Laboratory Experiment pubs.acs.org/jchemeduc
Integrating Biology into the General Chemistry Laboratory: Fluorometric Analysis of Chlorophyll a Meredith C. Wesolowski* Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States S Supporting Information *
ABSTRACT: A laboratory experiment that introduces fluorometry of chlorophyll a at the general chemistry level is described. The use of thin-layer chromatography to isolate chlorophyll a from spirulina and leaf matter enables quantification of small amounts of chlorophyll a via fluorometry. Student results were reasonably comparable to those found in the literature. This experiment uses relatively low volumes of organic solvents and an inexpensive fluorescence spectrometer (such as a Vernier SpectroVis Plus). The method could also be adapted for inquiry-based laboratories comparing chlorophyll a levels in plants, algae, and photosynthetic bacteria. KEYWORDS: First-Year Undergraduate/General, Biochemistry, Interdisciplinary/Multidisciplinary, Laboratory Instruction, Hands-On Learning/Manipulatives, Fluorescence Spectroscopy, Plant Chemistry, Thin Layer Chromatography iological sciences ranks as one of the top five majors nation-wide,1 and students pursuing these fields represent a significant proportion of enrollments in a typical general chemistry course. Calls for improved preparation of biological sciences majors recommend integration of biological concepts across the disciplines, including their chemistry instruction.2,3 Of the many spectroscopic techniques available to scientists, few have experienced as great a growth in biological application recently as fluorometry due to its versatility, sensitivity, and adaptability to both in vitro and in vivo use.4 Traditional general chemistry courses cover the fundamental principles involved in spectroscopy and fluorescence, placing fluorometry within conceptual reach of students if coupled with explicit discussion of the process of fluorescence. Because nature provides numerous fluorophores (such as chlorophyll a, Figure 1), practical application of fluorometry is highly accessible. Chlorophyll a (Chl a) is a highly conjugated pigment molecule responsible for photon absorption in the reaction
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center of most photosynthetic bacteria and plants.5 It also serves as the primary electron donor in the photosynthetic electron transport chain6 (Figure 2).
Figure 2. A schematic showing the photoinduced, electron transfer steps of photosynthesis.
Photoexcitation of Chl a facilitates electron transfer to quinone (Q), and water regenerates the reduced form of Chl a, providing a continuous flow of electrons through the photosynthetic pathway. However, in the presence of organic solvents, Chl a is unable to complete the electron-transfer process, trapping it in its reduced (neutral) form (Figure 3). Under these conditions, the pathways available for excited-state decay are restricted to thermal decay and fluorescence. In most photosynthetic organisms, Chl a is assisted by a number of accessory pigments, including chlorophyll b (Chl b; Figure 1) and carotenoids, which broaden the spectrum of light absorbed during photosynthesis.6 During initial development of this experiment, accessory pigment molecules (particularly carotenoids) were empirically found to quench the fluorescence of Chl a, an observation supported by the literature.7 However, these pigments can be resolved and selectively removed from plant extracts using thin-layer chromatography (TLC).
Figure 1. Chemical structure of chlorophylls a and b, which differ by only one R group. © XXXX American Chemical Society and Division of Chemical Education, Inc.
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cellulose capsules. Once isolated, Chl a concentrations can be determined via absorption spectroscopy (ε = 32 600 M−1 cm−1 at 660 nm in methanol9) and then used to prepare fluorescence standards.
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JUSTIFICATION The primary goal of this experiment is to introduce fluorometry as a biologically relevant technique to general chemistry students. A variety of related experiments can be found in the literature,10−14 but this experiment simplifies and combines procedures for use at the introductory level in a single 3 h laboratory session. This experiment also exposes students to a practical application of how polarity influences chemical behavior (via the separation of pigments by TLC), increases their awareness of how redox exchanges are important to biological systems, and provides a practical application of quantum topics (such as excited electron states and reactivity, electron decay, and molecular orbital theory). The level of sophistication can be modified for a variety of audiences, with the moderately detailed application described here used for an honors general chemistry course for biology and engineering majors.
Figure 3. Electron decay pathways available to photoexcited Chl a. Under physiological conditions, four decay pathways are available, but in the presence of organic solvents, only fluorescence and thermal decay are possible.
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MATERIALS
A general chemistry laboratory must have at least one analytical balance per section and one fluorescence spectrometer per student group of four. Each group should be supplied with at least 10 disposable fluorescence cuvettes with caps, scissors, enough volumetric glassware to perform the experiment, five plastic-backed TLC plates measuring 15 × 5 cm, and five developing jars. Approximately 50−100 mL each of acetone, petroleum ether, and methanol is required per group. For more information, see the Supporting Information.
Due to their similar chemical structures (Figure 1), some band overlap typically occurs between Chl a and Chl b when using TLC. Since the absorption spectra of Chl a and b also overlap in key regions (Figure 4A), using spectrophotometry to quantify the chlorophyll a that is purified via TLC typically involves some systematic error. However, Chl a and b can be fully resolved via fluorometry, as only Chl a will fluoresce if an excitation wavelength of 405 nm is used (Figure 4B). For quantitative applications of fluorometry, use of fluorescence standards is essential. Due to the high cost of purified Chl a from chemical suppliers, isolation of a Chl a standard by students is recommended. Some photosynthetic bacteria, such as Anthrospira platensis (spirulina), produce only the more evolutionarily distant Chl a, negating any issues of TLC band or absorption spectra overlap with Chl b. Spirulina is particularly attractive as a Chl a standard source as it is readily and inexpensively available at nutrition suppliers in the form of
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EXPERIMENTAL PROCEDURE The experiment has three main components: preparation of Chl a fluorescence standards from spirulina, chromatographic separation of pigments from leaf extracts, and quantification of Chl a from leaf extracts using fluorometry. The methods are described in detail in the Supporting Information and are summarized below.
Figure 4. Relative absorption (A) and fluorescence (B) spectra of Chl a and Chl b. The absorption spectrum of β-carotene is also provided (A). Absorption spectra were plotted using published molar extinction coefficients,8,9 and overlap in the 600−700 nm region can be observed for Chl a and Chl b. Fluorescence spectra were collected in methanol using an excitation wavelength of 405 nm. Fluorescence was observed only for Chl a under these conditions. B
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Isolation of Chl a from Spirulina
Using independently generated fluorescence standards and samples, students collected data that compared reasonably well from group to group, with the average reported concentration of Chl a across 16 types of mixed green leaves being 2.8 × 10−7 mol/g wet weight (SD = 2.7 × 10−7; n = 66 samples, with natural variation in the Chl a content of different leaf samples accounting for some of the variation observed). For a given leaf type, student averages compared more closely; for example, reported spinach Chl a concentrations averaged at 4.1 × 10−7 mol/g wet weight (SD = 3.4 × 10−7; n = 14, with the small sample size and outliers at the low end of the range accounting for some of the calculated variation). When data from three student groups with consistently low values were removed, the averages changed to 3.5 × 10−7 mol Chl a/g wet weight across 16 leaf types (SD = 2.66 × 10−7; n = 54 samples), and for spinach, changed to 5.1 × 10−7 mol Chl a/g wet weight (SD = 3.2 × 10−7; n = 11). Chl a concentrations determined by students also compared reasonably well to those found in the literature, which averaged around 1 × 10−6 mol Chl a/g wet weight for a variety of plant types.15−17 Yields from this method were also comparable to other quantitative Chl a experiments designed for introductory chemistry students.18 Given the small volumes and quantities of samples used, as well as natural variation of Chl a concentrations between types of leaves and varying distributions of Chl a within leaf structures, a broad range of results should be expected for this experiment. Some avoidable procedural errors that lead to low Chl a measurements were also noted, including loss of sample while cutting out the Chl a bands, use of too little spirulina and leaf extracts when preparing TLC plates, and not allowing sufficient time for water in the leaf extract evaporate before developing TLC plates with solvent. The primary objective of this experiment was to introduce students to fluorometry in a biologically relevant context, and this laboratory provided a hands-on introduction to the basic technique and application. To evaluate if students were able to understand the technique of fluorometry, students were asked to describe the essentials of the technique (including the underlying theory) as part of their laboratory report write-ups. Of the 62 reports evaluated for understanding of the technique, 77% accurately described the technique (by reflecting an understanding of the electronic transitions associated with absorption and emission wavelengths, as well as why the technique is sensitive and selective), 19% were able to partially describe the technique, and 3% were unable to correctly explain fluorometry. To evaluate their grasp of the biological relevance of the technique, students were also asked to propose an extension experiment. Over 90% proposed reasonable uses of fluorometry to answer questions of biological relevance (see Supporting Information for student examples, as well as for potential adaptations to accommodate inquiry instruction).
Photosynthetic pigments are extracted from powdered spirulina suspended in methanol. TLC is used to separate the pigments using a 75% petroleum ether/25% acetone by volume solvent system. The Chl a band is cut out of the TLC plate using scissors and resuspended in fresh methanol, and the resulting concentration of Chl a is determined at 660 nm using ε = 32 600 M−1 cm−1. Serial dilutions with methanol generate standard Chl a solutions ranging from 1 × 10−5 to 2.5 × 10−6 M. A standard fluorescence curve is generated using an excitation wavelength of 405 nm and emission wavelength of ∼670 nm. Separation of Plant Pigments Using TLC
Mixed salad green leaf extracts are transferred directly onto the silica surface of TLC plates by applying pressure with a flatsided coin. Leaf masses are measured before and after extract transfer (a 0.1 g difference is typical). Water present in the extract is allowed to evaporate before exposure to the solvent system described above. Each TLC plate is developed to separate photosynthetic pigments, with Chl a (Rf = 0.49) migrating farther up the plate than Chl b (Rf = 0.40). Determination of Chl a Concentrations Using Fluorescence Spectroscopy
The Chl a band is cut out from a given TLC plate, placed in a fluorescence cuvette, suspended in methanol, and capped. Fluorescence intensities of resulting Chl a solutions are measured as described above, and, when necessary, quantitatively diluted to achieve an intensity of 0.25 or lower. By comparing with the standard fluorescence curve, moles of Chl a per gram of leaf extract transferred are determined.
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HAZARDS
Acetone, methanol, and petroleum ether are extremely flammable in liquid and vapor forms, and may ignite if exposed to heat or open flame. Acetone and petroleum ether vapors are harmful to breathe and cause irritation to the respiratory tract. Liquid and vapor forms of acetone, petroleum ether, and methanol cause irritation to the skin or permanent damage to eyes and can affect the central nervous system; care should be taken to minimize physical contact and avoid breathing the vapors. Acetone, petroleum ether, and methanol should be handled with proper personal protection including safety goggles and gloves and use of a fume hood when handling.
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DISCUSSION This experiment was performed at the end of the first semester of an honors general chemistry course, concurrent with or after quantum theory, light, energy, oxidation−reduction reactions, and intermolecular forces were discussed in lecture. The goal of the experiment was to expose students to fluorometry and to relate course topics to a biologically relevant system. Students carried out the experiment in a standard 3 h laboratory period. The approximate times for each step were (i) preparing TLC plates for standards and samples, ∼30 min; (ii) developing TLC plates, ∼30 min; (iii) preparing samples for fluorescence measurements, ∼30 min; and (iv) collection of absorbance and fluorescence data, ∼40 min. Laboratory setup and cleanup accounted for the remaining time. These values are based on personal observations and teaching assistant (TA) reports from two years of using this experiment with a total of 100 students (∼15 students per laboratory section).
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CONCLUSION This experiment provided a method for introducing fluorometry of Chl a into a general chemistry laboratory course to increase the relevance of course topics for biological science majors. Support that this was achieved included an evaluation of student laboratory reports, student ranking of this experiment as one of the three best liked via an anonymous survey, as well as multiple students noting on course evaluations that the experiment made general chemistry more relevant to their major course of study. C
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(12) Diehl-Jones, S. M. Chlorophyll Separation and Spectral Identification. J. Chem. Educ. 1984, 61, 454−456. (13) Valverde, J.; This, H.; Vignolle, M. Quantitative Determination of Photosynthetic Pigments in Green Beans Using Thin-Layer Chromatography and a Flatbed Scanner as Densitometer. J. Chem. Educ. 2007, 84, 1505−1507. (14) Wigton, B. T.; Chohan, B. S.; Kreuter, R.; Sykes, D. The Characterization of an Easy-to-Operate Inexpensive Student-Built Fluorimeter. J. Chem. Educ. 2011, 88, 1188−1193. (15) Winter, H.; Robinson, D. G.; Heldt, H. W. Subcellular Volumes and Metabolite Concentrations in Spinach Leaves. Planta 1994, 193, 530−535. (16) Winter, H.; Robinson, D. G.; Heldt, H. W. Subcellular Volumes and Metabolite Concentrations in Barley Leaves. Planta 1993, 191, 180−190. (17) Cui, M.; Vogelmann, T. C.; Smith, W. K. Chlorophyll and Light Gradients in Sun and Shade Leaves of Spinacia oleracea. Plant, Cell Environ. 1991, 14, 493−500. (18) Silveira, A.; Koehler, J. A.; Beadel, E. F.; Monroe, P. A. HPLC Analysis of Chlorophyll a, Chlorophyll b, and Beta-Carotene in Collard Greens. J. Chem. Educ. 1984, 61, 264−265.
Students in an honors general chemistry course isolated Chl a from the photosynthetic bacterium spirulina to generate a fluorometric standard curve with which fluorescence intensities of Chl a solutions from plant leaf extract were compared. Student results were reasonably comparable between student groups and with those found in the literature. As many general chemistry students major in disciplines with a biological focus, this experiment helps address calls for improved training of biology students by forming an interdisciplinary connection between spectroscopic and quantum principles and photosynthesis.
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ASSOCIATED CONTENT
S Supporting Information *
Instructions for students; notes for the instructor with ideas for additional experiments; and grading notes including representative student data and answers to the laboratory questions. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS I would like to thank the Howard Hughes Medical Institute Undergraduate Science Education grant to the University of Delaware (grant number 52006947) for funding development of this work.
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REFERENCES
(1) Postsecondary Institutions and Price of Attendance in the United States: 2010−11; Knapp, L. G., Kelly-Reid, J. E., Ginder, S. A., Eds.; U.S. Department of Education: Washington, DC, 2011; p 19. (2) Vision and Change in Undergraduate Biology Education; Brewer, C., Smith, D., Eds.; AAAS: Washington, DC, 2011; pp 3, 15, 19. (3) Bio 2010; Whitacre, P. T., Ed.; NRC, National Academies Press: Washington, DC, 2003; pp 8−9, 12−14, 34−36. (4) Duysens, L. N. M. Introduction to (Bacterio)chlorophyll Emission: A Historical Perspective. In Advanced Fluorescence Reporters in Chemistry and Biology I: Fundamentals and Molecular Design; Demochenko, A. P., Ed.; Springer-Verlag: New York, 2010; pp 4−6. (5) The Chloroplast: Basics and Applications; Rebeiz, C. A., Ed.; Springer-Verlag: New York, 2010; p 214. (6) Gleason, F. K. Plant Biochemistry; Jones & Bartlett Learning: Sudbury, MA, 2012; pp 7−11. (7) Shinkarev, V. P.; Govindjee. Insight into the Relationship of Chlorophyll a Fluorescence Yield to the Concentration of Its Natural Quenchers in Oxygenic Photosynthesis. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 7466−7469. (8) University of Copenhagen, Department of Biology, Laboratory of Niels-Ulrik Friggard. http://www.bio.ku.dk/nuf/resources/scitab/ chlabs/index.htm (accessed Jun 2014). (9) Oregon Medical Laser Center, Laser Photomedicine and Biomedical Optics. http://omlc.org/spectra/PhotochemCAD/html/ 122.html (accessed Jun 2014). (10) Quach, H. T.; Steeper, R. L.; Griffin, G. W. Separation of Plant Pigments by Thin Layer Chromatography. J. Chem. Educ. 2004, 81, 385−387. (11) Curtright, R.; Emry, R.; Markwell, J. Student understanding of chromatography: A hands-on approach. J. Chem. Educ. 1999, 76, 249− 252. D
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