In the Laboratory
Chemical Analysis of an Endangered Conifer Environmental Laboratory Experiments Royce S. Woosley and David J. Butcher* Department of Chemistry and Physics, Western Carolina University, Cullowhee, NC 28723
Environmental chemistry is a topic that can be used to generate considerable student interest because of its familiarity and relevance. Several publications have described recent developments in the creation of links between environmental chemistry and chemical education (1–8). The Fraser fir (Abies fraseri) is a conifer native to highelevation sites in our geographic region, the Southern Appalachians. This species has recently suffered severe dieback that has been attributed to acidic deposition (9) and an exotic insect, the balsam woolly adelgid (BWA) (Adelges piceae) (10– 12). In addition to its presence at well-known sites such as the Great Smoky Mountains National Park and the Blue Ridge Parkway, this species has commercial significance in the region as a Christmas tree. We (13–17) have recently done research investigating the causes of the decline of the Fraser fir. This project led to the development of several undergraduate laboratory experiments using this species as a focal point, which include the determination of metals, volatiles, and chlorophylls in the fir’s foliage. Although this project is most appropriate for our geographical region, the procedures and concepts may be easily adapted to other plant species and tissues to address other environmental concerns. Elemental Analysis The companion tree of the Fraser fir is the red spruce (Picea rubens). Since 1970, a relatively mild decline of red spruce has been observed in the Southern Appalachians, which has been attributed to a depletion of soil calcium and magnesium and increased levels of aluminum induced by acidic deposition (18–20). A reduction in radial increment and crown condition has been observed at elevations exceeding 1800 m, where high levels of acidic deposition have been reported (21). The ratios of calcium to aluminum in the soil have been reduced to sufficiently low levels to limit uptake and retention of calcium, even though the concentrations of aluminum were below toxic levels (19, 22, 23). We hypothesized that acidic deposition may be a contributing factor, *Corresponding author.
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along with the BWA, to Fraser fir decline, and determined calcium, magnesium, and aluminum in foliage and soil at six sites in the Southern Appalachians. In general, our results did not support our hypothesis (15, 16). We have adapted our procedures to laboratory experiments for undergraduates. For example, a structured laboratory typically involves providing students with samples and detailed procedures on digestion and analysis procedures. Foliage is dissolved with 3 mL of nitric acid, 0.5 mL of sulfuric acid, and 0.5 mL of hydrogen peroxide. We have performed the digestions using test tubes with digestion blocks or Teflon microwave digestion vessels, although small beakers may be employed as well. Metals are extracted from the soil by 1 M ammonium acetate with shaking for one hour. We have done the analyses by atomic absorption spectrometry (AAS). Flame AAS (FAAS) is suitable for high concentration levels, such as aluminum (soil), calcium (soil and foliage), and magnesium (soil and foliage); graphite furnace AAS (GFAAS) was employed for aluminum in foliage. Analysis conditions are listed in Table 1. Alternatively, the analyses may be performed by other atomic spectrometric methods, such as inductively coupled plasma optical emission spectrometry (ICP–OES). Our senior-level instrumental analysis laboratory has recently been revised to consist entirely of projects designed to simulate analytical method development (24 ). The students are presented with an analytical chemistry problem (e.g., identify and determine volatiles in perfume), for which they research protocols in the literature, collect samples, implement methods, perform the analysis, and work up the data. In our experience, this appears to be an effective approach to learn method development and instrument optimization. Quality control procedures, including the use of standard reference materials and recovery checks, are incorporated to emphasize proper method development. The students spend a minimum of 50 hours on their projects (3 hours/week); many spend much more time than the minimum. They are graded on the basis of the quality of their method development, their results, and a written report. In addition to the acidic deposition projects described above, determination of other elements provides interesting Continued on page 1593
Journal of Chemical Education • Vol. 75 No. 12 December 1998 • JChemEd.chem.wisc.edu
In the Laboratory
Table 1. Conditions for Determination of Aluminum, Calcium, Magnesium, and Lead by FAAS and GFAAS Method
Sample
λ/nm
Calibration Range
Conditions
Al
GFAAS
Foliage
394.4
20– 200 ng/mL
Continuum source background correction; Mg chemical modifier
Al
FAAS
Soil
309.3
5– 60 µg/mL
N2O/C2H2 flame; no background correction
Ca
FAAS
Foliage, soil
422.7
1– 12 µg/mL
Air/C2H2 flame; no background correction
Mg
FAAS
Foliage, soil
285.2
0.2– 3 µg/mL
N2O/C2H2 flame; no background correction
Pb
GFAAS
Foliage
283.3
10– 50 ng/mL
Self-reversal background correction; Pd/Mg chemical modifier
Element
NOTE: See refs 15, 16, and 31.
student projects. For example, Bogle et al. (25) measured lead concentrations as high as 4.1 µg/g in Fraser fir foliage from the Great Smoky Mountains National Park in the mid-1980s. Student measurements from samples collected in 1994 at the same sites had lead levels below 0.5 µg/g. These results imply that reduced emissions from vehicles has caused a decrease in lead content of the conifers. Determination of Volatiles Fraser fir foliage is characterized by the presence of high concentrations (~5% of dry weight) of volatile compounds (26 ). On a weight percent basis, approximately 70% of the volatiles are composed of an oxygenated aromatic compound called maltol (C6H6 O3), 20% of various monoterpenes (e.g., α-pinene, β-pinene, camphene, 3-carene), 5% of oxygenated derivatives of monoterpenes (e.g., bornyl acetate), and the remainder of sesquiterpenes and other compounds. We have hypothesized that these volatiles may be involved in defending the conifer from insect predation; verification of this hypothesis is still in progress in our group (17, 26 ). As part of this research project, we have developed a simple method for the determination of volatiles in foliage (14 ). Foliage is removed from the stems with scissors, and 2 g is placed in a 2-dram vial. Methylene chloride (5 mL) is added with an internal standard (2-carene, 1 mg/g) for quantitative analysis, and the samples are shaken for 24 hours at room temperature. The resulting extract is then injected in a gas chromatograph–mass spectrometer (GC–MS) or GC for analysis. Our analysis conditions are listed in Table 2. If this project is adapted in undergraduate laboratories as a two-week experiment, the students are provided with samples, the extraction procedure, and pure samples of the volatiles to confirm identification and perform quantitative Table 2. Instrumental Conditions for Determination of Volatile Compounds in Foliage by GC– MS or GC Feature
Specification
Gas chromatograph
Hewlett Packard 5840A with flame ionization detector
Column
DB-1701, 60 m × 0.25 mm
Split ratio
100:1
Temperature program
15 min at 90 ⬚C; 10 ⬚C/min ramp to 200 ⬚C; hold for 5 min
Injector temperature
225 ⬚C
Detector temperature
250 ⬚C
Flow rate
1 mL/min
NOTE: See refs 14, 17, and 26.
analysis. GC–MS is used to identify the compounds, and quantitative analysis is performed on one or two compounds by GC or GC–MS. A semester-long project involves selection of relatively healthy and unhealthy native Fraser fir stands. The students are expected to consult the literature to find methods of sample preparation, analysis, and data treatment, as discussed in references 14 and 17. We find it instructive to suggest optimization of the solvent used for extraction by investigating hexane, methanol, etc., in addition to methylene chloride. In order to perform chemosystematic studies of conifers from different sites, we suggest the analysis of composite samples prepared by combining equal masses (e.g., 5 g) from each tree at a site. The use of composites allows more valid comparisons of differences between stands than averaging individuals, and also has the advantage that fewer GC analyses are required. Students employ quality control procedures (e.g., recovery checks) to verify the accuracy of the determinations. One advantage of this extraction procedure is its adaptability to other types of plant tissues, such as seeds (14). Determination of Chlorophylls The determination of chlorophylls a and b in foliage may be performed by column or thin-layer chromatography (27, 28), high-performance liquid chromatography (HPLC) (6 ), and solvent extraction followed by ultraviolet–visible (UV–vis) spectroscopy (29, 30). Compared with the chromatographic procedures, the extraction methods are simpler and can be performed with inexpensive spectrometers. We have adapted the procedures of Lichtenthaler and Wellburn (29) and Hiscox and Israelstam (30) for our quantitative analysis course. The experiment is designed to introduce students to the concepts of method development. The students are not provided with any specific analytical procedure except the Lichtenthaler and Hiscox citations. These methods allow the determination of chlorophylls a and b by making absorbance measurements at 645 and 663 nm. The students are provided with solvents described in the papers, including methanol, ethanol, acetone, and dimethyl sulfoxide (DMSO). After consulting the literature, most students employ the DMSO extraction method of Hiscox and Israelstam (30), which involves extraction of 100 mg of foliage with 7 mL of DMSO at 65 °C using a water bath. The students are instructed to optimize the time required for the extraction as an exercise in method development. This experiment provides an environmental application of simultaneous quantitative analysis of a two-component mixture that is more relevant to students than traditional systems (e.g., Co2+ and Cr3+). In addition, the fundamentals of method development are provided with low-cost instrumentation.
JChemEd.chem.wisc.edu • Vol. 75 No. 12 December 1998 • Journal of Chemical Education
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In the Laboratory
Conclusions Here we have discussed some examples of environmental chemistry experiments on a conifer species native to our area. These methods are readily adaptable for investigating other species and other environmental problems. Acknowledgments This work was supported in part by a Faculty Research Grant from the Western Carolina University Graduate School (1993–1994), an award from the Western Carolina University Off-Campus Scholar Assignment Program (1994–1995), and the Regional Development Fellows Program sponsored by the Center for Improving Mountain Living at Western Carolina University (1993–1994, 1994–1995). The graphite furnace atomic absorption spectrometer employed in this research was purchased with support from a Cottrell College Science Award of Research Corporation. We would like to acknowledge the donation of the gas chromatograph employed for this work by Louis R. Alexander of the Centers for Disease Control, Atlanta, GA, and the financial and logistical support of Richard C. Berne, Director of Summer Ventures in Science and Mathematics at WCU. Literature Cited 1. Ondrus, M. G. Environmental Chemistry: Experiments and Demonstrations, 2nd ed.; Wuerz: Winnipeg, AB, 1996. 2. Bennaars, A.; Musich, M.; Suchan, M.; Thompson, M. In Laboratory Modules; College–University Resource Institute: Washington, DC, 1995. 3. Burdick, I.; Ravich, T.; Bozzelli, J. W.; Kebbekus, B. B. In Laboratory Modules; College–University Resource Institute, Inc: Washington, DC, 1995. 4. Halstead, J. A.; Wagner, A. M.; Larner, V.; Yavuz, E.; Piepho, S. B. In Laboratory Modules; College–University Resource Institute, Inc: Washington, DC, 1995. 5. Fitch, A.; Wang, Y.; Mellican, S.; Macha, S. Anal. Chem. 1996, 68, 727A–731A. 6. Silveira, A.; Koehler, J. A.; Beadel, E. F.; Monroe, P. A. J. Chem. Educ. 1984, 61, 264–265. 7. Chittenden, D. M.; Dragenjac, M. E.; Wyatt, W. V. J. Chem. Educ. 1995, 72, 908. 8. Swan, J. A.; Spiro, T. G. J. Chem. Educ. 1995, 72, 967–970. 9. Bruck, R. I.; Robarge, W. P.; McDaniel, A. Water Air Soil Pollut. 1989, 48, 161–180. 10. Eagar, C. In The Southern Appalachian Spruce–Fir Ecosystem: Its Biology and Threats; White, P. S., Ed.; U.S. Department of the Interior, National Park Service: Washington, DC, 1984; Vol. Research/Resources Serial Report SER-71, pp 36–50.
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11. Hollingsworth, R. G.; Hain, F. P. Flor. Entomol. 1991, 74, 179– 187. 12. Johnson, K. Fraser Fir and the Balsam Woolly Adelgid: Summary of Information; The Southern Appalachian Research–Resource Management Cooperative: Asheville, NC, 1980. 13. Shepard, M. R.; Lee, C. E.; Woosley, R. S.; Butcher, D. J. Microchem. J. 1995, 52, 118–126. 14. Bowman, J. M.; Braxton, M. S.; Churchill, M. A.; Hellie, J. D.; Starrett, S. J.; Causby, G. Y.; Ellis, D. J.; Ensley, S. D.; Maness, S. J.; Meyer, C. D.; Sellers, J. R.; Hua, Y.; Woosley, R. S.; Butcher, D. J. Microchem. J. 1997, 56, 10–18. 15. Bryant, K. N.; Fowlkes, A. J.; Mustafa, S. F.; O’Neil, B. J.; Osterman, A. C.; Smith, T. M.; Shepard, M. R.; Woosley, R. S.; Butcher, D. J. Microchem. J. 1997, 56, 382–392. 16. Lee, C. E.; Cox, J. M.; Foster, D. M.; Humphrey, H. L.; Woosley, R. S.; Butcher, D. J. Microchem. J. 1997, 56, 236–246. 17. Sutton, B. A.; Woosley, R. S.; Butcher, D. J. Microchem. J. 1997, 332–342. 18. Dull, C. W.; Ward, J. D.; Brown, H. D.; Ryan, G. W.; Clerke, W. H.; Uhler, R. J. Evaluation of Spruce and Fir Mortality in the Southern Appalachian Mountains; USDA Forest Service Southern Region R8–PR13: Atlanta, 1988. 19. McLaughlin, S. B.; Andersen, C. P.; Hanson, P. J.; Tjoelker, M. G.; Roy, W. K. Can. J. For. Res. 1991, 21, 1234–1244. 20. Eagar, C.; Adams, M. B. In Ecological Studies; Billings, W. D., Golley, F., Lange, O. L., Olson, J. S., Remmert, H., Eds.; Springer: New York, 1992; Vol. 96, p 417. 21. Saxena, V. K.; Lin, N.-H. Atmos. Environ. A 1990, 24A, 329– 352. 22. McLaughlin, S. B.; Andersen, C. P.; Edwards, N. T.; Roy, W. K.; Layton, P. A. Can. J. For. Res. 1990, 20, 485–495. 23. McLaughlin, S. B.; Tjoelker, M. G.; Roy, W. K. Can. J. For. Res. 1993, 23, 380–386. 24. Wenzel, T. J. Anal. Chem. 1995, 67, 470A–475A. 25. Bogle, M. A.; Turner, R. R.; Baes, C. F. I. Environ. Int. 1987, 13, 235–246. 26. Carlow, S. J. A Comparison of Volatile Compound Concentrations in the Foliage of the Balsam Fir (Abies balsamea) and Fraser Fir (Abies fraseri); M.S. Thesis, Western Carolina University, Cullowhee, NC, 1997. 27. Moore, J. A.; Dalrymple, D. L.; Rodig, O. R. Experimental Methods in Organic Chemistry, 3rd ed.; Saunders: Philadelphia, 1982. 28. Pavia, D. L.; Lampman, G. M.; Kriz, G. S. Introduction to Organic Laboratory Techniques, 2nd ed.; Saunders: Philadelphia, 1982. 29. Lichtenthaler, H. K.; Wellburn, A. R. Biochem. Soc. Trans. 1983, 11, 591–592. 30. Hiscox, J. D.; Israelstam, G. F. Can. J. Bot. 1979, 57, 1332–1334. 31. Ohonjo, D. A. Highly Oriented Pyrolytic Graphite as a Platform for Atomic Absorption Spectrometry for the Determination of Lead, Copper, and Aluminum; M.S. Thesis, Western Carolina University, Cullowhee, NC, 1997.
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