Fitting It All In: Adapting a Green Chemistry Extraction Experimentfor Inclusion in an Undergraduate Analytical LaboratoryHeather 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 thisexperiment designed for use in the undergraduate analytical chemistry laboratory. Anestablished experiment of liquid CO2 extraction of D-limonene has been adapted toinclude a quantitative analysis by gas chromatography. This facilitates drop-inincorporation of an exciting experiment into an existing curriculum. The experimentprovides an introduction to natural product extraction, calibration curves, andinternal standards while simultaneously demonstrating alternative solvent selectionfor 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

As green chemistry becomes increasingly prevalent in bothacademic research and chemical industry, there are

ongoing calls for a “green” emphasis in undergraduatecurriculum.1 A new generation of scientists who embracethese principles and techniques as a natural part of doingchemical research will revolutionize how we think aboutchemistry in a way that is more difficult for establishedscientists.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 ofgreen chemistry curriculum.3−5 Students are engaged by theunconventional nature of the experiment, from the preparationof food products for extraction to the easily observedsimultaneous existence of carbon dioxide in solid, liquid, andvapor forms. However, a challenge that limits the widespreaduse of this and other innovative new experiments is the alreadyoverpacked nature of current introductory chemistry curricula.The addition of yet another idea or experiment often comes atthe expense of thorough coverage of fundamental material.In this contribution, we describe the adaptation of this green

chemistry laboratory experiment to meet teaching objectives inthe introductory analytical chemistry curriculum. The experi-ment has been used as a “drop-in” replacement for anothermore mundane analysis as part of the early stages of a broader-based revamp of our chemistry laboratory curriculum. It hasbeen implemented with a class of approximately 200 first-yearundergraduate chemistry majors. The experiment introducesthe preparation of a natural product sample for analysis byGC−FID (gas chromatography−flame ionization detector) andthe concepts of internal standards and calibration curves.

Students compare several methods of quantitation of anisolated natural product and are specifically asked to consideralternative assessment with the idea that greener choices can bemade when designing an analytical method.6

In addition to meeting the teaching objectives of analyticalchemistry, this experiment maintains the introduction of greenchemistry principles that motivated the original creation of theexperiment. Students are introduced to several of the 12principles of green chemistry,7 including pollution prevention,safer solvents, energy efficiency, renewable feedstocks, designfor degradation, and safer chemistry for accident prevention(Table 1). Relatively little work has been done in thedevelopment of green analytical chemistry for the under-graduate laboratory to-date;8−12 this experiment aims to helpfill this gap.

■ EXPERIMENTAL OVERVIEW

Extraction Procedure

This experiment is divided into two major parts: extraction andanalysis (Figure 1). The extraction takes approximately 1.5 h,and the sample preparation for analysis takes approximately 45min. Liquid CO2 is used to extract D-limonene from orangerind, which closely follows the procedure reported byMcKenzie et al.2 Grated orange rind is placed in a centrifugetube that is packed with dry ice. This tube is sealed and heatedenough to generate liquid CO2 in the tube; this serves as asolvent to extract orange oil and then evaporates completely.The orange oil is diluted with ethyl acetate and analyzed by

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GC−FID. The quantity of D-limonene in the extract isdetermined through the use of four types of calibration curves,whose relative reliability are compared in student analysis oftheir data. For this experiment, students work in pairs butperform their data analysis individually.

Preparation of a Sample for GC−FID Analysis

Full experimental details for this limonene extraction are givenin the student handout in the Supporting Information. Theextraction of approximately 1 g of orange rind isolates a smallquantity of orange oil in the bottom of a 15 mL polypropylenecentrifuge tube. This oil is dissolved in a 1.00 mL aliquot of astandard solution of anisole (0.25 g/L in ethyl acetate) andfiltered into a 5.00 mL volumetric flask.14 The centrifuge tube isrinsed with an aliquot of approximately 1 mL ethyl acetate andsimilarly filtered into the volumetric flask. The solution isdiluted to the 5.00 mL mark with ethyl acetate. Anisole is usedas an internal standard in the experiment.15 After thoroughmixing, an aliquot of this solution is transferred to a GC vialand submitted for analysis.Preparations of biologically derived natural products for

GC−FID are typically done with methylene chloride. Thisexperiment was attempted with both methylene chloride andethyl acetate and no difference was seen in the results. The useof ethyl acetate in the place of a more toxic chlorinated solventfollows principles five and twelve of the principles of green

chemistry by using a safer solvent. This can be highlighted indiscussion with students.

■ HAZARDSDry ice is very cold and is a frostbite hazard; most notablyvessels containing dry ice become very cold to the touch andshould be handled with insulating gloves. Providing test tuberacks to hold the centrifuge tubes also limits exposure ofstudents’ hands. Orange oil (predominantly D-limonene) andanisole are harmful if swallowed in quantity and can be a skin,eye, and respiratory irritants. Ethyl acetate, although lessharmful than many other solvents, is both harmful if ingested orif its vapors are inhaled. Ethyl acetate is also flammable andshould be handled in a fume hood when possible. Pressurizedcentrifuge tubes present a projectile hazard; heating should bedone in polypropylene (not glass) cylinders that are half full ofwater, which directs any projectiles directly upward. It is best towork behind a blast shield or the sash of a fume hood.However, with the recommended tubes, no ruptures occurredin our laboratories.

■ DATA ANALYSIS AND RESULTSFor an introductory analytical chemistry laboratory, alaboratory technician prepares and analyzes standard solutionsby GC−FID in advance; students are given the raw GC−FID

Table 1. Application of the 12 Principles of Green Chemistry7

Principle Application in this Experiment

1. Pollution Prevention Liquid CO2 is used in place of chlorinated solvents; gaseous CO2 is generated, but toxic waste is eliminatedAll of the waste generated in the extraction can be disposed in compost or regular trash

5. Safer Solvents andAuxiliaries

Ethyl acetate is used in place of methylene chloride

6. Design for EnergyEfficiency

No energy is required to evaporate solvent after extraction; life cycle analysis for the generation of dry ice vs methylene chloride must beconsidered13

7. Use of RenewableFeedstocks

CO2 can potentially be captured rather than produced from fossil fuel sources

10. Design forDegradation

Ethyl acetate biodegrades into ethanol and acetic acid

12. Inherently SaferChemistry

Less toxic solvents result in a safer laboratory environment due to reduced exposure risks; the explosion potential from pressurized liquidsand gases must be considered here

Figure 1. Extraction of D-limonene from orange rind using liquid CO2.

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chromatograms for further analysis (Figure 2). The samplessubmitted by students are also analyzed by GC−FID by a

technician, and the students are given the raw GC−FID trace oftheir own unknown. Details of the GC method employed aregiven in Table 2; the method was developed by modification of

a method published by Smith et al.16 and others17 to give goodseparation between D-limonene and anisole and to avoidthermal decomposition of the analyte.Each standard solution contains both D-limonene and anisole

in known quantities. From the chromatograms, students areable to construct two calibration curves. The first is a simplecalibration curve based only on the area of the D-limoneneversus concentration of D-limonene (ALim vs [Lim]). Thesecond is a calibration curve based on the ratio of the areas ofthe D-limonene and anisole peaks versus the ratio of theconcentrations of limonene and anisole (ALim/AAni vs [Lim]/[Ani]). Students also create both of these calibration curvesbased on the height of the peaks rather than the area (i.e., HLimvs [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 thisthey can determine the mass of limonene that they were able toextract from their sample of orange rind by considering thedilutions necessary to obtain the submitted solution.18 Typicalvalues were approximately 5 × 10−3 g limonene/g orange rind,with a range from 5 × 10−4 to 1.5 × 10−2. In general, peakheights gave a higher estimate of limonene content than peakareas by 10−15%; the differences between working with andwithout an internal standard were minimal for both peak

heights and peak areas. Error analysis at a level appropriate tothe course material allows the students to compare the resultsobtained by each of the calibration methods and discussionquestions prompt them to compare these methods to otheralternatives, such as directly weighing the extracts. Studentsnoted that the error associated with using peak heights wasgreater than that with using peak areas; they again foundminimal change in the error between direct creation ofcalibration curves and the use of an internal standard.

■ EXTENSION

In one laboratory section, students extracted the oil from othercitrus fruits (red and white grapefruit, ugli fruit, lemon, lime,and pomelo) and compared them to the extraction of orangerind.19 Measurable amounts of D-limonene were obtained withall fruits that were tested with the exception of lemons(technical difficulties were encountered here; lemon rind isknown to contain D-limonene). In this experiment, studentsalso noticed that there were other peaks present in theirchromatograms; however, the use of standard solutions made itstraightforward to identify which peak corresponded to D-limonene. It was commonly commented that anothercompound with the same retention time would confoundtheir data; this provided students with an opportunity to discussthe limitations of the GC−FID method.To better demonstrate the idea of alternative assessment, we

explored the possibility of having students perform theextraction of limonene both in the “traditional” method usingmethylene chloride and using this “green” method. The volumeof work proved to be excessive for a single-afternoon laboratoryexperiment, but we believe this additional experiment wouldhelp to emphasize the differences between the two approaches.Garnier and Garibaldi outline a traditional extraction withhexane that could complement the procedure presented here.20

Students can be guided to think about the green chemistryprinciples by asking questions about where the waste from theexperiment goes and whether they would hypothetically becomfortable putting the limonene in a beverage.The McKenzie et al. limonene extraction also has great

potential to be modified to teach green chemistry alongsideother scientific principles at a range of levels. One of theauthors has coupled the experiment with a discussion of thethree states of matter in an elementary school classroom; theextraction as presented in the original paper is suitable fordemonstration to a high school class.2 If the experiment is to beused early in the first semester of an undergraduate course,simpler calibration curves without an internal standard can alsobe used.Increasing responsibility can be transferred to students if this

experiment is introduced in upper division undergraduateanalytical chemistry courses. Students can run their own GC−FID samples and prepare their own standard solutions. Tofurther encourage independent work, the laboratory procedureprovided may go only so far as to suggest anisole as an internalstandard and to indicate the typical content of limonene in anorange rind, leaving students to determine appropriate dilutionfactors for their unknown and appropriate concentrations fortheir standard solutions based on their knowledge of GC−FIDinstrumentation.

Figure 2. Typical GC−FID trace showing elution of anisole at 3.2 minand limonene at 4.6 min.

Table 2. GC Settings for GC−FID Detection

Setting Value

Front InjTemp/°C

200

DetectorTemp/°C

260

Inj Volume/μL 1.0Split Ratio 20FID Analysis Agilent ChemStationOven 50 °C, 3 min hold; 250 °C, 30 °C/min; 1 min holdColumn Agilent HP-5 (5% phenyl, 95% methylpolysiloxane); Peak

heights and areas output

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■ CONCLUSIONSThis experiment provides an example of how a “classic” greenchemistry experiment can be adapted to serve as a drop-inreplacement for an undergraduate analytical laboratory. Itteaches all of the basic concepts surrounding quantitativeanalysis by GC−FID including calibration curves, internalstandards, and sample preparation, and also introduces greenchemistry principles and alternative assessment. The added“real-life” context and the fun of the experiment itself make theexperiment more relevant; this is a major goal of manychemistry curricula as we aim to educate scientists who aregood global citizens. The experiment presented here isappropriate for use in an introductory analytical chemistrycourse at the undergraduate level and with the extensionssuggested can be modified to suit a range of teaching outcomes.

■ ASSOCIATED CONTENT*S Supporting Information

Handout provided to students in preparation for the laboratory;notes for stockroom preparation; description of the GCmethod used; instructions on the use of the Excel spreadsheettemplate (docx file); GC−FID chromatograms for thelimonene/anisole concentrations used (pdf file); spreadsheetfor sample calculations of slope, error, and unknownconcentration (xlsx file). This spreadsheet was not providedto students, but was very useful to instructors for determiningwhether students obtained reasonable results. This material isavailable via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author

*E-mail: douskey@berkeley.edu.Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank the California Environmental Protection AgencyDepartment of Toxic Substances Control (EPA-DTSC), DOWFoundation, Berkeley Center for Green Chemistry, andstudents and graduate student instructors of Chem 4B spring2012. H.L.B. acknowledges the Fulbright International Science& Technology Fellowship.

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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 ofethyl 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 procedureis less quantitative than mixing in a volumetric flask, but simplifies theprocess. In either case, combination of the limonene sample andanisole internal standard prior to filtration allows for correction of totalvolume 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 orangerind at approximately 4.2 mg/g.19 Instructors could develop a guidedliterature 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.

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