In the Laboratory
Determination of Cinnamaldehyde in Cinnamon by SPME–GC–MS An Instrumental Analysis Experiment Yimin Wang, Jessica Ocariz, Jennifer Hammersand, Evan MacDonald, Ashley Bartczak, Frank Kero, Vaneica Y. Young, and Kathryn R. Williams* Department of Chemistry, University of Florida, Gainesville, FL 32611-7200; *
[email protected]
Experience with modern instrumentation is an essential component of the analytical chemistry curriculum, but in a working laboratory, sample preparation and control of matrix effects often dominate the analytical procedure. In 1989, Berladi and Pawliszyn (1) introduced a new method of sample preparation, solid-phase microextraction (SPME), that is most commonly used in conjunction with chromatographic analysis. The benefits of SPME include portability, speed, sensitivity, and compatibility with chromatographic instrumentation (2). But probably the most important feature is elimination of the toxic and environmentally hazardous solvents associated with liquid–liquid extraction. The experiment described in this work introduces undergraduate instrumental analysis students to SPME–GC–MS via the determination of trans-cinnamaldehyde in commercial cinnamon. The SPME quantitation experiments previously reported in this Journal involve meaningful chemical systems— nicotine in urine and sputum (3), bromoform in swimming pool water (4), and caffeine in beverages (5)—but two of them (3, 5) utilize expensive isotopically labeled compounds as internal standards, and the third (4) uses no internal standard at all. There are also serious safety issues associated with handling body fluids (3). The cinnamon analysis overcomes these drawbacks by utilizing readily available, low toxicity materials. The experiment also shows students a method for mimicking the sample matrix in the calibration standards.
mL in hexane, are pipetted directly onto the paper. To improve reproducibility, pure hexane is added to bring the total volume of liquid to 30 μL. After sealing, the vial is equilibrated for 5 minutes in a 40 °C bath, and then the SPME needle and fiber (50/30 μm divinylbenzene/Carboxen on polydimethylsiloxane) are inserted. The fiber is exposed to the headspace for 5 minutes; then the fiber and needle are withdrawn and inserted into a Hewlett-Packard 5890 GC-5971 MS. After desorption in the injection port at 60 °C for 5 minutes, the components are separated on an Alltech AT-1 column, 15 m × 0.25 mm using a 20 °C/min temperature ramp. The total GC runtime is 10.75 min. Including the extraction time, each run requires 20.75 min. This allows adequate time to repeat samples, if needed. The ground cinnamon sample (20 mg) is extracted in the same manner, but only the ethyl benzoate solution and hexane are added. Hazards Other than commercial cinnamon, the only compounds handled by the students are trans-cinnamaldehyde, ethyl benzoate, and hexane. All three compounds are classified as irritants, but the quantities handled by the students are very small (1 mg/mL solutions, less than 1 mL total volume). Hexane is also highly flammable and harmful by inhalation but again, the small quantities pose little fire or inhalation hazard (10).
Description of the Experiment
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The cinnamon analysis experiment has evolved over the past 15 years, originally as a liquid–liquid extraction with methylene chloride followed by GC–MS and later using SPME for the extraction step. Several internal standards were investigated, and finally ethyl benzoate was chosen, based on the structural similarity to trans-cinnamaldehyde and the excellent separation of the two compounds, as shown in the total ion chromatogram in Figure 1. Also, the literature data on the composition of various cinnamons do not include ethyl benzoate in the lists of components (6–9). Matching the matrix of cinnamon powder proved to be a challenge. Attempts to remove cinnamaldehyde entirely from commercial cinnamon by multiple extractions and steam distillation were time-consuming and of limited success. Instead, clean cellulose was chosen; cellulose powder can be used, but small pieces of filter paper work just as well and are very convenient. All extractions were performed in 3.7 mL glass vials having threaded caps with holes lined with Teflon-coated septa. For calibration, a piece of filter paper (ca. 0.2 cm2) is placed into a vial and known volumes of standard cinnamaldehyde (5, 10, 15, or 20 μL) and standard ethyl benzoate (10 μL), both about 1 mg/
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Time / min Figure 1. Total ion chromatogram of the headspace of a standard containing 0.01022 mg ethyl benzoate and 0.02090 mg cinnamaldehyde in hexane. Instrument: HP 5890 GC-5971 MS. Column: 15m x 0.25 mm AT-1 (Alltech Associates). Inlet pressure: 8 psi. Inlet temperature: 250 °C. Column temperature: start at 60 °C, hold 5 min, ramp to 215 °C at 20 °C/min.
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 85 No. 7 July 2008 • Journal of Chemical Education
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In the Laboratory
Results Figure 2 shows an internal standard curve containing data collected by the instructor and by two student groups. The peak areas were obtained by integrating the total ion chromatograms. Although there is some scatter among the data, the results are linear with a multiple R = 0.918. The linear regression slope and intercept are 0.78 ± 0.09 and ‒0.09 ± 0.12, respectively (uncertainties given as standard deviations). Analyses of McCormick Ground Cinnamon gave results ranging from 0.023% to 0.29% w/w cinnamaldehyde. According to Miller et al. (8), cinnamon contains 1–4% w/w steam distillable essential oil. Senanayake et al. (7) and Wijesekera et al. (6) obtained 75% w/w and 74% w/w, respectively, for the cinnamaldehyde content of the stem-bark oil of Cinnamomum zelanicum. Thus, ground bark of Cinnamomum zelanicum contains 0.75 to 3% w/w cinnamaldehyde. Since McCormick cinnamon is “ground bark of Cinnamomum cassia, Blume” (11), these values may not directly apply, but they show that the student experiment gives results in reasonable agreement, albeit somewhat low. The low results may be due to imperfect matrix-matching. Cinnamaldehyde pipetted onto the filter paper matrix may escape into the head space more efficiently than cinnamaldehyde in ground cinnamon. Conclusions The cinnamon analysis has been a popular experiment in the instrumental analysis laboratory for well over a decade, and the interest level has become even more evident with the addition of the SPME pretreatment. The student materials found in the online supplement also include a section on the further identification of trace components using a total ion chromatogram of cinnamon oil obtained and stored by the instructor. This part of the experiment helps students appreciate the benefits and limitations of computerized mass spectral library searches, as well as the importance of a solid background in organic chemistry. The entire experiment can be completed in a single 4-hour laboratory period. Acknowledgment vice.
The authors thank Matthew Booth for his technical ad-
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Cinn/EB Mass Ratio Figure 2. Internal standard curve showing pooled data from the instructors ( ) and two student groups ( ): Cinn is cinnamaldehyde and EB is ethyl benzoate.
3. Witter, A. E.; Klinger, D. M.; Fan, X.; Lam, M.; Mathers, D. T.; Mabury, S. A. J. Chem. Educ. 2002, 79, 1257–1260. 4. Hardee, J. R.; Long, J.; Otts, J. J. Chem. Educ. 2002, 79, 633–634. 5. Yang, M. J.; Orton, M. L.; Pawliszyn, J. J. Chem. Educ. 1997, 74, 1130–1132. 6. Wijesekera, R. O. B.; Jayewardene, A. L.; Rajapakse, L. S. J. Sci. Fd. Agric. 1974, 25, 1211–1220. 7. Senanayake, U. M.; Lee, T. H.; Wills, R. B. H. J. Agric. Food Chem. 1978, 26, 822–824. 8. Miller, K. G.; Poole, C. F.; Pawlowski, T. M. P. Chromatographia 1996, 42, 639–646. 9. Jayatilaka, A.; Poole, S. K.; Poole, C. F.; Chichila, T. M. P. Anal. Chim. Acta 1995, 302, 147–162. 10. The Physical and Theoretical Chemistry Laboratory Oxford University. http://ptcl.chem.ox.ac.uk/MSDS/#MSDS (accessed Mar 2008). 11. McCormick. http://www.mccormick.com/productdetail. cfm?ID=6422 (accessed Mar 2008).
Supporting JCE Online Material
http://www.jce.divched.org/Journal/Issues/2008/Jul/abs957.html Abstract and keywords
Literature Cited 1. Berladi, R. P.; Pawliszyn, J. Water Pollut. Res. J. Can. 1989, 24, 179. 2. Sigma Aldrich. http://www.sigmaaldrich.com/Brands/Supelco_ Home/Spotlights/SPME_central.html (accessed Mar 2008).
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Full text (PDF) Links to cited URLs and JCE articles Supplement
Student handout including prelab questions
Instructor notes
Journal of Chemical Education • Vol. 85 No. 7 July 2008 • www.JCE.DivCHED.org • © Division of Chemical Education
