In the Laboratory
Inquiry-Based Approach to a Carbohydrate Analysis Experiment Edward G. Senkbeil Department of Chemistry, Henson School of Science and Technology, Salisbury State University, Salisbury, MD 21801-6860
Chemical education needs to focus more attention on the learning process rather than the teaching process. The question of how students best learn has been an issue in several editorials in this Journal (1–3). I agree with the idea that students learn more by doing than by watching. This analysis of an unknown carbohydrate in an inquiry-based learning format has proven to be a valuable undergraduate biochemistry laboratory experiment, and incorporates many principles of carbohydrate chemistry normally taught in the lecture setting. This experience emphasizes the inquiry-based format (4 ) in which students are given an opportunity to pose their own questions, design and pursue their own investigations, analyze data, and present their findings. The basic wet chemistry procedures for carbohydrate analysis are well characterized in undergraduate laboratory manuals (5–7), and a recent publication in this Journal (8) emphasized utilizing several such procedures in prepared flow charts for students to follow in identification of carbohydrates. Although appropriate for nonmajor chemistry students, this approach is inadequate in providing upper-level science majors an opportunity to formulate their own experimental protocol to identify the unknown carbohydrate. In this experiment, students are required to gather information about carbohydrates in advance of the experiment and apply that information directly in the methods used to analyze the unknown. These methods may include not only wet chemistry but also instrumental techniques. Because of the number of possible identification methods, the 3-hour laboratory experience can be tailored to the available materials and instrumentation. Procedure The following experimental guidelines are given to the students two weeks prior to the lab. A solid sample (0.5 g) of an unknown carbohydrate will be provided for identification. The unknown will be one of the carbohydrates from the following list. arabinose fucose glucose fructose galactose mannose
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ribose xylose sorbose N-acetylglucosamine
1. Read Chapter 11, “Carbohydrates”, in the lecture text (9), being sure to emphasize information on structure and analysis. 2. Draw the structures of all the possible carbohydrates listed for the unknown. 3. Review the texts (5–7, 10, 11) on reserve in the library (or any other appropriate resources) relative to methods for classifying/identifying carbohydrates. 4. Study the structures of the different possible unknowns and formulate which identification tests to use for separation and identification of the unknown. 5. Make a flow chart of the protocol for identification of unknown (minimum of 5 possible tests/analyses). 6. Provide a list of needed reagents and equipment to the instructor one week in advance of laboratory period. These will be checked by instructor to assure availability of reagents and equipment, and note any special safety concerns. 7. During the scheduled laboratory period, do the analyses following the protocol of your flow chart. 8. The laboratory report should include objectives, procedure, structures of all possible unknowns, flow chart, data, results of analyses, chemical explanation of methods used, conclusions, and list of references.
Results and Discussion There are several advantages to running this type of guided inquiry lab. Students must acquire and assimilate the appropriate information to analyze and correctly identify the unknown carbohydrate. The open-ended format allows the students flexibility in designing the series of experiments to follow in their flow chart (see Fig. 1 for a cumulative example) for carbohydrate identification. This laboratory is conducted before the lecture discussion of carbohydrates and significantly decreases the lecture time required for this topic. Students are quite successful in the experiment (~88% correctly identify unknown) and have expressed a feeling of accomplishment upon completing the experiment. Incorrect unknown identification by students is commonly due to misinterpretation of experimental data.
Journal of Chemical Education • Vol. 76 No. 1 January 1999 • JChemEd.chem.wisc.edu
In the Laboratory arabinose, fructose, fucose, galactose, glucose, mannose, N -acetylgulcosamine, ribose, sorbose, xylose Seliwanoff's test for ketohexoses (5 ) (+)
(–) arabinose, fucose, galactose, glucose, mannose, ribose, xylose, N -acetylgulcosamine,
sorbose, fructose Identification by melting point or phenylosazone test (11)
Bial's test for aldopentoses (5)
(+)
(–)
arabinose, ribose, xylose Identification by melting point, chromatography (6), or phenylosazone test (11)
fucose, galactose, glucose, mannose, N -acetylgulcosamine Glucose oxidase Test (7 )
(–) fucose, galactose, mannose, N –acetylgulcosamine
(+) glucose
Morgan–Elson test (5 ) (–)
(+)
N -acetylgulcosamine
fucose, galactose, mannose 6-Deoxyhexose test (10 )
(+) fucose
(–) galactose, mannose Identification by galactose oxidase test (12 ), phenylosazone test (11), polarimetry (6 ), or melting point
Figure 1. Example flow chart for identification of unknown carbohydrate.
analysis. Melting points are always a method of choice, but close similarities in melting points of several sugars and varying melting points for the different forms of a sugar are limiting factors. The introduction of N-acetylglucosamine into the unknown list raises interesting questions about its reactivity and metabolic significance compared to glucose. Students quickly realize (by trial and error) the need to run blanks and standards for colorimetric or timed studies. A standard aldopentose, aldohexose, and ketohexose are provided for the experiment. Students must be reminded of safety factors when conducting some of the qualitative tests. There are a variety of experimental options that can be utilized for the identification of the unknown. Oligosaccharides or polysaccharides may be added to the list of possible unknowns, especially if the detection of reducing and nonreducing sugars is of interest. More instrumental techniques could be incorporated into the experiment depending on availability in the laboratory. Analysis of carbohydrates by HPLC would be a valuable technique, but would require additional lab time. Students may wish to use IR and NMR analysis, but results are limited owing to similarities of the monosaccharide structures. This inquiry-based laboratory experience provides a high degree of flexibility to both the instructor and students in the design of the experiment. This laboratory requires a considerable investment of time by the instructor. Instructors must carefully review the chemicals requested by students and respond in advance of the lab if there are any problems or concerns. There is a high degree of instructor–student interaction during the lab. Students often find new methods of analysis and this always adds interest to the lab. Students commonly comment that this is one of the most time-consuming labs, but also the most satisfying because of a sense of accomplishment and completion. Literature Cited 1. 2. 3. 4. 5. 6.
There are a variety of points and student pitfalls worth noting. Students will sometimes perform tests to indicate the presence of carbohydrates (such as Molisch test), which provide no helpful information. Students often perform Benedict’s test for reducing sugars. All results will be positive because all the possible unknowns are reducing monosaccharides. Paper chromatography is often used, but this only resolves some of the sugars in the limited setup and run time (approximately 3 hours). Polarimetry is often a method of choice by students, but the total size of unknown sample and type of available polarimeter might prohibit this method of
7. 8. 9. 10.
11. 12.
Moore, J. W. J. Chem. Educ. 1997, 74, 613. Moore, J. W. J. Chem. Educ. 1997, 74, 365. Moore, J. W. J. Chem. Educ. 1997, 73, A291. Willis, S. Curriculum Update 1995, 1–8. Robyt, J. F.; White, B. J. Biochemical Techniques: Theory and Practice; Wadsworth: Belmont, CA, 1987; pp 213–227. Clark, J. M.; Switzer, R. L. Experimental Biochemistry, 2 ed.; Freeman: New York, 1977; pp 147–158. Minch, M. J.; Experiments in Biochemistry: Projects and Procedures; Prentice Hall: Englewood Cliffs, NJ, 1989, pp 283-294. Malherbe, J. S.; Meyer, C. J. J. Chem. Educ. 1997, 74, 1304. Cox, M. M.; Nelson, D. L.; Lehninger, A. L. Principles of Biochemistry, 2nd ed.; Worth: New York, 1993; pp 299–321. Dische Z. In Methods in Carbohydrate Chemistry, Vol. 1; Whistler, R. L.; Wolfrom, M. L., Eds.; Academic: New York, 1962; p 501. Landgrebe, J. A.; Theory and Practice in the Organic Laboratory, 2nd ed.; Heath: Toronto, 1977; pp 489–490. Roth, H.; Segal, S.; Bertoli, D. Anal. Biochem. 1965, 12, 509.
JChemEd.chem.wisc.edu • Vol. 76 No. 1 January 1999 • Journal of Chemical Education
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