Laboratory Experiment pubs.acs.org/jchemeduc
Fitting It All In: Adapting a Green Chemistry Extraction Experiment for Inclusion in an Undergraduate Analytical Laboratory Heather L. Buckley, Annelise R. Beck, Martin J. Mulvihill, and Michelle C. Douskey* Department of Chemistry, University of California, Berkeley, Berkeley, California 94720, United States S Supporting Information *
ABSTRACT: Several principles of green chemistry are introduced through this experiment designed for use in the undergraduate analytical chemistry laboratory. An established experiment of liquid CO2 extraction of D-limonene has been adapted to include a quantitative analysis by gas chromatography. This facilitates drop-in incorporation of an exciting experiment into an existing curriculum. The experiment provides an introduction to natural product extraction, calibration curves, and internal standards while simultaneously demonstrating alternative solvent selection for pollution prevention and increased chemical safety.
KEYWORDS: First Year Undergraduate/General, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Calibration, Gas Chromatography, Food Science, Green Chemistry, Phases/Phase Transitions, Quantitative Analysis
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Students compare several methods of quantitation of an isolated natural product and are specifically asked to consider alternative assessment with the idea that greener choices can be made when designing an analytical method.6 In addition to meeting the teaching objectives of analytical chemistry, this experiment maintains the introduction of green chemistry principles that motivated the original creation of the experiment. Students are introduced to several of the 12 principles of green chemistry,7 including pollution prevention, safer solvents, energy efficiency, renewable feedstocks, design for degradation, and safer chemistry for accident prevention (Table 1). Relatively little work has been done in the development of green analytical chemistry for the undergraduate laboratory to-date;8−12 this experiment aims to help fill this gap.
s green chemistry becomes increasingly prevalent in both academic research and chemical industry, there are ongoing calls for a “green” emphasis in undergraduate curriculum.1 A new generation of scientists who embrace these principles and techniques as a natural part of doing chemical research will revolutionize how we think about chemistry in a way that is more difficult for established scientists. The liquid CO2 extraction of limonene from orange rind, published in 2004 by McKenzie et al.,2 has become a “classic” undergraduate laboratory experiment among early adopters of green chemistry curriculum.3−5 Students are engaged by the unconventional nature of the experiment, from the preparation of food products for extraction to the easily observed simultaneous existence of carbon dioxide in solid, liquid, and vapor forms. However, a challenge that limits the widespread use of this and other innovative new experiments is the already overpacked nature of current introductory chemistry curricula. The addition of yet another idea or experiment often comes at the expense of thorough coverage of fundamental material. In this contribution, we describe the adaptation of this green chemistry laboratory experiment to meet teaching objectives in the introductory analytical chemistry curriculum. The experiment has been used as a “drop-in” replacement for another more mundane analysis as part of the early stages of a broaderbased revamp of our chemistry laboratory curriculum. It has been implemented with a class of approximately 200 first-year undergraduate chemistry majors. The experiment introduces the preparation of a natural product sample for analysis by GC−FID (gas chromatography−flame ionization detector) and the concepts of internal standards and calibration curves. © XXXX American Chemical Society and Division of Chemical Education, Inc.
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EXPERIMENTAL OVERVIEW
Extraction Procedure
This experiment is divided into two major parts: extraction and analysis (Figure 1). The extraction takes approximately 1.5 h, and the sample preparation for analysis takes approximately 45 min. Liquid CO2 is used to extract D-limonene from orange rind, which closely follows the procedure reported by McKenzie et al.2 Grated orange rind is placed in a centrifuge tube that is packed with dry ice. This tube is sealed and heated enough to generate liquid CO2 in the tube; this serves as a solvent to extract orange oil and then evaporates completely. The orange oil is diluted with ethyl acetate and analyzed by
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Table 1. Application of the 12 Principles of Green Chemistry7 Principle 1. Pollution Prevention 5. Safer Solvents and Auxiliaries 6. Design for Energy Efficiency 7. Use of Renewable Feedstocks 10. Design for Degradation 12. Inherently Safer Chemistry
Application in this Experiment Liquid CO2 is used in place of chlorinated solvents; gaseous CO2 is generated, but toxic waste is eliminated All of the waste generated in the extraction can be disposed in compost or regular trash Ethyl acetate is used in place of methylene chloride No energy is required to evaporate solvent after extraction; life cycle analysis for the generation of dry ice vs methylene chloride must be considered13 CO2 can potentially be captured rather than produced from fossil fuel sources Ethyl acetate biodegrades into ethanol and acetic acid Less toxic solvents result in a safer laboratory environment due to reduced exposure risks; the explosion potential from pressurized liquids and gases must be considered here
Figure 1. Extraction of D-limonene from orange rind using liquid CO2.
chemistry by using a safer solvent. This can be highlighted in discussion with students.
GC−FID. The quantity of D-limonene in the extract is determined through the use of four types of calibration curves, whose relative reliability are compared in student analysis of their data. For this experiment, students work in pairs but perform their data analysis individually.
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HAZARDS Dry ice is very cold and is a frostbite hazard; most notably vessels containing dry ice become very cold to the touch and should be handled with insulating gloves. Providing test tube racks to hold the centrifuge tubes also limits exposure of students’ hands. Orange oil (predominantly D-limonene) and anisole are harmful if swallowed in quantity and can be a skin, eye, and respiratory irritants. Ethyl acetate, although less harmful than many other solvents, is both harmful if ingested or if its vapors are inhaled. Ethyl acetate is also flammable and should be handled in a fume hood when possible. Pressurized centrifuge tubes present a projectile hazard; heating should be done in polypropylene (not glass) cylinders that are half full of water, which directs any projectiles directly upward. It is best to work behind a blast shield or the sash of a fume hood. However, with the recommended tubes, no ruptures occurred in our laboratories.
Preparation of a Sample for GC−FID Analysis
Full experimental details for this limonene extraction are given in the student handout in the Supporting Information. The extraction of approximately 1 g of orange rind isolates a small quantity of orange oil in the bottom of a 15 mL polypropylene centrifuge tube. This oil is dissolved in a 1.00 mL aliquot of a standard solution of anisole (0.25 g/L in ethyl acetate) and filtered into a 5.00 mL volumetric flask.14 The centrifuge tube is rinsed with an aliquot of approximately 1 mL ethyl acetate and similarly filtered into the volumetric flask. The solution is diluted to the 5.00 mL mark with ethyl acetate. Anisole is used as an internal standard in the experiment.15 After thorough mixing, an aliquot of this solution is transferred to a GC vial and submitted for analysis. Preparations of biologically derived natural products for GC−FID are typically done with methylene chloride. This experiment was attempted with both methylene chloride and ethyl acetate and no difference was seen in the results. The use of ethyl acetate in the place of a more toxic chlorinated solvent follows principles five and twelve of the principles of green
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DATA ANALYSIS AND RESULTS For an introductory analytical chemistry laboratory, a laboratory technician prepares and analyzes standard solutions by GC−FID in advance; students are given the raw GC−FID B
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heights and peak areas. Error analysis at a level appropriate to the course material allows the students to compare the results obtained by each of the calibration methods and discussion questions prompt them to compare these methods to other alternatives, such as directly weighing the extracts. Students noted that the error associated with using peak heights was greater than that with using peak areas; they again found minimal change in the error between direct creation of calibration curves and the use of an internal standard.
chromatograms for further analysis (Figure 2). The samples submitted by students are also analyzed by GC−FID by a
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In one laboratory section, students extracted the oil from other citrus fruits (red and white grapefruit, ugli fruit, lemon, lime, and pomelo) and compared them to the extraction of orange rind.19 Measurable amounts of D-limonene were obtained with all fruits that were tested with the exception of lemons (technical difficulties were encountered here; lemon rind is known to contain D-limonene). In this experiment, students also noticed that there were other peaks present in their chromatograms; however, the use of standard solutions made it straightforward to identify which peak corresponded to Dlimonene. It was commonly commented that another compound with the same retention time would confound their data; this provided students with an opportunity to discuss the limitations of the GC−FID method. To better demonstrate the idea of alternative assessment, we explored the possibility of having students perform the extraction of limonene both in the “traditional” method using methylene chloride and using this “green” method. The volume of work proved to be excessive for a single-afternoon laboratory experiment, but we believe this additional experiment would help to emphasize the differences between the two approaches. Garnier and Garibaldi outline a traditional extraction with hexane that could complement the procedure presented here.20 Students can be guided to think about the green chemistry principles by asking questions about where the waste from the experiment goes and whether they would hypothetically be comfortable putting the limonene in a beverage. The McKenzie et al. limonene extraction also has great potential to be modified to teach green chemistry alongside other scientific principles at a range of levels. One of the authors has coupled the experiment with a discussion of the three states of matter in an elementary school classroom; the extraction as presented in the original paper is suitable for demonstration to a high school class.2 If the experiment is to be used early in the first semester of an undergraduate course, simpler calibration curves without an internal standard can also be used. Increasing responsibility can be transferred to students if this experiment is introduced in upper division undergraduate analytical chemistry courses. Students can run their own GC− FID samples and prepare their own standard solutions. To further encourage independent work, the laboratory procedure provided may go only so far as to suggest anisole as an internal standard and to indicate the typical content of limonene in an orange rind, leaving students to determine appropriate dilution factors for their unknown and appropriate concentrations for their standard solutions based on their knowledge of GC−FID instrumentation.
Figure 2. Typical GC−FID trace showing elution of anisole at 3.2 min and limonene at 4.6 min.
technician, and the students are given the raw GC−FID trace of their own unknown. Details of the GC method employed are given in Table 2; the method was developed by modification of Table 2. GC Settings for GC−FID Detection Setting Front Inj Temp/°C Detector Temp/°C Inj Volume/μL Split Ratio FID Analysis Oven Column
EXTENSION
Value 200 260 1.0 20 Agilent ChemStation 50 °C, 3 min hold; 250 °C, 30 °C/min; 1 min hold Agilent HP-5 (5% phenyl, 95% methylpolysiloxane); Peak heights and areas output
a method published by Smith et al.16 and others17 to give good separation between D-limonene and anisole and to avoid thermal decomposition of the analyte. Each standard solution contains both D-limonene and anisole in known quantities. From the chromatograms, students are able to construct two calibration curves. The first is a simple calibration curve based only on the area of the D-limonene versus concentration of D-limonene (ALim vs [Lim]). The second is a calibration curve based on the ratio of the areas of the D-limonene and anisole peaks versus the ratio of the concentrations of limonene and anisole (ALim/AAni vs [Lim]/ [Ani]). Students also create both of these calibration curves based on the height of the peaks rather than the area (i.e., HLim vs [Lim] and HLim/HAni vs [Lim]/[Ani], respectively). Students use each of the four calibration curves to determine the concentration of D-limonene in their solution. From this they can determine the mass of limonene that they were able to extract from their sample of orange rind by considering the dilutions necessary to obtain the submitted solution.18 Typical values were approximately 5 × 10−3 g limonene/g orange rind, with a range from 5 × 10−4 to 1.5 × 10−2. In general, peak heights gave a higher estimate of limonene content than peak areas by 10−15%; the differences between working with and without an internal standard were minimal for both peak C
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(8) Hjeresen, D. L.; Boese, J. M.; Schutt, D. L. J. Chem. Educ. 2000, 77, 1543−1547. (9) Armenta, S.; De la Guardia, M. J. Chem. Educ. 2011, 88, 488−491. (10) Zhu, J.; Zhang, M.; Liu, Q. J. Chem. Educ. 2008, 85, 256−257. (11) Goldcamp, M. J.; Underwood, M. N.; Cloud, J. L.; Harshman, S.; Ashley, K. J. Chem. Educ. 2008, 85, 976−979. (12) Chemat, F.; Perino-Issartier, S.; Petitcolas, E.; Fernandez, X. Anal. Bioanal. Chem. 2012, 404, 679−682. (13) Koel, M.; Kaljurand, M. Pure Appl. Chem. 2006, 78, 1993−2002. (14) Alternatively, the 1 mL aliquot of standard solution and 4 mL of ethyl acetate can be added directly to the centrifuge tube; in this case, an aliquot of the solution can be filtered into a GC vial. This procedure is less quantitative than mixing in a volumetric flask, but simplifies the process. In either case, combination of the limonene sample and anisole internal standard prior to filtration allows for correction of total volume error. (15) Williams, K. R.; Pierce, R. E. J. Chem. Educ. 1998, 75, 223−226. (16) Smith, D. C.; Forland, S.; Bachanos, E.; Matejka, M.; Barrett, V. Chem. Educator 2001, 6, 28−31. (17) Cartoni, E. B. G. R.; Ravazzi, E. C. E.; Chimica, D.; La, R.; Moro, R. A. Chromatographia 1991, 31, 489−492. (18) Literature values put the concentration of D-limonene in orange rind at approximately 4.2 mg/g.19 Instructors could develop a guided literature search around this data point. (19) Lavoie, J.-M.; Chornet, E.; Pelletier, A. J. Chem. Educ. 2008, 85, 1555−1557. (20) Garner, C. M.; Garibalidi, C. J. Chem. Educ. 1994, 71, A146.
CONCLUSIONS This experiment provides an example of how a “classic” green chemistry experiment can be adapted to serve as a drop-in replacement for an undergraduate analytical laboratory. It teaches all of the basic concepts surrounding quantitative analysis by GC−FID including calibration curves, internal standards, and sample preparation, and also introduces green chemistry principles and alternative assessment. The added “real-life” context and the fun of the experiment itself make the experiment more relevant; this is a major goal of many chemistry curricula as we aim to educate scientists who are good global citizens. The experiment presented here is appropriate for use in an introductory analytical chemistry course at the undergraduate level and with the extensions suggested can be modified to suit a range of teaching outcomes.
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ASSOCIATED CONTENT
S Supporting Information *
Handout provided to students in preparation for the laboratory; notes for stockroom preparation; description of the GC method used; instructions on the use of the Excel spreadsheet template (docx file); GC−FID chromatograms for the limonene/anisole concentrations used (pdf file); spreadsheet for sample calculations of slope, error, and unknown concentration (xlsx file). This spreadsheet was not provided to students, but was very useful to instructors for determining whether students obtained reasonable results. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the California Environmental Protection Agency Department of Toxic Substances Control (EPA-DTSC), DOW Foundation, Berkeley Center for Green Chemistry, and students and graduate student instructors of Chem 4B spring 2012. H.L.B. acknowledges the Fulbright International Science & Technology Fellowship.
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REFERENCES
(1) Anastas, P. T.; Wood-Black, F.; Masciangioli, T.; Eds. Exploring Opportunities in Green Chemistry and Engineering Education: A Workshop Summary to the Chemical Sciences Roundtable; National Research Council (US); Washington, DC, 2007. (2) McKenzie, L. C.; Thompson, J. E.; Sullivan, R.; Hutchison, J. E. Green Chem. 2004, 6, 355−358. (3) Paar, L.; Dlbert, J.; Manfredi, K. Organic Chemistry Laboratory Experiments for Organic Chemistry Laboratory; University of Northern Iowa: Cedar Falls, IA, 2008. (4) Raynie, D. Laboratory Evaluation for Liquid CO2 Extraction of Dlimonene from Orange Peel Source; South Dakota State University: Vermillion, SD, 2006. (5) Case, M.; Sadlowski, C. Extraction of Limonene Using Liquid CO2; Chem143 Lab Manual University of Vermont: Burlington, VT, 2009. (6) Namies, J.; Tobiszewski, M.; Mechlin, A. Chem. Soc. Rev. 2010, 39, 2869−2878. (7) Anastas, P. T.; Warner, J. C. Green Chemistry: Theory and Practice; Oxford University Press: New York, 1998. D
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