Communication pubs.acs.org/jchemeduc
Spectroscopy 101: A Practical Introduction to Spectroscopy and Analysis for Undergraduate Organic Chemistry Laboratories Lucas A. Morrill, Jacquelin K. Kammeyer, and Neil K. Garg* Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095-1569, United States S Supporting Information *
ABSTRACT: An undergraduate organic chemistry laboratory that provides an introduction to various spectroscopic techniques is reported. Whereas organic spectroscopy is most often learned and practiced in the context of reaction analyses, this laboratory experiment allows students to become comfortable with 1H NMR, 13 C NMR, and IR spectroscopy, in addition to mass spectrometry. The laboratory experiment also teaches students how to perform thin-layer chromatography. The specific compound analyzed is isoamyl acetate, commonly known as “banana oil”. With the skills acquired from completing this laboratory experiment early in a given course, students are well-prepared to perform spectroscopic analyses in subsequent experiments encountered in their organic chemistry laboratory course.
KEYWORDS: Second-Year Undergraduate, Organic Chemistry, Laboratory Instruction, Inquiry-Based/Discovery Learning, Spectroscopy, Chromatography, NMR Spectroscopy, IR Spectroscopy, Mass Spectrometry
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PEDAGOGICAL GOALS Spectroscopy is critical to organic chemistry research, but often can be daunting to students in introductory organic chemistry. This laboratory experiment was designed to introduce students to practical spectroscopic techniques in the laboratory classroom in a low stress, pleasant environment. The specific learning goals for the students are as follows: • To provide a forum for students to practice a number of fundamental laboratory techniques, including pipetting and preparing samples for spectroscopic analysis. • To teach students the key concepts and practical aspects associated with performing thin-layer chromatography, including compound visualization and analysis.
pectroscopy is an integral component of undergraduate chemistry education.1 In lectures, students are commonly taught the theory behind various spectroscopic methods and how to analyze data. This provides students with an important opportunity to hone their problem-solving skills. However, the transition from this “on-paper” understanding of spectroscopy to performing spectroscopic analysis in the laboratory is daunting for most students. We therefore questioned if there was a simple way to help students practice the hands-on use of spectroscopy and other analytical methods in a way that would help them excel for the duration of an organic chemistry laboratory course. Although examples of an introduction to a particular spectroscopic technique have been reported,2 a laboratory concept that introduces the combination of sample preparation, chromatography, and multiple spectroscopic techniques at the beginning of an organic chemistry laboratory course remains underdeveloped. To address this notion, we have developed a laboratory experiment in which students perform several forms of analysis of isoamyl acetate, otherwise known as “banana oil” (Figure 1). The experiment teaches students how to prepare samples for 1 H NMR and 13C NMR, IR, and GC−MS analyses. Additionally, the students perform analysis by TLC, a tool routinely used by organic chemists. Finally, the students have the opportunity to predict 1H NMR spectra using online software, thus exposing them to the power of modern computational chemistry.3 The experiment provides students with the fundamental analytical skills and confidence they need at the beginning of a term to perform more advanced experiments involving chemical reactions throughout the duration of the term. © XXXX American Chemical Society and Division of Chemical Education, Inc.
Figure 1. Analyses of isoamyl acetate. Received: April 11, 2017 Revised: July 20, 2017
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DOI: 10.1021/acs.jchemed.7b00263 J. Chem. Educ. XXXX, XXX, XXX−XXX
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• To help students learn to confidently interpret NMR, IR, and GC−MS data. • To expose students to common instrumentation and practices used by organic chemists. • To showcase the growing power of computational chemistry, by having students perform a spectroscopic simulation using accessible online software. • To promote a spirit of confidence and collaboration among students, with the aim of helping them excel when conducting more challenging experiments.
(b) Had diagnostic IR stretching frequencies representative of at least one common functional group. (c) Was sufficiently volatile to undergo GC−MS analysis, and (d) Was sufficiently nonvolatile to be analyzed using TLC. As such, isoamyl acetate (“banana oil”) was selected (bp 142 °C). Although the experiment would be performed in a ventilated fume hood, the pleasant and familiar smell of isoamyl acetate was deemed a benefit. The optimized experimental protocol was first implemented during one term of an introductory undergraduate organic chemistry laboratory course and repeated three times thereafter. Students had previously been taught the theory behind various spectroscopic techniques and chromatography in lecture. However, they had minimal (or variable) prior experience with such techniques in a laboratory setting. The students were required to read the laboratory handout, which covered experimental details and various background information on spectroscopy and chromatography. Students completed a prelaboratory worksheet, which was designed to make sure students understood the key concepts, the experimental protocol, and safety considerations. Students were required to complete the prelaboratory worksheet prior to carrying out the experiment. Students were placed in pairs, although the experimental protocol could easily be performed individually. With isoamyl acetate available, the main aspects of this experiment included preparing samples for analysis, performing TLC analysis, and analyzing spectroscopic data after data collection. In addition to using the IR spectrometer, students had the opportunity to see NMR and GC−MS instrumentation in person (data collection for NMR and GC−MS spectra was performed by a teaching assistant, although it could be performed by students if facilities allow). A required postlaboratory worksheet was used to facilitate data analysis and further expand on the key chemical concepts from this experiment. Included in the postlaboratory was an opportunity for students to use online software to predict NMR spectra. All 69 students who initially carried out the experiment were able to successfully acquire the data needed for analysis (student spectra are given in the Supporting Information). However, in a few cases, data analysis was nondetrimentally hampered by dilute samples. With regard to analysis, the following statistics were noted: (a) 68 students (99%) presented reliable TLC data and correctly calculated Rf values. (b) 56 students (81%) correctly assigned their 1H NMR data. (c) 30 students (43%) correctly assigned their 13C NMR data. (d) 51 students (74%) correctly assigned their GC−MS data (i.e., identification of isoamyl acetate and the acetate fragment). (e) 66 students (96%) correctly assigned their IR data, specifically denoting the ester carbonyl present in isoamyl acetate. In addition, as part of the postlaboratory assignment, students were asked about the spectroscopic techniques they had utilized and asked to comment on their utility. The average grade on this question was 3.1 out of 4 possible points. These data collectively demonstrate significant student learning and understanding. Moreover, in some cases (e.g., NMR data interpretation), the results show that even further practice would be valued. Student comments regarding this experiment are available in the Supporting Information.
OVERVIEW OF LABORATORY EXPERIMENT This laboratory experiment is designed for an introductory undergraduate organic chemistry laboratory course. The experimental protocol requires 2−3 h to finish and can be performed over a single laboratory period. Prior to the laboratory period, students complete a prelaboratory worksheet to ensure they understand the experiment and any safety concerns. Completion of a postlaboratory worksheet further promotes student understanding and helps the students perform a critical analysis of their results. Experiment
Students work in pairs to complete this laboratory experiment. Students add five drops of isoamyl acetate to a test tube containing 1.5 mL of CDCl3. A portion is then transferred into an NMR tube. Students label their samples, and the samples are submitted to the instructor. The remaining isoamyl acetate solution is then used for thin-layer chromatography and IR analysis. For TLC analysis, two silica gel TLC plates are spotted with the sample. The two plates are developed using different mobile phases of varying polarity (1:2 and 1:4 ethyl acetate/hexanes). After TLC analysis and allowing the plates to dry, each TLC plate is visualized by anisaldehyde staining. The remaining solution of isoamyl acetate in CDCl3 can be used for IR spectroscopic analysis. One drop of the sample is loaded into the IR spectrometer equipped with ATR attachment. After evaporation of the CDCl3, an IR spectrum is acquired. To prepare samples for the GC−MS, a drop of isoamyl acetate is added to a standard GC−MS vial. The sample is then diluted with methanol (1 mL) and submitted for analysis. A detailed description of the experiment is described in the Supporting Information.
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HAZARDS Closed-toed shoes, long pants covering the ankles, safety glasses, gloves, and flame-resistant laboratory coats should be worn at all times. All hazardous materials should be handled and disposed of in accordance with the recommendation of the materials’ safety data sheets and EH&S. Isoamyl acetate is an irritant. Ethyl acetate and hexanes are flammable and are volatile organic solvents. The n-hexane in hexanes is a neurotoxin. Deuterated chloroform (CDCl3) is a cancer suspect agent and mutagen. Methanol is flammable and toxic by inhalation, contact with skin, or ingestion. Anisaldehyde is an irritant.
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RESULTS AND DISCUSSION For development of this experiment, the choice of compound used for analysis was critical. More specifically, we sought a compound that (a) Would provide interesting, yet not unreasonably complex 1 H and 13C NMR spectra. B
DOI: 10.1021/acs.jchemed.7b00263 J. Chem. Educ. XXXX, XXX, XXX−XXX
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Discussion Topics
ACKNOWLEDGMENTS We thank the students and teaching assistants (Emma BakerTripp, Shane Bradner, Junyong Kim, Marco Messina, Jin Park, Ethan Rosser, Bryan Simmons, Robert Susick) of the Chem 30BL organic chemistry laboratory course for their feedback on this experiment. L.A.M. thanks the Foote family for financial support. The authors are grateful to the University of California, Los Angeles, for financial support. These studies were supported by shared instrumentation grants from the NSF (CHE-1048804) and the National Center for Research Resources (S10RR025631).
A range of important topics relevant to undergraduate organic chemistry lecture material and modern laboratory practices were discussed. In addition to the critical discussion of laboratory safety,4 students were taught about the entire spectrum of electromagnetic radiation.5 This provides a segue to discuss nuclear magnetic resonance and infrared spectroscopy from a theoretical and practical standpoint. A discussion of proper sample preparation methods should not be overlooked, as these are new to most students. Similarly, the theory and practice of mass spectrometry was discussed, including explanations of commonly used instrumentation and accessories (e.g., GC−MS, DART). Given that students perform TLC, along with GC−MS, this laboratory provides ample opportunity to discuss the principles and practical aspects associated with chromatography. The discussion concerning TLC also helps to prepare students for later discussions pertaining to column chromatography using silica gel, which is another technique commonly used and taught at the undergraduate level. Finally, the applications and impact of spectroscopic methods in modern society can be discussed. Examples include MRI scans,6 athletic drug testing,7 and analysis of trace drug contaminants on currency.8 Nobel Prizes awarded that pertain to spectroscopy also serve as excellent discussion topics.9 In addition, the use of a pleasant smelling chemical, isoamyl acetate, facilitates the discussion of the many applications of organic chemistry, specifically the flavor/fragrance industry.
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CONCLUSIONS A simple experimental protocol intended to help students acclimate to undergraduate organic chemistry instructional laboratories was developed. The protocol has students analyze isoamyl acetate using 1H NMR, 13C NMR, and IR spectroscopy. In addition, GC−MS and TLC analyses are performed. By performing this laboratory, students are exposed to a variety of common laboratory techniques and spectroscopic methods. Having completed this introductory experiment, students will have gained knowledge, analytical skills, and confidence, without the added complexity of performing a chemical reaction. As such, students will be better positioned to excel in their future organic chemistry laboratory coursework. ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.7b00263. Detailed student handout, a prelaboratory worksheet, a postlaboratory worksheet, notes for instructors, and spectra of isoamyl acetate, including spectra from students (PDF, DOCX)
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REFERENCES
(1) Zubrick, J. W. The Organic Chem. Lab Survival Manual: A Student’s Guide to Techniques, 9th ed.; John Wiley and Sons: Hoboken, NJ, 2012; Chapter 33. (2) (a) Baer, C.; Cornely, K. Spectroscopy of Simple Molecules. J. Chem. Educ. 1999, 76 (1), 89−90. (b) Shine, T. D.; Glagovich, N. M. Organic Spectroscopy Laboratory: Utilizing IR and NMR in the Identification of an Unknown Substance. J. Chem. Educ. 2005, 82 (9), 1382−1384. (c) Liotta, L. J.; James-Pederson, M. Identification of an Unknown Compound by Combined Use of IR, 1H NMR, 13C NMR, and Mass Spectrometry: A Real-Life Experience in Structure Determination. J. Chem. Educ. 2008, 85 (6), 832−833. (d) Chamberlain, P. H. Identification of an Alcohol with 13C NMR Spectroscopy. J. Chem. Educ. 2013, 90 (10), 1365−1367. (3) For a recent example of an undergraduate computational chemistry laboratory, see: Esselman, B. J.; Hill, N. J. Integration of Computational Chemistry into the Undergraduate Organic Chemistry Laboratory Curriculum. J. Chem. Educ. 2016, 93 (5), 932−936. (4) For a discussion on safety in the undergraduate laboratory, see: Alaimo, P. J.; Langenhan, J. M.; Tanner, M. J.; Ferrenberg, S. M. Safety Teams: An Approach to Engage Students in Laboratory Safety. J. Chem. Educ. 2010, 87 (8), 856−861. (5) Gans, D. M. The Electromagnetic Spectrum as an Analytical Tool. J. Chem. Educ. 1944, 21 (9), 421−429. (6) Hull, L. A. A Demonstration of Imaging on an NMR Spectrometer. J. Chem. Educ. 1990, 67 (9), 782−783. (7) Werner, T. C.; Hatton, C. K. Performance-Enhancing Drugs in Sports: How Chemists Catch Users. J. Chem. Educ. 2011, 88 (1), 34− 40. (8) Heimbuck, C. A.; Bower, N. W. Teaching Experimental Design Using a GC-MS Analysis of Cocaine on Money: A Cross-Disciplinary Laboratory. J. Chem. Educ. 2002, 79 (10), 1254−1256. (9) (a) Vestling, M. M. Using Mass Spectrometry for Proteins. J. Chem. Educ. 2003, 80 (2), 122−124. (b) Cavagnero, S. Using NMR to determine Protein Structure in Solution. J. Chem. Educ. 2003, 80 (2), 125−127. (c) Fry, C. G. The Nobel Prize in Medicine for Magnetic Resonance Imaging. J. Chem. Educ. 2004, 81 (7), 922−932.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Neil K. Garg: 0000-0002-7793-2629 Notes
The authors declare no competing financial interest. C
DOI: 10.1021/acs.jchemed.7b00263 J. Chem. Educ. XXXX, XXX, XXX−XXX
