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Steam Distillation of Essential Oils from Cloves

Synthetic FFR #1

Kevin Chen 11/14/13 Chem 213 Section #101 TA: Michael Banales

Clove has long been used as an analgesic in dentistry and a variety of herbal medicines, but researchers have just recently begun to study its molecular mechanism of action.1, 2 While some effects have been attributed to cloves high antioxidant content, the essential oils of clove, eugenol and caryophyllene, account for most of its biological potency.1,3 The major component of clove oil is eugenol, but caryophyllene has also been found to possess some analgesic effects.4 Recent discoveries surrounding eugenol promise deeper insights into medically relevant fields. Eugenols similarity to certain opioid drugs such as morphine and diazepam could help elucidate more details of their effects.5 Eugenol also acts on Na+ and Ca2+ ion channels related to sensation of pain.6 Exploration of eugenol in this area could help researchers understand nociceptor pathways. In addition, eugenol has proven to be a potent analgesic itself, as it was orally active in pain tests with mice.7 Reports of eugenols anti-oxidant and anti-fungal effects could lead to its use in a wider variety of health benefits in the future.8, 9 Eugenol is also used for its aroma in a variety of settings. Cloves are of course used as spice in food, and it is the essential oils eugenol and caryophyllene which provide the flavor. However, many cosmetic products, such as lotions, also utilize eugenol as a natural source of aroma.10 Each of these processes requires that eugenol be isolated and purified. This experiment, distillation of eugenol from clove, is one process that allows the compound to be applied to the medical, research, and cosmetic uses described above. The experiment consists of three steps, steam distillation of essential oils from ground clove, extraction of oils from distillate, and identification of oil using GC and GC-MS. Of these techniques, steam distillation is the most notable. The high boiling point of eugenol, at 253C, prevents the use of simple distillation, as this could burn the sample. Because oil is immiscible
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with water, the partial pressures of the two fractions add and the mixture boils when their sum reaches atmospheric pressure. At temperatures below 100C, waters vapor pressure is almost at atmospheric pressure and oils only need to add a small amount more pressure to boil. Thus, the essential oils distill at a much lower temperature than their boiling points.11 Commercially, the main use of steam distillation is isolation of essential oils from leaves, bark, and other plant material. Steam distillation can be used industrially to remove pollutants, such as hydrocarbons, from wastewater. Volatile non-polar compounds may also be the desired product of organic reactions, so industrial and research chemists can apply this technique. The second technique used to purify the essential oils was extraction. Because the oils were co-distilled with water, the organic layer they formed needed to be separated from the aqueous layer. Dichloromethane, a nonpolar solvent, was used to achieve this separation. The purpose of this experiment was to isolate the essential oils of clove using steam distillation. Oils were extracted from water in the distillate using nonpolar solvent. Oil obtained from these processes was characterized by GC and GC-MS. Organic chemists could find this experiment useful because the major component of clove oil, eugenol, has several biological and cosmetic purposes.

Cloves (4 g) were ground and codistilled with water (50mL) using simple distillation. Distillation was stopped when distillate became clear. Distillate was extracted with dichloromethane (3 x 15mL). The organic layers were combined and dried with anhydrous sodium sulfate. The solvent was evaporated to yield a white, scented oil (240mg, 6%).

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GC (40-250C, 10C/min) RT % Composition 13.51 14.02 93.68 6.32

MS Compound Eugenol Caryophyllene Major Peaks 77, 103, 131, 149, 164 69, 91, 93, 105, 133, 204 Molecular Mass (literature) 164.20 g/mol 204.35 g/mol

Ground cloves were codistilled with water. The high boiling point of the desired oils did not allow simple distillation, as this would have burned or degraded the sample. Resulting distillate was extracted using dichloromethane and pure oil was obtained after evaporation of solvent. The oils identities were confirmed with GC and GC/MS. Purified oils were first analyzed using GC. Although the retention times of the two compounds identified were slightly less than those of the compendium, the relative positions and sizes of the peaks were almost identical. The oil appeared very pure, with no contaminant peaks present besides the solvent, which appeared at 1.79 and 1.84. Percent composition also agreed with literature values. Eugenol accounted for 93.68% of the mixture. A similar study involving the steam distillation of Turkish clove buds found the percent composition of eugenol to be 87%. This value is similar to the value obtained, and, when combined with the value for eugenyl acetate, rises to 95.01%, which is even closer.1 Caryophyllene accounted for 6.32% of the mixture. This was also similar to the studys value of 3.56%. A different form of cloves may
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have been used in the study versus the experiment performed, as eugenyl acetate was not obtained in this experiment. However, the similarity between the values suggests that the experiment obtained reasonable results. The GC performed as part of the GC-MS analysis differed from the first analysis in that the retention times of 13.51 and 14.02 were slightly greater, and closer to compendium values. This may be due to errors in loading the first GC or inaccuracies resulting from using an older model. However, these differences are not significant. Analysis using mass spectrometry confirmed the identities of the oils. Software utilized mass spectrometry data to identify the two compounds as eugenol and caryophyllene. Manual assignment of fragments to MS peaks also found that the structures and MS spectrums of eugenol and caryophyllene fit well. Key peaks for eugenol included a 76.95 m/z peak corresponding to the benzene ring and 164.02 m/z for the molecular ion. Additional peaks correspond to the benzene ring with various constituents, such as benzene plus methoxy group at 102.99 m/z, benzene plus propene and hydroxyl group at 130.98 m/z, and benzene plus propene and methoxy group at 149.00 m/z. Caryophyllene displayed a more complex spectrum with strong peaks at 93.00 m/z corresponding to cyclobutane plus three methyl groups and at 133.03 m/z corresponding to the nine carbon loop attached to the cyclobutane plus its methyl and methylene substituents. The molecular ion formed a small peak at 204.16 m/z. This experiment produced a mixture of oils containing only eugenol and caryophyllene, therefore achieving its purpose of isolating and purifying cloves essential oils. Because the GC identified only two peaks, both of which were attributed to desired products, the oils were successfully purified. The resemblance of the GC to compendium data and the analysis of MS software confirmed that the intended oils had been isolated. The percent recovery, at 6%,

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compared favorably to a similar study involving steam distillation of cloves.9 This yield, combined with the purity of the oils, implies that the technique was performed successfully. However, if this area were to be examined for possible improvements, it does seem that manual grinding of the clove buds may have been inadequate. Mechanical grinding or pressing of the cloves could have resulted in a higher yield and perhaps allowed some of the less concentrated oils to be detected. Purification of active compounds from natural sources is an essential step in using these substances for medicinal, research, and industrial purposes. Clove oil, in particular eugenol, has a variety of notable biological effects and must be isolated if these effects are to be fully examined. Steam distillation proved to be an effective method of achieving this purification, as the identity and purity of the products from this experiment were confirmed by GC and GC/MS analysis.

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1. Alma, M. et al. BioResources 2007, Vol 2, No 2. 2. Lin, X.; Chen, C. Zhongguo Zhong Yao Za Zhi 2007, 32, 186191. 3. Dragland, S. et al. J. Nutr. 2003, 133, 1286-1290. 4. Klauke, A. et al. European Neuropsychopharmacology. DOI:10.1016/j.euroneuro.2013.10.008. Published Online: Nov 8, 2007. 5. Dal B, W. et al. Fundamental & Clinical Pharmacology. 2013, 27, 517525. 6. Seo, H. et al. J Pharmacol Exp Ther 2013, 347, 310317.

7. Park, S.-H. et al. Arch. Pharm. Res. 2011, 34, 501507. 8. Lee, K.-G.; Shibamoto, T. Food Chemistry 2001, 74, 443448. 9. Martini, H et al. Italian Journal of Food Science. 1996, 8, 63-67. 10. Luce, C. PCT Int. Appl. 2013, 101. 11. Rummel, S. Lab guide for Chemistry 213; Hayden McNeil: Plymouth, MI, 2014; pp 182-183

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