734
Energy & Fuels 2006, 20, 734-737
Essential Oils Investigated by Size Exclusion Chromatography and Gas Chromatography-Mass Spectrometry Trevor J. Morgan,† William E. Morden,‡ Eiman Al-muhareb,† Alan A. Herod,*,† and Rafael Kandiyoti† Department of Chemical Engineering, South Kensington Campus, Imperial College London, London SW7 2AZ, U.K., and LGC Ltd., Runcorn, U.K. ReceiVed NoVember 8, 2005. ReVised Manuscript ReceiVed January 9, 2006
Three essential oils, tea tree oil from Melaleuca alternifolia and two lavender oils (LaVandula angustifolia and LaVandula intermedia laVandin) produced by steam distillation, have been examined by gas chromatography-mass spectrometry (GC-MS) and by size exclusion chromatography (SEC) using 1-methyl-2pyrrolidinone (NMP) as the eluent. This mode of operation of SEC normally produces bimodal chromatograms for petroleum residues, asphaltenes, and coal-derived liquids. Because these volatile oils consist of relatively small molecules, they were seen as a test of the SEC mechanism in eluting terpene molecules and their oxygenated derivatives as small molecules rather than through the formation of aggregates that may masquerade as large molecules. GC-MS confirmed the small molecular types, and the oils eluted from the SEC column only as small molecules. The inclusion of oxygen favored early elution from SEC but not into the excluded region.
1. Introduction work1
Our recent has involved the development of size exclusion chromatography (SEC) for aromatics using 1-methyl2-pyrrolidinone (NMP) as the eluent. This work showed that SEC for aromatics in coal liquids and petroleum residues could be calibrated using polystyrenes or poly(methyl methacrylate)s and could provide a good estimate of molecular mass ranges for aromatics in these materials up to masses of a few thousand mass units. In addition, chromatograms from petroleum residues, asphaltenes, and various coal-derived liquids and their fractions have been observed as being bimodal, with a peak eluting in the excluded region of the chromatogram.2-7 The excluded peak has been alleged to consist of nanoaggregates in the case of asphaltenes,8 but the comparison of laser-desorption mass spectra and SEC indicates that a change in size with increasing mass might be responsible.5,6 For bisphenol resins used in epoxy resins, calibration with polysaccharides gave an acceptable estimate of number and weight-average molecular masses as measured against several other methods of estimation.9 * To whom correspondence should be addressed. E-mail:
[email protected]. Fax: 44-207-594-5604. † Imperial College London. ‡ LGC Ltd. (1) Karaca, F.; Islas, C. A.; Millan, M.; Behrouzi, M.; Morgan, T. J.; Herod, A. A.; Kandiyoti, R. Energy Fuels 2004, 18, 778. (2) Li, W.; Morgan, T. J.; Herod, A. A.; Kandiyoti, R. J. Chromatogr., A 2004, 1024, 227. (3) Suelves, I.; Islas, C. A.; Millan, M.; Galmes, C.; Carter, J. F.; Herod, A. A.; Kandiyoti, R. Fuel 2003, 82, 1. (4) Morgan, T. J.; Millan, M.; Behrouzi, M.; Herod, A. A.; Kandiyoti, R. Energy Fuels 2005, 19, 164. (5) Millan, M.; Morgan, T. J.; Behrouzi, M.; Karaca, F.; Galmes, C.; Herod, A. A.; Kandiyoti, R. Rapid Commun. Mass Spectrom. 2005, 19, 1867. (6) Millan, M.; Behrouzi, M.; Karaca, F.; Morgan, T. J.; Herod, A. A.; Kandiyoti, R. Catal. Today 2005, 109, 154. (7) Ascanius, B. E.; Merino-Garcia, D.; Andersen, S. I. Energy Fuels 2004, 18, 1827. (8) Badre, S.; Goncalves, C. C.; Norinaga, K.; Gustavson, G.; Mullins, O. C. Fuel 2006, 85, 1.
Essential oils are aromatic substances (i.e., giving an aroma and not being benzene derivatives) produced by certain plants, once considered to be the “essence” of the plants. Many of these oils, which are extracted, concentrated, and used as perfume scents or food flavorings, have been known since ancient times. Chemically complex, they are a mixture of organic compounds, primarily terpenes. Because the essential oils selected here were produced by steam distillation of plant materials, their molecular components would be limited to small volatile molecules. They would be suitable for examination by SEC and GC-MS to confirm that they (a) consisted of only small molecules and (b) did not elute in the excluded region of SEC. In particular, small molecules in SEC should not form aggregates and appear in SEC to be of a much larger size than their molecular masses would indicate.10 Therefore, these essential oils that are soluble in NMP would present an unequivocal test of SEC in the elution of small molecules. Although these types of molecules are not typical of those expected to be found in asphaltenes or coal liquids, none of the small standard molecules already examined by SEC1 have been observed to form aggregates in the excluded region of the chromatograms. Essential oils form an additional source of small molecules to test the SEC mechanism, because molecules typical of asphaltenes are not available as standards. Two types of essential oils were investigated: tea tree oil from the leaves of the tree Melaleuca alternifolia in Australia and lavender oils from the United Kingdom. Tea tree oil contains about 100 components,11 mainly terpenes and sesquiterpenes and their alcohols; most of the components have antimicrobacterial properties. It was expected that the GC-MS analysis would detect all of the components of each type of oil and confirm that only small molecules were injected into the SEC (9) Podzimek, S. Int. J. Polym. Anal. Charact. 2005, 9, 305. (10) Karaca, F.; Behrouzi, M.; Morgan, T. J.; Herod, A. A.; Kandiyoti, R. Energy Fuels 2005, 19, 187. (11) Hammer, K. A.; Carson, C. F.; Riley, T. V. J. Appl. Microbiol. 2003, 95, 853.
10.1021/ef050364i CCC: $33.50 © 2006 American Chemical Society Published on Web 02/04/2006
Essential Oils InVestigated by SEC and GC-MS
methods in these samples. GC-MS analysis is the normal method of analysis for these essential oils to determine the presence of only those components characteristic of the products. 2. Experimental Section 2.1. Samples. Tea tree oil was obtained from Holland and Barrett (U.K.) as batch number 16212 exp 6/06. Lavender oils were purchased from Snowshill Lavender, Hill Barn Farm, Snowshill, Broadway, Worcs WR12 7JY, as LaVandula angustifolia batch 0108 and LaVandula intermedia laVandin batch 02-04. 2.2. Size Exclusion Chromatography. Details of the methods used with NMP as the eluent have been described elsewhere;1 the Mixed-A column was used in this work. For aromatics detected by UV absorbance at several wavelengths (280, 300, 350, 370, and 450 nm) and evaporative light scattering (ELS), the small molecules (200 000 u) elute early, before 14 min, as defined by polymer standards. Therefore, a clear-cut experimental result was expected for these oils consisting of only small, volatile molecules unless the small polar molecules aggregated to form apparently large molecules. The tea tree oil and lavender oils were examined using NMP as the eluent, with a flow rate of 0.5 mL min-1 at room temperature and detection by both UV absorbance and ELS. 2.3. Mass Spectrometry. Tea tree oil has been examined by GC-MS using a Kratos Concept 1S double focusing mass spectrometer with a CB8 column, 50 m length and 0.25 µm film thickness, with a temperature from 42 °C held for 5 min raised to 250 °C at 5 °C min-1; the carrier gas pressure was 18.5 psi with a 75 m/m split, and the injection temperature was 225 °C. The injection volume was 1 µL. The lavender oils were examined using an Agilent 6890/5972 GC-MS system with a CB8 column, 50 m length and 0.25 µm film thickness, with a temperature from 50 °C held for 5 min and ramped to 250 °C at 10 °C min-1; the carrier gas pressure was 12 psi with a flow of 70 mL/m split. The injection volume was 1 µL. 2.4. UV Fluorescence Spectroscopy. The procedure has been described elsewhere.12 The Perkin-Elmer LS50 luminescence spectrometer was set with a slit width of 25 nm, to scan at 240 nm min-1; synchronous spectra were acquired at a constant wavelength difference of 20 nm. A quartz cell with a 1 cm path length was used. The spectrometer featured automatic correction for changes in source intensity as a function of wavelength. Emission, excitation, and synchronous spectra of the samples were obtained in NMP; only synchronous spectra are shown. Solutions were diluted with NMP to avoid self-absorption effects: dilution was increased until the fluorescence signal intensity began to decrease.
3. Results and Discussion 3.1. GC-MS Analyses. Tea tree oil is a readily available natural oil produced by steam distillation of the leaves of the Australian tree Melaleuca alternifolia. The major components of the oil are known,11 and those of the present sample have been determined by GC-MS. The chromatogram is not shown, but the list of components, relative peak areas, and retention times is shown in Table 1. The components identified correspond to 84.2% of the total signal recorded. The major component is terpinen-4-ol and includes some aromatic and aliphatic molecules, with the aliphatics being terpene structures. The aliphatics of Table 1 are mainly C10H16. However, they correspond in structure to terpenes and sesquiterpenes, incorporating a six-membered ring and a three- or four-membered ring as well. These structures incorporate three-dimensional ring systems, in contrast to the essentially linear alkane structures. They remain as small molecules, however, and because they (12) Li, C.-Z.; Wu, F.; Cai, H.-Y.; Kandiyoti, R. Energy Fuels 1994, 8, 1039.
Energy & Fuels, Vol. 20, No. 2, 2006 735 Table 1. Tea Tree Oil Data peak area peak time peak scan 0.85 14:22.14 2.37 14:36.99 0.29 16:03.94 0.66 16:10.31 0.45 16:40.00 0.36 17:08.63 6.37 17:34.08 6.23 17:49.99 1.55 17:59.53 3.82 18:04.83 15.76 18:59.98 2.56 20:00.42 0.11 20:24.81 34.03 22:52.21 2.63 23:15.54 0.22 28:38.98 0.38 29:35.18 0.40 29:52.15 1.52 30:22.90 0.55 30:56.83 0.33 31:11.68 1.29 31:46.67 1.29 32:23.79 0.21 32:39.69 sum of peak areas 84.23
247 261 343 349 377 404 428 443 452 457 509 566 589 728 750 1055 1108 1124 1153 1185 1199 1232 1267 1282
identity
formula
a-thujene a-pinene sabinene b-pinene b-myrcene a-phellandrene a-terpinene p-cymene b-phellandrene + limonene eucalyptol g-terpinene a-terpinolene possibly linalool terpinen-4-ol a-terpineol copaene a-gurjunene caryophyllene probably an aromadendrene alloaromadendrene probably a cadinene isomer ledene + unresolved coeluter d-cadinene cadina-1,4-diene
C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H18O C10H16 C10H16 C10H18O C10H18O C10H18O C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24
are detected by GC-MS, the structures are fairly volatile. Molecular masses of these components range from 136 to 204. Table 1 shows a total of 26 components as the major molecules in the present sample. Reproducibility tests of a tea tree oil by two-dimensional gas chromatography13,14 indicated a total of 42 components that were not identified; clearly, the 15.8% of the signals not identified in the present study corresponds to at least 16 different minor compounds in the 2-D study and many more minor components11 known to be in the typical oil. The lavender extracts were examined by GC-MS, and although the total ion chromatograms are not shown, the components detected and their relative peak areas and retention times are listed in Tables 2 and 3 for the LaVandula angustifolia and LaVandula intermedia laVandin oils, respectively. The proportion of signal identified in the tables is about 95% in each case. The components are terpenes, sesquiterpenes, acetates, and alcohols of small molecular mass only, up to about 204 mass units. The proportion of oxygenated molecules in these lavender oils (about 76 and 86%, respectively, for L. angustifolia and L. intermedia) was greater than the proportion of oxygenates in the tea tree oil (about 38%), mainly terpinenol. 3.2. Size Exclusion Chromatography. The calibration of the Mixed-A column with polystyrene and polysaccharide standards and small molecules is shown in Figure 1. The elution of oxygenates is defined as happening earlier than that of hydrocarbons, with no obvious reason for the difference in behavior. There is clearly some difference in the size of oxygenated molecules in solution in NMP that may be associated with an increased solvation of the oxygen function, enhancing the apparent size of the oxygenated molecules. Thus, acetone with a mass of 58 u elutes earlier than benzene with a mass of 78 u, whereas pyrogallol of mass 110 u elutes earlier than rubrene with a mass of 532 and equivalent to polystyrene with a mass of about 1 000 u and polysaccharide with a mass of about 700 u. The calibration shows the total exclusion volume at about (13) Marriott, P. J.; Massil, T.; Hu¨gel, H. J. Sep. Sci. 2004, 27, 1273. (14) Shellie, R.; Marriott, P.; Leus, M.; Dufour, J.-P.; Sun, K.; Winniford, B.; Griffith, J.; Luong, J.; Mondello, L.; Dugo, G. J. Chromatogr., A 2003, 1019, 273.
736 Energy & Fuels, Vol. 20, No. 2, 2006
Morgan et al. Table 3. Pure English Lavender LaWandula intermedia laWandin Data
Figure 1. Calibration of the Mixed-A column with polystyrene (PS) standards, polysaccharide (PSAC) standards, and small molecules. (1) Pyrogallol; (2) acetone; (3) the elution range of lavender oils; and (4) the elution range of tea tree oil. Table 2. Pure English Lavender LaWandula angustifolia Data peak area peak time peak scan 0.14 0.23 0.03 1.38
9:48.75 9:59.41 10:21.70 11:13.07
590 612 658 764
1.17 11:20.34 0.37 11:26.64 0.06 11:40.70 0.23 11:48.93 0.10 11:57.17 0.32 12:06.87 1.34 12:13.65 0.13 12:17.04 6.00 12:21.89 2.34 12:35.94 0.40 12:51.94 0.02 13:09.87 0.30 13:28.77 23.78 13:38.94 1.87 13:50.09 0.64 14:04.63 0.09 14:37.58 0.18 14:56.48 0.70 15:00.85 13.47 15:11.99 4.14 15:25.56 25.40 16:25.65 3.35 16:58.61 0.63 18:08.88 0.80 18:26.33 1.35 19:09.94 1.75 19:16.24 0.28 19:22.06 1.33 19:32.72 0.44 20:08.10 area sum 94.76
779 792 821 838 855 875 889 896 906 935 968 1005 1044 1065 1088 1118 1186 1225 1234 1257 1285 1409 1477 1622 1658 1748 1761 1773 1795 1868
identity a-thujene a-pinene camphene 5-methyl-3-heptanone or 3-octanone b-myrcene probably a 3-octanol probably a-phellandrene 3-carene a-terpinene p-cymene limonene plus b-phellandrene eucalyptol b-cis-ocimene b-trans-ocimene g-terpinene linalool oxide a-terpinolene linalool 1-octenyl acetate 3-octanyl acetate camphor lavandulol endo-borneol terpinen-4-ol a-terpineol linalyl acetate lavandulyl acetate neryl acetate geranyl acetate a-santalene trans-b-caryophyllene a-bergamotene b-farnesene germacrene D
formula C10H16 C10H16 C10H16 C8H16O C10H16 C8H18O C10H16 C10H16 C10H16 C10H16 C10H16 C10H18O C10H16 C10H16 C10H16 C10H20O2 C10H16 C10H18O C10H20O2 C10H20O2 C10H16O C10H20O C10H18O C10H18O C10H18O C10H18O C12H22O2 C12H20O2 C12H20O2 C15H24 C15H24 C15H24 C15H24 C15H24
14 min, as expected,15 and increasing the molecular mass produces no further reduction in elution time. The permeation limit is expected to form a similar sharp cutoff of the smallmolecule elution time,15 in which the elution time does not increase further as the molecular mass decreases. This is clearly not the case for the small standard molecules examined,1 for which elution times do continue to increase as the molecular mass decreases, forming a “tail”, as in Figure 1, out to about 28 min. 3.2.1. Tea Tree Oil. The sample was examined by SEC using the Mixed-A column with NMP as the eluent to determine the (15) Malawer, E. G. In Handbook of Size Exclusion Chromatography; Wu, C., Ed.; Chromatographic Science Series Vol. 69, Marcel Dekker, Inc.: New York, 1995; Chapter 1.
peak area
peak time
peak scan
identity
0.35 0.18 0.06 0.42 0.59 0.22 0.08 0.98
9:59.39 10:21.68 10:57.54 11:02.87 11:20.80 11:49.40 12:07.81 12:13.63
612 658 732 743 780 839 877 889
5.40 1.42 0.48 0.31 31.57 8.46 3.69 2.00 3.55 26.58 2.30 0.56 0.72 0.27 1.35 0.16 0.68 0.73 0.40
12:17.02 12:22.35 12:36.89 13:28.74 13:38.92 14:36.59 15:00.34 15:11.97 15:25.54 16:25.63 16:58.59 18:08.86 18:26.30 19:09.92 19:16.22 19:22.04 19:32.70 20:08.07 20:14.86
896 907 937 1044 1065 1184 1233 1257 1285 1409 1477 1622 1658 1748 1761 1773 1795 1868 1882
0.37 20:22.13 0.59 20:33.76 0.33 20:35.70 0.06 20:37.64 0.29 22:15.05 area sum 95.15
1897 1921 1925 1929 2130
a-pinene camphene sabinene b-pinene b-myrcene 3-carene p-cymene limonene + possibly a trace of b-phellandrene eucalyptol b-cis-ocimene b-trans-ocimene a-terpinolene linalool camphor endo-borneol terpinen-4-ol a-terpineol linalyl acetate lavandulyl acetate neryl acetate geranyl acetate a-santalene caryophyllene a-bergamotene b-farnesene germacrene D neryl or geranyl propanoate b-bisabolene g-cadinene b-sesquiphellandrene probably d-cadinene cadinol isomer
formula C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H18O C10H16 C10H16 C10H16 C10H18O C10H18O C10H18O C10H18O C10H18O C12H20O2 C12H20O2 C12H20O2 C12H20O2 C15H24 C15H24 C15H24 C15H24 C15H24 C13H22O2 C15H24 C15H24 C15H24 C15H24 C15H24O
behavior of the aromatic and terpene types in the sample. The tea tree oil was completely soluble in NMP. The SEC chromatogram at two wavelengths of UV absorbance is shown in Figure 2. The detectors gave peaks at long times (after 23 min)
Figure 2. SEC chromatograms of tea tree oil on Mixed-A column; curves are UV absorbance at 280 and 300 nm.
representing relatively small molecules with UV absorbance at only two wavelengths, 280 and 300 nm, indicating the absence of large aromatic ring systems, as expected. There was no ELS signal, again as expected for small volatile molecules, because the majority of the sample would evaporate with the NMP solvent and fail to register with the light-scattering detector. The polystyrene calibration indicates masses for these peaks of less than 200 mass units and corresponds approximately to the elution times of benzene and toluene, in agreement with the mass spectrometry. There was no early eluting peak, indicating that no aggregation occurred in solution in NMP, as expected.10
Essential Oils InVestigated by SEC and GC-MS
Figure 3. SEC chromatograms of LaVandula angustifolia on Mixed-A column with detection by UV absorbance at 280, 300, 350, and 370 nm and by ELS.
Figure 4. SEC chromatogram of LaVandula intermedia laVandin on Mixed-A column with detection by UV absorbance at 280, 300, 350, and 370 nm and by ELS.
3.2.2. Lavender Oils. Both lavender oils were completely soluble in NMP. The SEC chromatograms on the Mixed-A column showed only late-eluting peaks, with no evidence of peaks at the exclusion limit, indicating the absence of aggregate formation10 despite the presence of oxygenated and polar molecules in the oils. Figures 3 and 4 show the chromatograms of the respective oils. Both chromatograms are very similar, with the most intensity shown at 300 nm (the 280 nm absorbance wavelength is partly absorbed by the NMP solvent) and only low-intensity absorbance shown at 370 nm. Both samples showed some signal by the ELS detector, corresponding to a mass equivalent in polystyrene standards of about 340 mass units. The majority of each sample lies at lower masses, down to benzene and toluene, that elute between 24.5 and 25 min on this column.1 The lavender oils both contain larger size molecules than the tea tree oil, eluting from 20 min (Figures 3 and 4) rather than from 23 min, as in Figure 2. The increased content of oxygenated molecules in the lavender oils compared
Energy & Fuels, Vol. 20, No. 2, 2006 737
Figure 5. Synchronous UV fluorescence spectra of essential oils in methanol solvent. (1) Tea tree oil; (2) lavender oil LaVandula angustifolia; and (3) lavender oil LaVandula intermedia laVandin (bold line).
to that of the tea tree oil (which were mainly alcohols) may be responsible for the earlier elution of lavender oils. The calibration shown in Figure 1 indicates that the standard oxygenates, polysaccharides, and pyrogallol eluted earlier than their molecular masses would indicate in comparison with polystyrenes. It is presumed the same is true for the essential oils studied here, given that the compound types in all three oils are similar, but with an increased proportion of acetates in the lavender oils. 3.3. Synchronous UV Fluorescence Spectra. The spectra shown in Figure 5 indicate that the tea tree oil fluoresces at shorter wavelengths than the lavender oils, which are very similar to each other. All three oils had relatively weak fluorescence compared to that of the aromatic (benzene derivative) molecules. The shift to longer wavelengths for the lavender oils may be a result of having an oxygen content higher than that in tea tree oil. 4. Conclusions The comparison of GC-MS and size exclusion chromatography in NMP has shown that the molecules of three steamdistilled essential oils are of small size and molecular mass and produce no peaks in the excluded region of SEC caused by aggregated polar molecules in solution in NMP. This agreement is reached despite the Mixed-A column being described as the best for macromolecules by the manufacturer, Polymer Laboratories. There is evidence that the inclusion of oxygen in the oils has caused the lavender oils to elute at earlier times than the tea tree oil, but they still correspond to small molecules, not aggregates. Fluorescence of the three oils is weak, but differences are evident between tea tree oil and lavender oils. Acknowledgment. We thank Mahtab Behrouzi for help with some of the experimental work. EF050364I
