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Extending the Marine Microcosm Laboratory Hal Van Ryswyk,* Eric W. Hall, Steven J. Petesch, and Alice E. Wiedeman Department of Chemistry, Harvey Mudd College, Claremont, CA 91711-5990; *
[email protected] The marine microcosm laboratory developed by Kenneth D. Hughes (1) has been adopted in a range of analytical chemistry laboratory courses. Originally devised as a means to promote appreciation, understanding, and historical awareness of classical analysis while bringing real-world motivation into the instructional laboratory, the marine microcosm laboratory allows students to consider the entire analytical process from beginning to end within the context of a complex, self-contained laboratory ecosystem. Many instructors use marine microcosm experiments, either as the core of their laboratory or as a supplement to other offerings. The marine microcosm laboratory is an excellent example of a robust tradition within analytical chemistry of bringing the outside world into the instructional laboratory. Walters has used role-playing throughout the entire process of chemical analysis to provide context and motivation and to build communication and management skills (2–5). Both geological (6) and environmental topics (7) lend themselves nicely to a problem-based laboratory approach and present excellent opportunities to employ cooperative learning techniques (8). All of these exercises answer the call for chemists to be trained in the entire analytical process, not just bench wet chemistry (9). Others have successfully extended an aquarium-based approach to the general chemistry laboratory (10), chemical analysis modeling (11), the instrumental methods of analysis laboratory (12), and interdisciplinary instruction in a high school setting (13). We describe here three extensions to the traditional range of marine microcosm experiments that extend the chemistry
illustrated to include additional wet analytical, instrumental, and biochemical methods, while mitigating chemical hazards associated with traditional methods of analysis. Specifically, we incorporate the determination of ammonia via ion-selective electrode using the method of standard additions; the determination of sulfate via ion chromatography with an internal standard; and the determination of nitrate via enzymatic reduction with spectrophotometric detection. Variations on a Theme The marine microcosm laboratory can be staged successfully in anything from a 60 to 300 gal saltwater aquarium, employing filtration and lighting systems ranging from simplistic to sophisticated. The particularly hardy and cheap yellow-tailed blue damsels (Chromis xanthurus) are ideal microcosm inhabitants. Advanced microcosms can support invertebrates and living rock, as well. In the original conception of the laboratory, students monitor ten analytes in seawater. The laboratory covers the four chemical venues shown in Table 1 using techniques drawn largely from standard methods for the analysis of water (14). The degree of independence granted the student varies with instructor. Hughes provides a laboratory manual based on standard methods, modified slightly for use with seawater (15). At this college, students are responsible for the entire analysis from conception through integrated analysis and reporting. Students draw the name of their analyte out of a hat during the first meeting of the laboratory, then spend up
Table 1. Venues, Alternate Methods, and the Extensions To Study Ten Analytes in Seawater Venue
Beer’s law and the nitrogen cycle
Electrochemistry and ecosystem stability
Gravimetry and the sulfur cycle Titrimetry and reef structure
Analyte
Classical Method/Alternatives
Extensions
Ammonia/ ammonium
Spectrophotometric determination via the Nessler method/phenate method
Ion-selective electrode using the method of standard additions
Nitrite
Spectrophotometric determination with Griess’s reagent
---
Nitrate
Column-based reduction followed by spectrophotometric determination with Griess’s reagent
Enzymatic reduction followed by spectrophotometric determination with Griess’s reagent
Chloride (salinity)
Mohr method (argentometric titration with chromate indicator)/potentiometric detection with silver electrode of the second kind
---
Alkalinity (buffer capacity)
Potentiometric titration
---
Dissolved oxygen
Winkler method (iodometric titration)
---
Sulfate
Gravimetric determination via barium precipitation
Ion chromatography with an internal standard
Calcium
Complexometric titration with EDTA
----
Magnesium
Complexometric titration with EDTA
----
Phosphate
Spectrophotometric determination via the stannous chloride ---method/ascorbic acid method
NOTE: Venues are described by Hughes in the marine microcosm laboratory (1).
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to six weeks researching, developing, testing, calibrating, and validating a method after performing a literature search and consulting a range of primary and secondary sources in either print or electronic form (16–20). Students are responsible for making all solutions, locating the appropriate glassware for all operations, and the overall design and setup of their experimental stations. The instructor acts as a consultant during this process, providing opinions, asking questions, and providing “just-in-time” instruction on the fine points of various analytical techniques. The final product after these initial six weeks is a student-authored procedure that is published electronically for the rest of the class to use. These student-authored procedures typically parallel those described in Table 1 and usually draw heavily on references such as Standard Methods for the Analysis of Water and Wastewater (14), but invariably provide more details, practical advice, and pertinent background information than is found in secondary literature references. In addition, the student authors are forced to make informed judgments regarding expected interferences, standard concentrations, dynamic range, and so forth, as pertains to the seawater matrix. By the end of the six-week development period, students consistently obtain good baseline data on their analyte in the microcosm without fish present. In the seventh week the fish arrive and students move to different analytes, spending two successive weeks working individually on the determination of a single analyte using a peer’s procedure. The two-week period is opportune in that if there are no viable results generated from the first week of work, students will undoubtedly do much better the subsequent week when they are more familiar with the chemistry. Of course, if viable results are obtained both weeks, this increases the quantity of information in the semester-long data stream for that analyte. Each student submits the results of their work to the peer who wrote the procedure, along with an abstract describing the results and their significance. The instructor is copied on all abstracts. The peer receiving the data is responsible for validating the data and updating a running plot of this analyte concentration. All of these raw data are archived electronically on the course Web site. The instructor assigns analytes from the seventh week onward such that each student sees a wide range of analytical chemistry over the course of the semester. In this fashion, every student sees a potentiometric method, performs a titration utilizing complexometric, redox, or precipitation chemistry, and constructs a calibration curve for a spectrophotometric determination. The lab confers weekly to determine whether the class should intervene in the marine microcosm for the sake of fish well being based upon the results available to date and the students’ collective understanding of the overall microcosm chemistry. Typical concerns include what to do about rising nitrate and phosphate concentrations, and whether depleted water should be replaced with deionized water or new seawater. At the end of the semester, each student writes a report describing the determination of their analyte throughout the semester. This report places the analyte in the context of the complex chemistry and biology operating within the microcosm and describes statistical analyses to determine whether significant changes occurred in this chemistry during the sewww.JCE.DivCHED.org
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Figure 1. Standard addition plot for the determination of ammonia in seawater via ion selective electrode. V0 and Vs are the volumes of sample and spike, respectively; E is the observed cell potential; and s the slope per decade concentration of the electrode response curve (23). These data correspond to an ammonia concentration of 0.4 ± 0.1 ppm at the 95% confidence level.
mester. A course Web site facilitates the electronic exchange of procedures, raw data, running plots, and abstracts (21). Extensions In the course of using the marine microcosm approach for eleven years, we have developed many extensions to the original chemistry. Three of these extensions include determining: • Ammonia via ion-selective electrode using the method of standard additions • Sulfate via ion chromatography with an internal standard • Nitrate via enzymatic reduction
The first two extensions allow instrumental techniques to be added to the range of analysis options, thereby extending the chemistry that can be explored in this suite of experiments. Other established instrumental methods experiments suitable to the seawater matrix, such as the determination of strontium by atomic absorbance spectroscopy (12), or heavy metals by graphite furnace (22), can be added to the suite as well. Determining ammonia by ion-selective electrode introduces a second potentiometric method into the suite of existing experiments. While there are challenges to measuring sub-part-per-million quantities of ammonia in seawater, this technique gives relatively rapid and accurate results such that in the course of an afternoon laboratory period three replicate measurements can be made. In addition, determining ammonia with an ion selective electrode (ISE) allows for the introduction of standard addition techniques into the marine microcosm. Finally, determining ammonia via an ISE method allows for the removal of the mercuric iodide waste stream associated with the Nessler method. Example data for the determination of ammonia by ISE are shown in Figure 1.
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Figure 2. Ion chromatogram of seawater diluted 250-fold and treated with a 10 ppm bromide internal standard. The chromatogram shows eluant conductance in microSiemens versus retention time for (1) chloride, (2) bromide, and (3) sulfate corresponding to a sulfate concentration in undiluted seawater of 2500 ppm. These data were obtained on a Dionex 4500i ion chromatograph employing an AS-14 column, a mobile phase of 3.5 mM sodium carbonate/1.0 mM sodium bicarbonate at a flow rate of 1.1 mL min᎑1, and a sulfuric acid-based suppression column.
Figure 3. Calibration curve for nitrate in seawater as determined by reduction with corn leaf reductase followed by conversion of the resulting nitrite to an azo dye with Griess’ reagent. The absorbance is measured at 540 nm.
The determination of sulfate in seawater by precipitation, one of the original experiments in the suite, is a long, arduous affair that cannot be completed in a single afternoon laboratory period. In addition, the desirability of platinum crucibles to achieve precise results makes this an expensive prospect. Ion chromatography is an extremely useful technique in analytical environmental science (24–26). While it is still not possible with current technology to analyze undiluted seawater samples by this technique owing the high concentration of chloride ion present (∼0.6 M), it is possible to do accurate and fast analyses of chloride and sulfate with diluted seawater samples. Literature from the petroleum industry on brackish water and brines shows that approximately 250-fold dilution of seawater will allow samples to be analyzed via ion chromatography without overloading the column (27). (Freshwater samples are another matter—ion chromatography allows the simultaneous determination of fluoride, chloride, bromide, nitrate, nitrite, phosphate, and sulfate in undiluted samples.) An example ion chromatogram is shown in Figure 2. The third extension substitutes corn leaf nitrate reductase (28) for the traditional cadmium column used to reduce nitrate to nitrite. This substitution allows enzymatic techniques to be included in the laboratory and promotes skills in working with microliter volumes while eliminating the need for a cadmium-based reduction column. In addition, integration of biochemical concepts into core courses is one way to meet American Chemical Society Committee on Professional Training guidelines for instruction in biochemistry (29). A commercially-available kit containing the nitrate reductase, NADH cofactor, and all necessary buffers allows 100 samples to be run over a period of up to two months for
roughly $1 per sample. The chemistry used once nitrate has been reduced to nitrite is identical to that already employed for nitrite determination (albeit on a much smaller sample size). A typical calibration curve for nitrate in seawater by enzymatic reduction is shown in Figure 3.
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Evaluation Graduating seniors in confidential, one-on-one exit interviews with the department chair cite the marine microcosm as one of the most fulfilling and engaging instructional laboratories in the chemistry curriculum. They particularly appreciate the opportunity for self-direction on a problem with no single correct solution. Equipment • VWR SympHony ammonia ion selective electrode and SB series pH/ISE meter. • Dionex 4500i ion chromatograph fitted with an AS14 column, cation suppression column, and conductivity detector. • Nitrate laboratory test kit L-NTK-203 (30)
Hazards Griess’s reagent (a two-component system consisting of sulfanilamide in 3 M hydrochloric acid and naphthylethylenediamine hydrochloride in water) and concentrated sodium hydroxide (10 M) should be handled with gloves to prevent skin exposure. Both hydrochloric acid and sodium
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hydroxide are corrosive and sulfanilamide and naphthylethylenediamine hydrochloride are possible mutagens. Conclusions The marine microcosm laboratory is an ideal environment in which to teach the entire analysis process, from conception through planning, execution, analysis, and reporting, in an interesting matrix. It lends itself nicely to a studentcentered approach with opportunities for collaborative learning, and provides many openings to develop critical communication skills. The extensions described here allow an instructor to bring pedagogically useful chemistry emphasizing material typically taught in the chemical analysis, instrumental methods, and biochemistry courses to bear while mitigating chemical hazards. These extensions broaden the horizon of chemistry in an already exciting venue. W
Supplemental Material
Instructions for the students and notes about the details of the three extensions are available in this issue of JCE Online. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Hughes, K. D. Anal. Chem. 1993, 65, 883A–889A. Walters, J. P. Anal. Chem. 1991, 63, 977A–985A. Walters, J. P. Anal. Chem. 1991, 63, 1077A–1084A. Walters, J. P. Anal. Chem. 1991, 63, 1179A–1191A. Jackson, P. T.; Walters, J. P. J. Chem. Educ. 2000, 77, 1019– 1025. Phillips, D. N. Anal. Chem. 2002, 74, 427A–430A. Wenzel, T. J.; Austin, R. N. Env. Sci. Tech. 2001, 35, 326A– 331A. Wenzel, T. J. Microchim. Acta 2003, 142, 161–166. Grasselli, J. G. Anal. Chem. 1977, 49, 182A–192A. Selco, J. I.; Roberts, J. L., Jr.; Wacks, D. B. J. Chem. Educ. 2003, 80, 54–57. Grguric, G. J. Chem. Educ. 2000, 77, 495–498. Gilles de Pelichy, L. D.; Adams, C.; Smith, E. T. J. Chem. Educ. 1997, 74, 1192–1194.
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13. Calascibetta, F.; Campanella, L.; Favero, G.; Nicoletti, L. J. Chem. Educ. 2000, 77, 1311–1313. 14. Standard Methods for the Examination of Water and Wastewater, 18th ed.; American Public Health Association, American Water Works Association, Water Environment Federation: Washington, DC, 1992. 15. Hughes, K. D. Marine Microcosm Laboratory Manual. Personal communication. 16. Chemical Analysis of Inorganic Constituents of Water; CRC Press: Boca Raton, FL, 1982. 17. Strickland, J. D. H. A Practical Handbook of Seawater Analysis, 2nd ed.; Fisheries Research Board of Canada: Ottawa, Canada, 1972. 18. Vogel, A. I. Vogel’s Textbook of Quantitative Inorganic Analysis, 4th ed.; Longman Scientific & Technical (Wiley): New York, 1987. 19. Boltz, D. F. Colorimetric Determination of Nonmetals, 2nd ed.; Wiley: New York, 1978. 20. Fresenius, W.; Quentin, K. E.; Schneider, W. Water Analysis: A Practical Guide to Physico-chemical, Chemical, and Microbiological Water Examination and Quality Assurance; Springer– Verlag: New York, 1988. 21. Van Ryswyk, H. The Aquarium Project. http://www4. hmc.edu:8001/chemistry/109 (accessed Oct 2006). 22. Quigley, M. N.; Vernon, F. J. Chem. Educ. 1996, 73, 671–675. 23. Harris, D. C. Quantitative Chemical Analysis, 6th ed.; W. H. Freeman: New York, 2003. 24. Sinniah, K.; Piers, K. J. Chem. Educ. 2001, 78, 358–362. 25. Xia, K.; Pierzynski, G. J. Chem. Educ. 2003, 80, 71–75. 26. Whelan, R. J.; Hannon, T. E.; Zare, R. N. J. Chem. Educ. 2004, 81, 1299–1302. 27. Singh, R. P.; Pambid, E. R.; Abbas, N. M. Anal. Chem. 1991, 63, 1897–1901. 28. Patton, C. J.; Fischer, A. E.; Campbell, W. H.; Campbell, E. R. Env. Sci. Tech. 2002, 36, 729–735. 29. American Chemical Society Committee on Professional Training. Undergraduate Professional Education in Chemistry: Guidelines and Evaluation Procedures, 2003. 30. Available from the Nitrate Elimination Co. Inc., 334 Hecla Street, Lake Linden, MI 49945. 1-888-648-7283 http:// www.nitrate.com (accessed Oct 2006).
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