APPLICATION BRIEF Gas Chromatography/ Mass Spectrometry Authors: David Scott PerkinElmer, Inc. Shelton, CT Separation and Characterization of Indian and Australian Sandalwood Oils Introduction Sandalwood (Santalum album L.) is a desired wooden base note for many fragrances in perfume and other scented products including incense. The major component is alpha-santalol with distilled oil usually containing approximately 5. The alpha-santalol is a weak odor compound with beta-santalol being the stronger odor compound more associated with sandalwood oil. Typically the tree is grown for 12 years before the total harvest of trunk, branch and root. There are many related species giving rise to counterfeiting and confusion among consumers. The yield of the wood will change over time with a transition from beta- to alpha-santalol reported. There are more than 234 compounds 1 that have been identified in sandalwood extract to add to the overall experience with ongoing research still identifying others. 2 Not all compounds have an odor that contributes to the overall scent and it is possible to identify the tree source by the ratio of compounds present. There is also a synthetic santalone that is available and present in commercial fragrances. The addition of synthetic materials can be somewhat controversial as there is a school of thought that such synthetics take away the magic from a fragrance. The alternative viewpoint is that the synthetic material is not subject to natural disasters or simply weather impacting supply and there are fewer compounds present which may cause allergic reactions.
The application of Gas Chromatography-Olfactometry (GCO) and mass spectrometry are considered routine and are frequently the techniques of choice for analyzing extracts from these fragrant woods. Experimental Three oil samples were obtained from a commercial aromatic oil source. Separation was achieved with a 6 m wax column for the increased number of theoretical plates, though a shorter 3 m column could also be used for a faster separation. Post column, an S Swafer device was used to split the effluent between the SNFR olfactometry accessory for olfactometry and the Clarus SQ8 MS for identification. Due to the sensitivity of the mass spectrometer, the samples were diluted with 1 drop added to 1 ml of methanol. Two injection techniques were performed. A simple wet needle injection, and headspace (HS) trapping with the TurboMatrix HS trap. The wet needle injection demonstrates the analysis of a distilled oil, and the TurboMatrix headspace (HS) trap describes the possible analysis of wood prior to distillation. No wood samples were available for this work however the excellent trapping efficiency of the TurboMatrix and 22 ml vial size suggests that a representative sample of wood chips could be analyzed prior to distillation. Figure 2. PerkinElmer SNFR olfactometry accessory. Table 1. TurboMatrix HS11 Trap. Needle 21 C Oven 19 C Transfer Line 21 C Trap Low 3 C Trap High 35 C Vial Pressurization 1 min Vial Desorb 3 min Dry Purge 1 min Trap Hold 5 min Desorb.1 min Thermostat 25 min GC Cycle 14 min Figure 1. Schematic of the separation. For wet needle analysis the headspace system was removed and the capillary injector was used instead. Carrier Pressure Desorb Pressure Vial Pressure 35 psi 35 psi 4 psi The Swafer configuration allows manipulation of the column flow rate and separation without impacting the active split between the mass spectrometer and olfactometry port. The split ratios and flows are a result of the selected transfer line geometries and carrier gas pressure. The addition of the second pressure source regulates the splitting and maintains the engineered configuration independent of the column head pressure. The independence from the column head pressure increases the options available to the chemist with respect to flow rate in the separation. The separation is critical from an identification standpoint and for optimum human interaction. It is all too easy for the human to be overwhelmed with multiple peaks and the odors rolling over one another making an accurate description troublesome. Therefore a 6 m x.32 id wax column was used starting at 4 ºC and ramping 2 ºC/min to 24 ºC. The full instrument conditions are given in Tables 1 through 3. Table 2. GC Conditions. Injectors Both at 25 C Oven Program 4 C (hold 2 min) and ramping 2 C/min to 24 C (hold 15 min) Column 6 m x.32 id x 1. µm Table 3. MS Conditions. Transfer Line Temperature 2 C Source Temperature 18 C Mass Range 3 3 m/z Scan.2 secs Interscan Delay.1 secs Ionization Mode EI+ Run 18 min 2
Results The compounds present in sandalwood oils are a direct result of the distillation/extraction of the oil from the wood. A recent patent application 3 concerning the therapeutic uses of different sandalwood oils describes the different compositions of Santalum spicatum (West Australian) and Santalum album (East Indian) sandalwoods. Among the major differences are the higher alpha and beta santalol content in the Indian Sandalwood and the presence of several key components in Australian Sandalwood that are absent or significantly reduced in Indian Sandalwood oil. 1 95.38 7.26e9 Alpha-santalol Beta-santalol 99.12 Alpha-santalene Epi-beta-santalene Beta-santalene 56.71 87.6 95.59 98.38 11.2 13.41 46. 51. 56. 61. 66. 71. 76. 81. 86. 91. 96. 11. 16. Figure 3. Chromatogram of Indian Sandalwood oil showing the santalene compounds and the large alpha- and beta-santalol content. It is readily apparent that the Indian Sandalwood has significant alpha- and beta-santalol content but there are other compounds present in the Australian sample that are absent in the Indian oil. The Australian Sandalwood oil has less of the alpha- and betasantalol but more significant is the increase in several other compounds that could be considered as indicative of Australian Sandalwood oil. The dendrolasin, (E)-nerolidol, the beta-bisabolol, apha-bisabolol and nuciferol are easily identified as distinct from the Indian Sandalwood. 1 Dendrolasin (E)-nerolidol 79.9 Beta-bisabolol 73.66 85.37 Alpha-bisabolol 88.68 Farnesol 95.2 Alpha-santalol Bergamotol Beta-saantalol 95.55 97.51 99.3 11.58 99.52 Nuciferol 13.41 4.18e9 46. 51. 56. 61. 66. 71. 76. 81. 86. 91. 96. 11. 16. Figure 4. Chromatogram of Australian Sandalwood oil with the distinctive compounds indicated. 3
Analysis of the third oil sample gave strong indications that the sample was not an authentic sandalwood oil. 1 Limonene 25.41 Linalyl acetate 5.74 Isobornyl acetate Isolongifolene 83.59 79.9 37.32 53.12 63.89 47.22 8.35 Diethyl phthalate 88.62 95.1 Benzyl benzoate 97.9 13.41 1.2 19.86 14.16 5.99e8 23. 28. 33. 38. 43. 48. 53. 58. 63. 68. 73. 78. 83. 88. 93. 98. 13. 18. 113. Figure 5. Chromatogram of Sandlewood oil showing the synthetic ester compounds and lack of alpha- and beta-santalol. The presence of several synthetic esters and diethyl phthalate to fix them in the oil is strongly suggestive that the "sandlewood" sample is not an oil distilled from a species of Santalum. The isolongofolene and the benzyl benzoate are both woody tones that have been used to mimic the sandalwood experience. The limonene would appear to have been added to balance out the tones. The common components are easily identified with a wet needle experiment and the TurboMatrix HS Trap was used to increase the sample volume without solvent column overloading. Four microliters of the sample were then injected into a headspace vial for a total evaporation experiment. The increased mass on column assists in the identification of smaller peaks and further highlights the differences in the two sandalwood species. 1 5.59e9 Beta-curcumene Teresantalol 1 5.59e9 Beta-himachalene Alpha-longipinene Alpha-curcumene 47. 51. 55. 59. 63. 67. 71. 75. 79. 83. 87. 91. 95. Figure 6. Chromatograms of Indian and Australian Sandalwood oil with TurboMatrix HS-Trap to elucidate small differences in the two samples. 4
Conclusion The GCO system discussed successfully separated alpha- and betasantalol in two commercially obtained sandalwood samples. Qualitative differences were identified demonstrating the capability of adulterant recognition. There is still active research in identifying new scents from botanical sources making GC-olfactometry an important tool in the quest for new sources of fragrance as well as preventing adulteration and counterfeiting. References 1. N. Baldovini et al., Flavour Fragr. J. 211, 26, 7 26 2. T. Hasagewa et al., Flavour Fragr. J. 211, 26, 98 1 3. Ian Clements, Paul Castella, Corey Levenson, Application number PCT/US211/2676, 211. PerkinElmer, Inc. 94 Winter Street Waltham, MA 2451 USA P: (8) 762-4 or (+1) 23-925-462 www.perkinelmer.com For a complete listing of our global offices, visit www.perkinelmer.com/contactus Copyright 215, PerkinElmer, Inc. All rights reserved. PerkinElmer is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. 1221_1 PKI