In the Laboratory
Identification of Secondary Metabolites in Citrus Fruit Using Gas Chromatography and Mass Spectroscopy Jean-Michel Lavoie* and Esteban Chornet Département de Génie Chimique, Faculté de Génie, Université de Sherbrooke, Sherbrooke, Québec, J1K 2R1; *
[email protected] André Pelletier Département de Chimie et Biochimie, Université de Moncton, Moncton, Nouveau-Brunswick, Canada, E1A 3E9
It is now well-known that the observable differences in smell, color, and texture of different citrus fruit (lemons, limes, grapefruits, oranges, etc.) are also significant at a molecular level. The abundance of secondary metabolites in citrus fruit peels allows an easier understanding of the close relationship between chemistry and our senses of taste and smell (1). Many molecular families are found in these fruits, among which terpenes in general and monoterpenes in particular are abundant (1–3). This article describes a new undergraduate laboratory experiment that allows students to see that the differences in the aroma and fragrance of common fruits such as lemons, limes, grapefruits, and oranges are directly related to their chemical composition (Figure 1).
Figure 1. Fruit used in the experiment.
Background One of the very first analyses of citrus fruit, accomplished in 1917, investigated the composition of grapefruits (4). Zoller isolated the molecules using classical column chromatography before identifying them using their physical properties such as boiling point, refractive index, and so forth. The next fruit to be investigated through similar procedures about a decade later was a variety of orange: Citrus aurentium (5). As the methods of analyses were getting more efficient, researchers acquired increasing interest in locally grown species, identifying the major molecular compounds found in the different plant tissues. One of the first analyses using a GC–MS was made in 1982 and investigated the mutagenicity of citrus fruit juices (6). Since then, many citrus species from every corner of the planet have been studied using GC–MS protocols. In 2001, Smith et al. (7) described an academic-level experiment on citrus fruit, which gave insight on the composition of the essential oils from a few common citrus fruit. Their procedures were oriented toward the qualitative analysis of the extracts, as opposed to the experiment described here that allows their quantitation. The work by Smith and co-workers showed the identification of the volatile compounds present in the essential oils without, however, trying to compare their quantity. The comparison of the molecules is therefore only related to the occurrence or the absence of an arbitrarily selected compound. There were also noticeable differences in the extraction techniques such as the use of a cheese grater to remove the rind, provoking the unwanted evaporation of monoterpenes and the restrictive use of the rind as opposed to the entire peel.1 Another experiment, published by Greenberg in 1968 (8), characterized (R)-limonene in orange oil using NMR, IR, GC, and the refractive index. It mostly showed the difference between different analytical techniques whereas the experiment described here offers a comparison between secondary metabolites from citrus
fruit. An experiment suggested by Glidewell also mentions the extraction of (R)-limonene from orange peels but instead of comparing the extracts to other citrus fruit, the content of (R)limonene was compared to terpenes present in caraway seeds and cumin seeds, two terpene-rich plants (9). Work by Williams and co-workers also allowed a quantitation of (R)-limonene in orange oil without, however, aiming at a comparison between the citrus fruit as suggested in the present experiment (10). Since the procedures must be accomplished during a laboratory period of 4 to 5 hours, it is important to use an extraction technique that will be efficient and fast. This experiment could also be performed during two laboratory periods of 3 hours, the first period used for the extraction and the second for the GC–MS analysis. Many techniques have been used to obtain the essential oils from citrus peels, one of them being the classical hydrodistillation technique (7) although it has the major inconvenience of being time-consuming. Modern techniques include (but are not limited to) supercritical fluid extraction (8), microwave extraction (9), cold pressing (1), and ultrasonic-assisted extraction (10). Among the few used in literature, cold pressing is the easiest to reproduce at an academic level. Although the professional apparatus may be expensive and hard to acquire, the technique can also be done with common household items as depicted below. Experimental Extraction of Essential Oils Remove the whole skin of the citrus fruit and cool it in a bath filled with water and ice (about 100 mL in a 250 mL beaker) for 20 minutes. It is necessary to have the peels submerged in icy water to avoid an unwanted evaporation and loss of the
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 85 No. 11 November 2008 • Journal of Chemical Education
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In the Laboratory
targeted monoterpenes. Add 100 mL of a previously prepared brine (250 mL of glacial acetic acid and 60 g of NaCl in 1 L of water). The brine provides an acidic and ionic solution to help solubilize non-terpenic and more polar secondary metabolites that could be found in the fruit peels. Grind the whole mixture in a kitchen blender for approximately 4.5 minutes (three periods of 1.5 minutes to avoid increasing the temperature of the solution). Vacuum-filter the mixture using a 100 mL Büchner funnel and set aside the aqueous part (be careful to weigh the filter beforehand). Put the filter and its content in an oven at 100 oC for 24 hours to get the dry mass of the extracted peels. Put the aqueous mixture in a 500 mL separatory funnel and extract three times with 30 mL of methylene chloride (3 × 30 mL) measuring the volume in a 50 mL graduated cylinder. Dry CH3
CH2
CH3
H3C
CH2
CH2
H3C
OH
H 3C H2C
CH3
(R)-limonene
H 3C B-pinene
CH3
C-myrcene
H3C
CH3
(R)-linalool
Figure 2. Structure of the four most common monoterpenes found in citrus fruit.
Table 1. Concentration and Standard Deviation of (R )-Limonene in Lime, Orange, and Grapefruit Dried Peels Fruit
Yield/(mg g–1)
Standard Deviation/ (mg g–1)
Lime
7.43
±0.03
Orange
4.21
±0.03
Grapefruit
5.91
±0.23
the organic phase with 5 g or more of anhydrous MgSO4 and then vacuum-filter the mixture again using the same glassware as before to remove the hydrated MgSO4. Keep the organic phase (methylene chloride) for the following GC–MS analysis. All chemicals used when this experiment was initially developed were purchased from Sigma (Mississauga, Ontario). Standard Calibration Curve The most concentrated secondary metabolite in the extracts of citrus fruit is usually (R)-limonene, which is a good compound to be quantitated by students for further comparison with other fruits. They might also be able to quantitate other monoterpenes such as α-pinene, β-myrcene, or (R)-linalool. The standard calibration curve should be done with concentrations varying from 2 to 10 mg per liter. Some of the most common monoterpenes found in the essential oils from citrus fruit that can be used as standards are presented in Figure 2. GC–MS Analysis Prior to injection, citrus peel extracts were diluted a hundred-fold (1/100) to ensure that the signal was within the quantitation limit of the method. Hazards Acetic acid is strongly corrosive, causes serious burns, is harmful if swallowed, and is a lachrymator. Methylene chloride is harmful if swallowed or inhaled, is an eye and skin irritant, and is a suspected carcinogen. (R)-Limonene is harmful if swallowed and is also a skin, eye, and respiratory irritant. α-Pinene is an eye, skin, and respiratory irritant and may be absorbed through the skin. β-Myrcene is a skin, eye, and respiratory irritant. (R)-Linalool is a skin, eye, and respiratory irritant. It is thus important to conduct all manipulations under a fume hood to prevent accidental exposure. Hazard data were obtained from the MSDS database Web site (11). Result and Discussion
Table 2. GC Retention Time and Mass Spectrum of the Suggested Standards Used for the Analysis of Citrus Fruit Peels Standard
Retention Time/min
α-Pinene
5.80
136(45), 121(45), 107(36), 95(12), 94(40), 93(100), 92(35), 91(33), 81(13), 80(16), 79(46), 77(26), 68(95), 67(74), 53(20).
β-Myrcene
6.58
136(5), 121(6), 107(3), 94(10), 93(100), 92(12), 91(20), 80(8), 79(14), 77(12), 70(4), 67(10), 65(4), 53(7), 51(4)
(R)-Limonene
7.23
136(45), 121(45), 108(11), 107(36), 105(11), 95(12), 94(40), 93(100), 92(35), 91(33), 81(13), 80(16), 79(46), 77(26), 68(95), 67(74), 53(20).
(R)-Linalool
8.22
154(1), 136(12), 121(30), 109(10), 107(10), 96(10), 94(12), 93(89), 92(17), 91(12), 83(18), 81(11), 80(32), 79(15), 71(100), 69(39), 68(12), 67(19), 55(43), 53(10).
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Mass Spectrum/[m/z (%)]
For quantitation, it is necessary for students to calculate an extraction yield of (R)-limonene (or any of the previously mentioned monoterpenes). To do so, they will need the mass of dried citrus fruit peels, keeping in mind that the initial solution had a volume of 90 mL and was diluted a hundred-fold before injecting in the GC–MS. By using the calibration curve made with the external standard, the students find a corresponding concentration of the (R)limonene. This concentration can easily be converted to a total mass if the original dilutions are considered. The total mass of (R)-limonene is then divided by the total mass of dry peel used for this experiment. This will provide a quantity of (R)-limonene (or any other monoterpene) per mass of citrus fruit peels. The students working on different citrus fruit are then invited to compare and discuss their results. They should be able to observe a consistency between the concentration of (R)-limonene and the natural aroma of the different fruit. Results shown in Table 1 present an example of the (R)-limonene concentration in limes, grapefruits, and oranges. It is also possible to investigate the fragmentation of the molecules in a mass spectrometer. The retention times and actual fragmentation report of the four suggested molecular structures are shown in Table 2.
Journal of Chemical Education • Vol. 85 No. 11 November 2008 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory
Conclusion This experiment allows students to have a better understanding of natural product chemistry by analyzing quantitatively up to four monoterpenes in the essential oils from citrus fruit. It will also allow them to put into practice their knowledge of analytic-organic chemistry. The main goal of the experiment is to show that the variations that can be observed by our senses (in this case the senses of smell and taste) are directly related to chemistry. This laboratory also familiarizes the students with the concept of chemotaxonomy.
7. Smith, D. C.; Forland, S.; Bachanos, E.; Matejka, M.; Barrett V. Chemical Educator 2001, 6, 28–31. 8. Greenberg, F. H. J. Chem. Educ. 1968, 45, 537–538. 9. Glidewell, C. J. Chem. Educ. 1991, 68, 267–269. 10. Williams, K. R.; Pierce, R. E. J. Chem. Educ. 1998, 75, 223– 226. 11. Oxford University MSDS page. http://ptcl.ch em.ox.ac.uk/MSDS (accessed Aug 2008).
Supporting JCE Online Material
http://www.jce.divched.org/Journal/Issues/2008/Nov/abs1555.html
Note
Abstract and keywords
1. The rind is the thin layer outside the fruit and is usually a couple of millimeters thick. The whole peel surely contains the rind but also contains a white fiber that is close to a centimeter thick. This whole fiber has a lesser concentration of terpenes in it but by using the whole peel the students will not lose essential oils isolating the rind solely.
Full text (PDF) Links to cited URLs and JCE articles Figure 1 in color
Literature Cited 1. Njoroge, S. M.; Ukeda, H.; Sawamura, M. J. Agr. Food Chem. 1996, 44, 550–556. 2. Torssell, K. B. G. Natural Product Chemistry; John Wiley and Sons: New York, 1983; pp 177–189. 3. Bhat, S. V.; Nagasampagi, B. A.; Sivakumar, M. Chemistry on Natural Products; Narosa: New Delhi, 2005; pp 125–136. 4. Zoller, H. F. J. Ind. Eng. Chem. 1917, 10, 364–373. 5. Ikeda, T.; Fujita, Y. Nippon Kagaku Kaishi 1930, 51, 349–354. 6. Mazaki, M.; Ishii, T.; Uyeta, M. Mutat Res. 1982, 101, 283– 291.
Supplement Information for the instructor including specifications on the standard deviation curves, the complete GC–MS programs, results obtained by students, common fragmentation patterns, and pre- and postlab questions and answers JCE Featured Molecules for November 2008 (see p 1584 for details) Structures of some of the molecules discussed in this article are available in fully manipulable Jmol format in the JCE Digital Library at http://www.JCE.DivCHED.org/JCEWWW/Features/ MonthlyMolecules/2008/Nov/. JCE Cover for November 2008 This article is featured on the cover of this issue. See p 1459 of the table of contents for a detailed description of the cover.
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