiology of these organisms and their likely evolutionary relationships. In this lab students will extract photosynthetic pigments from red, brown, and green algae, as well as from a cyanobacterium ("blue-green alga") and from an angiosperm (flowering plant). They then separate the pigments by means of thin-layer chromatography and interpret the patterns they obtain to make ecological and evolutionary inferences using the information and questions at the end of the exercise as a guide.
Centrifuge, small clinical (1 per lab)
Flask, side-arm, fitted with long-stem funnel and attached to aspirator at sink (1 or 2 per lab)
Flask, side-arm, fitted with Buchner funnel and attached to vacuum line (1 or 2 per lab)
Jar for chemical waste to receive used chromatography samples and extracts (1 per lab)
Each group (46 students) will need the following:
Graduated cylinders, 10 ml (2 or 3)
Thin-layer chromatography (TLC) strips in glass jar or other lidded container (1520 strips in each of 2 or 3 jars)
Test tube racks (3)
Test tubes, 15 mm diameter (5/student)
Stoppers, #0, rubber (5/student)
Acetone in labeled 125-ml squirt (wash) bottle, with cap for nozzle (1)
TLC solvent in labeled 125-ml squirt (wash) bottle, with cap for nozzle (1)
Capillary tubes (1 vial with 810 tubes)
Spinach leaves, fresh (1/student)
Coins (2 or 3, optional)
Each group of students extracts pigments from a single alga for use by all groups in the class. Materials specific for each group are listed below.
Cyanobacteria (Spirulina) group
Red alga (Porphyra) group
Brown alga (Fucus, Ascophyllum, or Sargassum) group
Green alga (Ulva or Spirogyra) group
Pigment Extraction Procedures
Listed below are the procedures for extracting pigments from an assortment of species. Each lab group will prepare extracts from one kind of alga (or the cyanobacterium). For the red alga and Spirulina groups, each person in the group will carry out only one of the extraction procedures (either an aqueous or acetone extraction), as directed by your instructor. The non-polar (acetone) extracts will be shared with other groups to enable everyone in the lab to analyze a sample from each species.
The cyanobacteria contain both water-soluble (polar) and
water-insoluble (non-polar) photosynthetic pigments. Both types can readily
extracted from Spirulina, a marine species. Conveniently for our
purposes, it is cultivated in large saltwater ponds, where it is collected
and dried for sale in powdered form as a nutrient supplement.
To extract the polar (water-soluble) pigment of Spirulina proceed as follows.
To extract the non-polar (acetone-soluble) pigments of Spirulina proceed as follows:
Rhodophyta (Red Algae)
The red algae also contain both water-soluble and water-insoluble photosynthetic
pigments. These will be extracted from Porphyra (laver), an edible
To extract the polar (water-soluble) pigments from Porphyra proceed as follows:
To extract the non-polar (acetone-soluble) pigments from Porphyra proceed as follows:
Phaeophyta (Brown Algae)
To extract pigments from a brown alga you will use either Fucus or Ascophyllum, two intertidal seaweeds from cold waters, or Sargassum, a warm-water genus. Proceed as follows:
Chlorophyta (Green Algae)
As an example of the green algae, you will use either Ulva (sea lettuce) or a freshwater, filamentous species such as Spirogyra. Proceed as follows:
The acetone extracts contain a mixture of pigments, which you will separate by thin layer chromatography, or TLC. This process uses a thin plastic plate coated with silica gel. A sample of a mixture of compounds is deposited near the bottom of the plate, and the plate is then placed vertically in a suitable solvent. As the solvent is wicked up, it passes the sample and starts to carry the compounds upward with it. Different compounds dissolved in the solvent adsorb to the silica gel to different degrees; the more polar a molecule, the more strongly it is adsorbed. (Note: Adsorb means adhere to the surface of another compound without forming a chemical bond.) As a result, some compounds, the relatively more polar ones, remain near the bottom of the plate while other, less polar ones are carried by the solvent nearer the top.
Each student will make separate chromatograms from the acetone extracts of the cyanobacterium, red, brown, and green algae, and from leaves of a representative flowering plant, spinach. You will not make chromatograms from either of the aqueous extracts. Your instructor will use these for a demonstration when you are ready to interpret your results.
Figure 6.1. Diagram showing application of acetone extract to thin-layer chromatography (TLC) strips.
Identifying Photosynthetic Pigments
There are three broad classes of photosynthetic pigments: chlorophylls,
carotenoids, and phycobilins. Each class includes several pigments with similar
chemical structures. Different pigments absorb light at different wavelengths,
and hence differ in color. (Why?) Because they differ in chemical structure,
they also vary in their solubility in water and in their degree of polarity
and adsorbance to silica gel. This information is summarized in
Table 6.1. Characteristics of photosynthetic pigments.
|Pigment||Color||Water soluble?||Position on TLC strip*||Observed in:|
|xanthophylls||yellow, orange||no||moderate to low|
|chlorophyll a||bluish green||no||high|
|chlorophyll b||yellow green||no||slightly lower than chl. a|
|chlorophyll c||light green||no||very low|
* Note: A high position (nearer the top of the chromatogram) means the pigment adsorbs weakly to the silica, while a low position (nearer the origin) means it adsorbs strongly.
Sketch the banding patterns you obtained on your chromatograms onto the diagram in Figure 6.2. You should do this as soon as you remove the strips from the tubes because some of the fainter bands may fade quickly, especially under fluorescent lights. Compare your chromatograms with those of other members of your group, especially if any of your strips are faint or blurry. Using the information in Table 6.1, identify the kind of pigment responsible for each band. Label the bands you have sketched, then fill in the right-hand column of the Table 6.1. Your instructor will discuss the expected results with the class and give you additional information about the pigments.
Use Figure 6.2 and the summary of the distribution of
photosynthetic pigments in different groups of organisms in
Table 6.1 to help you answer the questions in the
Figure 6.2. Patterns of photosynthetic pigments on TLC strips for acetone extracts from five different sources. Sketch the bands you observed on your chromatograms, and label them based on the information contained in Table 6.1 and provided by your instructor.
Interpreting Patterns in Photosynthetic
Isolation of photosynthetic pigments from many species clearly shows that the pigments do not occur in different groups at random. Knowing this, you can use your chromatogram results to (1) infer evolutionary relationships among groups, (2) detect evidence of possible multiple origins of photosynthetic capability in eukaryotes, and (3) evaluate the ways different groups have solved the physiological problem of obtaining light for photosynthesis in different habitats. Refer to your chromatograms and Table 6.1 to answer the following questions.
What you are seeing is fluorescence. The light absorbed by the pigment molecules in solution excites electrons that subsequently fall back to their resting energy level, and in doing so emit photons of light. It is these electrons that in an intact cell transfer the energy of the light to the chlorophyll a molecules.
As indicated by the student Introduction and the questions at the end, the exercise as presently written emphasizes algal diversity. It takes about 1.5 to 1.75 hours to complete and thus can be effectively complemented by providing live and preserved specimens for students to observe. These can include not only the species used in the chromatography (live Spirulina and Spirogyra are both delightful under the microscope) but additional taxa to illustrate other growth forms, for example, unicellular green algae, crustose and filamentous red algae, and large-bladed brown algae such as kelps. Specimens of other prominent algal groups, such as diatoms, dinoflagellates, and Euglena, could also be included for even greater variety.
Another way to use the exercise is to adapt it for a photosynthesis lab to
emphasize the variety and properties of accessory photosynthetic pigments.
For example, the pigment bands on the TLC strips can be readily cut apart
and the pigments individually re-solubilized in acetone. Bands from about
1520 strips placed in 4 or 5 ml of acetone yield a colored solution
sufficiently concentrated to measure an absorption spectrum with a
spectrophotometer. To save time in the lab the extracts can be prepared ahead
of time for the students and stored in a refrigerator in tightly sealed,
dark containers. (Note: Because of the fire hazard with volatile
organic solvents it is not advisable to make acetone extracts of pigments
in a blender.) If there is time to use only one pigment source other than
an angiosperm, a brown alga is probably best because of the distinctive
chlorophyll c and the strikingly orange fucoxanthin band.
Students should be divided into groups, with each group responsible for one alga. Groups should include enough students that they can supply acetone extract to all of the groups in the lab; for a lab of 24 students four groups of six students works well. The amount of extract one student should produce is somewhat more than a group of 46 students would need to spot their TLC strips, but having more than the minimum number of students making an extract provides insurance against spills or weak solutions. If groups sizes are unequal or the total number of students is small, the Spirulina and Porphyra groups should be larger because these groups produce both acetone and aqueous extracts. (In the green and brown alga groups all students produce acetone extracts.) Because the finished aqueous extracts are delivered to the instructor for demonstration purposes, no more than two students per Spirulina and Porphyra group need to be involved in their production.
Individual TLC strips are 812 mm wide and 8387 mm long and are cut with scissors from precoated 20 cm × 20 cm sheets (see Appendix A for sources). The "Silica Gel 60 plastic backed TLC plates" manufactured by E. Merck work well. To avoid contaminating the sheets, the people cutting the strips should wash their hands first and/or wear thin cotton gloves. Cut TLC strips are stored in double zip-locked bags before lab to reduce hydration of the silica gel. If absorption of atmospheric moisture is likely to be a problem, the bagged strips should probably be kept in a desiccator. Fresh strips are put out as necessary for each day of the lab.
Chromatogram tubes are emptied of solvent (in the designated chemical waste jar) but are not washed with water at the end of lab because of the difficulty in drying them completely before the next lab period. Percale cloth filters are rinsed and reused.
The most important points to watch for in this exercise are: (1) grinding the algae sufficiently vigorously to produce strongly-colored extracts, (2) ensuring that all extracts are carefully decanted so that they are not contaminated with water or sediment, and (3) controlling the size of the spots applied to the TLC strips by letting spots dry between applications and by keeping a finger firmly over the end of the capillary tube so that only a small amount of solvent is drawn out onto the strip. It is especially important to make the extracts as darkly colored as possible. In particular, obtaining strongly colored extracts of the brown alga and the red algae requires vigorous grinding, especially if dried Porphyra is used. To a certain extent, dilute extracts can be compensated for by spotting the TLC strips more frequently (the higher applications rate shown on Figure 6.1for the red and brown algae reflects the sometimes weaker concentration of these extracts), but applying more spots requires patience and increases the difficulty of controlling the spot sizes. Applying the spots goes more quickly if students adopt an "assembly line" approach to their strips, applying the extract from each kind of alga in succession rather than completely spotting one kind of strip before starting the next one.
Other problems that students may have with the procedures include: strips that are too wide and wedge into the bottom of the tube (the pigments form streaks along the edge of the strip); too much TLC solvent in the test tube or spots placed too low on the strip (the spots become immersed in the solvent and dissolve off the strip); and using acetone instead of the TLC solvent in the chromatogram tubes (the pigments do not separate).
Solvent Preparation and Safety Notes
A total of about 200250 ml of acetone is needed per lab section (24 students) for extraction of polar pigments. Allow about 0.7 ml of TLC solvent for each chromatogram. With 5 chromatograms per student and 24 students per lab, a total of about 100 ml of solvent is sufficient for each section. Three TLC solvent combinations can be used; each has advantages and disadvantages as described below. Because they are mixtures of compounds with different volatilities, and the ratio of parts is critical for the chromatography, they should be made up fresh (ideally no more than a few weeks in advance of the lab) and stored in tightly sealed containers.
Answers to Discussion Questions
Brief answers to the questions are given below. For an informative but not overly technical discussion of algae consult Raven et al. (1992). A more thorough treatment of algal photosynthetic pigments is given in Rowan (1984). For general information on the characteristics of photosynthetic pigments and on photosynthesis consult Salisbury and Ross (1992), a standard plant physiology text.
The procedures using Spirulina are adapted from an exercise developed by Kathleen Nolan of Columbia University. Her original work is gratefully acknowledged.
Lewis, R. J. 1991. Hazardous chemicals desk reference. Second edition. Van Nostrand Reinhold, New York, 1579 pages.
Raven, P. H., R. F. Evert, and S. E. Eichorn. 1992. Biology of plants. Fifth edition. Worth Publishers, New York, 791 pages. [ISBN 0-87901-532-2]
Rowan, K. S. 1984. Photosynthetic pigments of algae. Cambridge University Press, Cambridge, 334 pages.
Saffo, M. B. 1987. New light on seaweeds. Bioscience, 37:654664.
Salisbury, F. B., and C. W. Ross. 1992. Plant physiology. Fourth edition. Wadsworth Publishing, Belmont, California, 682 pages. [ISBN 0-534-15162-0]
The "Silica Gel 60 plastic backed TLC plates" manufactured by E. Merck are available from EM Science (480 Democrat Rd., Gibbstown, NJ 08027, 609/354-9200) and are also sold by Alltech Associates, Inc. (address for main office: 2051 Waukegon Rd., Deerfield, IL 60015-1899, 708/948-8600). A box of 25 sheets (20 cm × 20 cm) costs about $85.00 US and should yield over 1,000 strips.
Spirulina powder is readily available from health food stores and is used just as is.
A convenient source of Porphyra is dried "laver" (Porphyra umbicalis) from Maine Coast Sea Vegetables (Franklin, ME 04634, 207/565-2907). The most economical package is one pound for about $15.00 US, an amount sufficient for many hundreds of students. (Note: The thin sheets of dried "nori" that are sold in the Oriental food section of groceries are not recommended as the pigments have been too degraded during processing.) To save time grinding the Porphyra in the mortars, the dried pieces are first chopped briefly in a blender into fragments several millimeters across. These are then rehydrated at the start of the lab by adding just enough water for the dried pieces to absorb without becoming dripping wet.
Ascophyllum or Fucus can sometimes be obtained free from local seafood markets because these seaweeds are used as a packing material for lobsters, clams, and mussels shipped from New England. It is best to check availability with several merchants well ahead of the lab period. Both species can be stored for several weeks in a refrigerator in buckets of sea water, including "Instant Ocean" mix, with no ill effects. An alternative source for Fucus (and possibly Ascophyllum) is the Marine Biological Laboratory's Department of Marine Resources (Woods Hole, Massachusetts 02543, 508/548-3705, ext. 375). An approximately 3 liter bucket of either genus costs about $30.00 US (but with a minimum order charge of $50.00 US) and is available year round. For pigment extraction purposes this volume should be enough for 45 sections of 24 students; the amount needed for one section is roughly one large handful. Fresh Sargassum can be substituted for Fucus or Ascophyllum -- it is also a member of the order Fucales and produces a strong fucoxanthin pigment band -- but it seems to be more susceptible to contamination by epiphytes in other algal divisions. Dried kelp, Laminaria, does not work well.
Spirogyra is common in farm ponds, although it is a good idea to check on potential sources in advance as the abundance in a given location may shift unexpectedly. It should be collected fresh for the lab, especially in warmer months, cleaned of mud or any dead plant debris, and stored in a refrigerator for at most a few days to a week. Although other filamentous green algae should also work, Spirogyra is easy to recognize and collect because of its bright green color and slippery texture. (It is also aesthetically pleasing for students to observe under the microscope.) Fresh Ulva (sea lettuce) can be substituted for a freshwater, filamentous form, although care must be taken to avoid including epiphytic algae from other divisions. It does not store in a refrigerator as well as the brown algae -- a week to 10 days is about the maximum -- and is tougher to grind than filamentous forms. Ulva is also available from the Marine Biological Laboratory in two forms: as food (for sea urchins) and as student observation material. The latter is more expensive ($30.00 US a bucket) but is sorted to avoid obvious contamination with other species. For both the Spirogyra and Ulva the amount needed for a section of 24 students is a large handful, or about the quantity that could be wadded into a 250 ml beaker.
Recently, Carolina Biological Supply Company (2700 York Rd., Burlington, NC 27215-3398, or PO Box 187 Gladstone, OR 97027-0187, 800/334-5551) has included live seaweed in their catalog, including Ascophyllum, Fucus, Ulva, and Porphyra. They offer a "generous portion, freshly shipped from Maine" for $25.00 US. The fresh Porphyra can presumably be treated like Ulva for extracting acetone-soluble pigments, although this has not yet been tested. It also been suggested that excess fresh marine algae can be frozen for later use; this happens to northern intertidal species naturally and should not affect the photosynthetic pigments.
Three types of pigments are extracted in this exercise: phycobilins, carotenoids, and chlorophylls. The phycobilins occur only in the cyanobacteria and the red algae. In contrast to the carotenoids and chlorophylls, the phycobilins are polar and water-soluble. When they are extracted they remain associated with the proteins that help determine their absorbance characteristics and hence their color. The two most abundant phycobilins are phycocyanin (blue) and phycoerythrin (red). They occur together in the same organism, although as the names of the groups imply, phycocyanin predominates in cyanobacteria and phycoerythrin predominates in red algae. This pattern is evident in the aqueous extracts from Spirulina and Porphyra. No attempt is made in the exercise to separate different kinds of phycobilins in the aqueous extracts.
The carotenoids are long chain hydrocarbons and are of two types: the carotenes and xanthophylls. The only carotene of importance in photosynthesis is the widely occurring betacarotene, the familiar source of color in carrots. Xanthophylls are much more diverse than carotenes, and all are distinguished from carotenes by having at least one oxygen attached to them. A particularly common xanthophyll (prominent in plants and green algae) is lutein. Like many xanthophylls it is yellow. A distinctive, more orange-colored xanthophyll is fucoxanthin, the pigment that gives the brown algae and related groups (such as the diatoms) their characteristic colors.
The chlorophylls are the dominant non-polar photosynthetic pigments. (Notice that all of the acetone extracts, including those from cyanobacteria and red alga, are distinctly shades of green.) Chlorophylls consist of a magnesium-containing porphyrin group typically attached to a long hydrocarbon chain, the phytol tail (see Salisbury and Ross (1985) for illustration). Chlorophyll a is the form that participates most directly in the light reactions of photosynthesis and is found in all of the groups. Chlorophyll b is found in plants and green algae, and is very similar to chlorophyll a, differing only in the substitution of an aldehyde for a single methyl group on the porphyrin ring. This small change is sufficient to alter the absorption properties of the molecule and makes chlorophyll b appear yellow-green while chlorophyll a looks blue-green. Chlorophyll c, which occurs in the brown algae and related groups, is identical to chlorophyll a except that it lacks the long phytol tail.
Figures 6.3 and 6.4 below show the expected banding patterns for TLC strips developed in the toluene/acetone and cyclohexane/propanol mixtures, respectively. Note that the order in which the pigments separate is the same for both solvent mixtures. At the top is betacarotene, the most non-polar and fastest moving pigment. In the toluene/acetone mixture betacarotene forms a distinct thin, orange line that travels with the solvent front. In the cyclohexane/propanol it forms a more diffuse yellow band slightly behind the front. Chlorophyll b comes out just below chlorophyll a, a consequence of the slightly greater polarity conferred by the substitution of the aldehyde for the methyl group. The various xanthophylls, with the oxygens attached to the ends of their chains making them more polar than the betacarotene, sort out below the chlorophylls. (The topmost xanthophyll in the green alga and spinach is lutein, but otherwise it is not feasible with the chromatogram information alone to try to identify the different xanthophylls, except of course for fucoxanthin.) Toward the bottom, nearest the origin, is chlorophyll c. Because it lacks the long hydrocarbon tail of the other chlorophylls, it is much more polar and thus is strongly adsorbed to the silica gel.
A major difference between the two solvent systems is that the chlorophylls and xanthophylls do not migrate as fast relative to the betacarotene in cyclohexane/propanol as they do in toluene/acetone, and as a result the bands in cyclohexane/propanol come out closer together. Nevertheless, the separation between chlorophyll a and chlorophyll b in this system is usually quite sharp, with less of the blurring below the trailing edge that sometimes occurs with toluene/acetone. Other differences in results between the two solvent systems are that the xanthophyll bands in cyclohexane/propanol are sometimes a paler yellow and harder to distinguish while the bands of the breakdown products of chlorophyll (described below) are more obvious.
In addition to the colored photosynthetic pigments diagrammed in Figures 6.3 and 6.4, some TLC strips may also contain bands formed by the breakdown products of chlorophyll. When a chlorophyll molecule loses the magnesium from its porphyrin ring it becomes pheophytin, which on a chromatogram appears as a gray band just above chlorophyll a. If the chlorophyll loses both the magnesium and the non-polar phytol tail it becomes pheophorbide, which appears as a gray band at about the same height on the TLC strip as chlorophyll c. The gray pheophytin and pheophorbide bands are most likely to occur in the extracts of Spirulina and dried Porphyra because both of these pigment sources have been more subjected to the conditions likely to degrade the chlorophyll (including heating and drying) than the fresh materials. For more information about chlorophyll degradation products see Rowan (1984).
The patterns illustrated in Figures 6.3 and 6.4 also assume no contamination of the pigments sources by algae in other divisions. However, TLC is a very sensitive technique, and faint but noticeable fucoxanthin and chlorophyll c bands may appear in extracts of Ulva or fresh Porphyra as a result of diatoms that are too few in number to form a film visible to the naked eye. Also, the chromatograms from Spirulina occasionally have a very light green band in the chlorophyll b position. It is much fainter than the chlorophyll b band on the green alga or spinach strips and may caused by small amounts of green algae harvested with the Spirulina.
Figure 6.3. Expected separation of non-polar photosynthetic pigments on silica-gel thin layer chromatography strips using toluene/acetone as the developing solvent.
Figure 6.4. Expected separation of non-polar photosynthetic pigments on silica-gel thin layer chromatography strips using cyclohexane/propanol as the developing solvent.
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