Chemical Education Today edited by
NSF Highlights
Susan H. Hixson
Projects Supported by the NSF Division of Undergraduate Education
National Science Foundation Arlington, VA 22230
Richard F. Jones
Biochemical Applications in the Analytical Chemistry Lab
Sinclair Community College Dayton, OH 45402-1460
by Cynthia Strong* and Jeffrey Ruttencutter
The application of analytical techniques to complex biological systems is one of the fastest growing areas of analytical chemistry. According to a recent compilation (1), seven of the 10 most-cited papers in Analytical Chemistry concern biochemical applications. At Cornell College, we find that many of our chemistry students have a strong interest in biochemistry. On the national level, the American Chemical Society’s Committee on Professional Training (CPT) has adopted a biochemistry requirement for ACS-certified majors, which we will fulfill by integrating biochemical topics throughout the chemistry curriculum. The project described in this report focuses on the sophomore-level analytical lab. We identified an HPLC and a UV–visible spectrophotometer as instruments that would help us incorporate more biologically-relevant experiments into the course, in order to increase our students’ understanding of selected biochemistry topics and enhance their ability to apply an analytical approach to biochemical problems. With funding from the NSF, we have acquired the HPLC1 and UV–vis;2 here we report our progress with bringing a biochemical emphasis to the analytical course. We are developing or adapting three experiments for the analytical course.3 In the first experiment, students use the UV–visible spectrophotometer and the Bio-Rad protein assay (2) to determine total protein. The Bio-Rad assay is based on the Bradford method; we chose it because it is quick, inexpensive, and widely used. For the unknown, we use a sample that was right under our students’ noses: powdered cocoa mix. (The analytical course at Cornell is always taught during the January term, and we provide a pot of hot water and a steady supply of cocoa mix in a study room adjacent to the lab.) Students prepare a dilute solution of cocoa mix, use micropipettors to prepare a series of standards and add the dye reagent, and then record the visible spectra. The dye reagent changes from red to blue when it binds to protein; the absorbance is read at 595 nm. Students record and overlay the visible spectra for their standards and samples, rather than simply recording the absorbance at a single wavelength, and it is immediately obvious whether or not the standards were prepared with sufficient care. (If precisely the same amount of dye reagent is added to each standard, there will be a clear isosbestic point.) The students realize that automatic pipettors, just like burets, pipets, and analytical balances, must be used properly in order to provide reproducible results. This experiment was designed to replace the determination of iron in breakfast cereal, in which the absorbance was monitored at a single wavelength. Like the old experiment, the new experiment teaches students about spectrophotometric methods of analysis and the use of a standard 1706
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curve with linear regression. In addition, the new experiment gives students experience with applying analytical chemistry to a biochemical problem. In the second experiment for the analytical course, students apply a combination of the FMOC (fluorenylmethyl chloroformate) and OPA (o-phthalaldehyde) precolumn derivatization methods (3) to the determination of amino acids in a powdered protein nutritional supplement. The technique of amino acid analysis is of critical importance in biochemical research as well as in medicinal chemistry and the food industry. Students hydrolyze the protein powder in 6 M HCl, derivatize the amino acids, and analyze the mixture by HPLC on a C18 column, with UV detection. Each student or pair of students determines the position of one or two amino acids in the chromatogram and the concentration of those amino acids; the results of the class are combined to provide a complete analysis of the protein powder. Using an autosampler, students are able to load many samples into the HPLC and run them overnight. Figure 1 shows a chromatogram for a mixture of 18 amino acids. This experiment was tested with the advanced analytical chemistry class; we plan to use it in the sophomore-level analytical class next year. A third new experiment is an adaptation of one published in this Journal (4), in which students develop a method to separate a mixture of several biological molecules using lowpressure gel filtration and ion exchange columns. The compounds used exhibit a range of colors, sizes, and charges. After separating the components, our students measure the UV– visible spectrum of each component and compare it to the spectrum of the pure compound to obtain a qualitative mea-
Figure 1. HPLC separation of 18 amino acid standards. Sample: 0.30 mM each amino acid; injection volume 0.5 L. Column: Zorbax Rx-C18, 4.6 ⫻ 150 mm. Eluents: A = 40 mM sodium phosphate, pH 7.8; B = 45% acetonitrile, 45% methanol, 10% water; 2 mL/min; gradient of 0 to 57% B in 20 minutes. Detection at 338 nm. Column temperature 40 ⬚C.
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Chemical Education Today
sure of the effectiveness of the separation. This experiment could be made quantitative by providing students with molar absorptivity values for the components and asking them to calculate the percent recovery of each component. The three experiments described above involve amino acids, peptides, and proteins. Pre-lab and post-lab discussions can include a review of amino acids, peptide bond hydrolysis, and protein structure. Students learn about protein primary structure in organic chemistry and about enzymes as catalysts in general chemistry; thus, these new experiments in the analytical lab build on students’ existing knowledge. Our new experiments complement existing biologicallyrelevant experiments in the analytical course. These include the potentiometric determination of the pKa values of the side chain and amino end of an amino acid and the iodometric determination of the concentration of vitamin C in orange juice. In addition, we have replaced our previous gas chromatography lab with the determination of ethanol in (theatrical) blood (5). In this last experiment, students gain experience with the important technique of headspace analysis of a volatile analyte in a complex biological matrix. Although the sophomore-level analytical course is the focus of this project, our effort to include biochemical topics in analytical chemistry has extended to the advanced analytical course as well. We are developing a spectroelectrochemistry experiment in which students will use the UV–visible spectrophotometer to determine the reduction potential of cytochrome c. In addition, students will use the HPLC to develop a method to separate a mixture of proteins on small, inexpensive plastic columns containing ion exchange or gel filtration media. Through this project, we sought to bring more biochemistry into the analytical course, in response to the increasing importance of biochemical applications in modern analytical chemistry and the high level of interest in biochemistry among our students. While it would be difficult to remove material from the lecture portion of the analytical course in order to incorporate more biochemistry, we have found that it is feasible to teach certain biochemical concepts in the context of the analytical chemistry lab. Biochemical examples in the analytical course help our students understand the relevance of analytical chemistry to biochemistry and the
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importance of performing biochemical experiments with precision and accuracy. In addition, chemistry students who do not plan to take the biochemistry course will develop a greater understanding of selected biochemical concepts. Acknowledgments The authors thank the National Science Foundation’s Division of Undergraduate Education for their support in purchasing the HPLC system and UV–visible spectrophotometer (Award #DUE-0126921, CCLI-A&I). Matching funds were generously provided by Cornell College. Notes 1. Agilent 1100 HPLC system with diode array detector and ChemStation software. 2. Varian Cary 50 spectrophotometer. 3. Find additional details about these experiments at http:// www.cornellcollege.edu/chemistry/cstrong/bioanalyt.shtml (accessed Oct 2004). The Web site lists references for bioanalytical lab experiments found in this Journal and other sources.
Literature Cited 1. Riordan, J.; Zubritsky, E.; Newman, A. Anal. Chem. 2000, 72, 324A–330A. 2. Bio-Rad Protein Assay, Bulletin 9004; Bio-Rad Laboratories, 1994. 3. (a) Henderson, J. W.; Ricker, R. D.; Bidlingmeyer, B. A.; Woodward, C. Application note #5980-1193E, Agilent Technologies, 2002; (b) Einnarson, S.; Josefsson, B.; Lagerkvist, S. J. Chromatogr. 1983, 282, 609–618; (c) Clapp, C. H.; Swan, J. S.; Poechmann, J. L. J. Chem. Educ. 1992, 69, A124–126. 4. Gorga, F. R. J. Chem. Educ. 2000, 77, 264–265. 5. Zabzdyr, J. L.; Lillard, S. J. J. Chem Educ. 2001, 78, 1225– 1227.
Cynthia Strong is in the Department of Chemistry, Cornell College, Mount Vernon, IA 52314; cstrong@ cornellcollege.edu; Jeffrey Ruttencutter is now a D.D.S./ Ph.D. student at the University of Illinois at Chicago.
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