extraction and variation of the essential oil from western

Extraction and Variation of the Essential Oil from Western

Australian Sandalwood (Santalum spicatum)

Paul Moretta, B.Sc. (Hons)

This thesis is presented for the degree of

Doctor of Philosophy

The University of Western Australia

Department of Chemistry

2001

This thesis is presented for the degree of Doctor of Philosophy to The University of

Western Australia.

The work described in this thesis was carried out by the author in the Departments at

The University of Western Australia under the supervision of Dr Robert. D. Trengove

and Associate Professor Emilio. L. Ghisalberti. Unless duly referenced, the work

described is original.

Paul Moretta

June 2001

ii

Acknowledgements

This study could not have been performed without the help of many people, all whom I

owe a great deal of thanks.

I would firstly like to thank my supervisors Robert Trengove and Emilio Ghisalberti for

the opportunity to undertake a PhD, and their never-ending enthusiasm, support and

advice over the duration.

I could not go without thanking my fellow lab members, Brendan, Courtney, Anthony

and Gavin, along with numerous honours students, who have constantly helped with

numerous problems and provided me with a wealth of knowledge, along with numerous

years of entertainment. I must especially thank Brendan who always took time out of his

'busy' schedule for advice on writing and countless computer solutions.

The Department of Conservation and Land Management (CALM) now Forest Products,

need to be thanked for their support of the project, in particular Syd Shea, Don Keene,

and Peter Jones. Special thanks must be directed towards Dave Evans, Ben Sawyer, Jon

Brand and Kim Phillips from CALM, and Anthony who helped in the collection of

samples. I must also thank Westcorp for providing bulk samples of sandalwood for large

scale extraction.

To George, Nigel, Darko, and Bruce from the workshop who were always available to

manufacture anything on demand, no matter how obscure the design, and provide

numerous tools to process the wood, many thanks.

iii

Mike Whitby, Brendan Grierson, Rod Minnet and Dennis Gere from Agilent

Technologies must also be thanked for their assistance and generosity in relation to the

equipment and consumables used for the project.

Finally I would like to thank my parents for the oppurtunities they have given

throughout my life, and Simonne for her understanding and support. -

iv

Abstract

Western Australian sandalwood (Santalum spicatum) is a small tree that contains

fragrant oil within the heartwood. Even though sandalwood was one of WA's first

industries, and has continued to supply the world with wood for over 150 years, little

focus has been placed into research and development of the industry, compared to East

Indian sandalwood (Santalum album), particularly in the field of chemistry. S. album

contains an oil content of over 6% and santalol content of over 90%. Much conjecture

exists in the literature as to the amount and chemical composition of sandalwood oil

found in Santalum spicatum.

The essential oil from Western Australian sandalwood was extracted by supercritical

carbon dioxide. Solid bed trapping conditions were optimised by using a standard

mixture of compounds representative of the components of sandalwood oil. It was found

that the type of trapping material, trap temperature, flow rate and rinse solvent all had an

effect on the recoveries. Quantitative recoveries of the standards were obtained when

Isolute diol was used as the trapping material at 20°C, using polar rinse solvents such as

ethanol, ethyl acetate or methyl-tert-butyl ether.

The effects of the extraction parameters including density, time and particle size on the

extraction of real sandalwood samples were examined. An extraction density of 0.75

g/mL for 30 minutes was sufficient to completely remove the volatile components of the

sandalwood oil from the wood, leaving behind the larger molecular weight compounds

not responsible for the odour of the oil. The SFE extraction method was up-scaled from

7mL to 300 mL with no effect on oil yield. The oil extracted using these optimal

V

trapping and extraction conditions was found to differ in both yield and composition to

the oil extracted using hydrodistillation and solvent extraction (hexane and ethanol).

Variations in the yield and composition of extracted sandalwood oil were found to exist

for different sections of the tree. More oil and higher santalol contents were found in the

roots of sandalwood trees, and these values decreased moving further up the tree. The

oil content was also found to vary across the diameter of the tree. More oil was found in

the centre of the wood (heartwood) compared to the outer sapwood.

To aid in the knowledge of sandalwood, a study was conducted to examine whether

differences existed in oil yield and composition due to geographic location. A total of 87

trees from 12 geographic locations were sampled. Average oil yields were found to

differ significantly between geographic locations, ranging between 2.0 and 4.6%. The

oil composition was also found to vary between geographic locations, with the average

santalol content varying between 3.3 and 66.6%. An investigation was also conducted to

see if differences in oil arose due to seasonal variation. No significant variation was

found to exist in oil yield and composition from trees samples during summer, autumn,

winter or spring.

vi

Table of Contents

1. Introduction 1

1.1. Essential Oils 1

1.2. Sandalwood 2

1.2.1. Sandalwood Species 2

1.2.2. S. spicatum 4

1.2.3. Uses ~ 12

1.2.4. Sandalwood Oil 14

1.2.5. Factors affecting Sandalwood Oil Quantity and Quality 17

1.3. Supercritical Fluid Extraction 24

1.3.1. What is a Supercritical Fluid? 24

1.3.2. Supercritical Fluid Extraction 27

1.4. Optimisation of SFE 32

1.4.1. Extraction Conditions 33

1.4.2. Collection Conditions 39

1.4.3. Adsorption 42

1.4.4. Desorption 43

1.5. Extraction of Essential Oils 46

1.5.1. Steam and Hydrodistillation of Essential Oils 46

1.5.2. Solvent Extraction of Essential Oils 50

1.5.3. Other Extraction Techniques 51

1.5.4. SFE for the Extraction of Essential Oils 51

1.6. Analysis of Essential Oils 54

1.6.1. Gas Chromatography 54

1.6.2. Liquid Chromatography 55

1.6.3. Supercritical Fluid Chromatography 55

1.7. Aims of Project 57

2. Experimental 58

2.1. Equipment 58

2.2. Optimisation of Trapping Conditions 59

2.2.1. Materials 59

2.2.2. Time Course Extraction of Standard Mixture 60

2.2.3. Trapping Conditions 62

2.2.4. Hydrodistillation of Standard 65

2.3. Optimisation of Sandalwood Extraction 66

2.3.1. Materials 66

2.3.2. Calculations of Percentage Yield, Volatiles and Composition 67

2.3.3. Exhaustive Extraction 68

2.3.4. Effect of Density on Percentage Yield, Percentage Volatiles, and Composition 69

vii

2.3.5. Particle Size 70

2.4. Large Scale Extraction 73

2.4.1. Materials 73

2.4.2. Instrument Modification 74

2.5. Comparison of Extraction Techniques 76

2.5.1. Materials 76

2.5.2. Methods 77

2.6. Sandalwood Survey 78

2.6.1. Geographic - 78

2.6.2. Seasonal Variation 85

2.6.3. Sectional Examination 86

3. Results and Discussion 88

3.1. Optimisation of Trapping Conditions 88

3.1.1. Extraction Time Course of Components of Standard Mixture 88

3.1.2. Trapping 91

3.1.3. Inert Trapping Material 93

3.1.4. Non-polar traps 97

3.1.5. Polar Trapping Material 101

3.2. Desorption 105

3.2.1. Inert Trapping Material 106

3.2.2. Non-Polar Trapping Material 106

3.2.3. Polar Trapping Material 110

3.2.4. Rinse Temperatures 114

3.3. Hydrodistillation of Standard Mixture 114

3.4. SFE Extraction Conditions for Sandalwood Samples 116

3.4.1. Extraction Time 117

3.4.2. Density 120

3.4.3. Particle Size 123

3.5. G C - M S Identification of Components in Volatile Sandalwood Oil 128

3.6. Comparison of Extraction Techniques 129

3.7. Scale up of Extraction 133

3.8. Section of Tree 135

3.9. Geographic Variation 150

3.10. Seasonal Variation in Oil Yield and Volatile Composition 162

4. Conclusion Error! Bookmark not defined.

5. References 170

6. Appendix 183

Vlll

Table of Figures

1.1. Santalum spicatum 4

1.2. Distribution of S. spicatum in Western Australia 6

1.3. Distribution of sandalwood species in Australia 8

1.4. Major components of sandalwood oil 16

1.5. Phase (pressure-temperature) diagram of a pure substance 25

1.6. Variation of the reduced density of a pure component in the vicinity of its critical point 27

1.7. Schematic diagram of basic SFE apparatus ._ 31

1.8. Liquid solvent SFE trapping device 40

1.9. Solid bed trapping device 41

1.10. Conversion of sabinene in acidic conditions 49

2.1. Structure of compounds used in the standard mixture 60

2.2. Modification of the SFE apparatus to allow scale-up of the extraction vessel to 300 m L 75

2.3. Large-scale trapping device 75

2.4. Coring of sandalwood trees 79

2.5. Drillhole filled with Sellys polyfiller post core sampling 80

2.6. Sampling locations throughout Western Australia 85

2.7. Exposed roots of a sandalwood tree 87

3.1. Cumulative percentage of individual components of the standard mixture extracted over 80

minutes at a C 0 2 density of 0.90 g/mL 90


minutes a t a C 0 2 density of 0.65 g/mL 90


minutes at a C 0 2 density of 0.30 g/mL 91

3.4. Phases bonded to solid silica supports used in the study

93

3.5. Recoveries of the components of the standard mixture using stainless steel beads at various trap

temperatures 95

3.6. Recoveries of the components of the standard mixture using stainless steel beads at various flow

rates 97

3.7. Recoveries of the components of the standard mixture using Hypersil O D S at various trap

temperatures 98

3.8. Recoveries of the components of the standard mixture using Isolute CI 8 at various trap

temperatures 99

3.9. Recoveries of the components of the standard mixture using Hypersil O D S at various flow rates 100

3.10. Recoveries of the components of the standard mixture using Isolute C18 at various flow

rates 101

3.11. Recoveries of the components of the standard mixture using Isolute silica at various trap

temperatures 103

3.12. Recoveries of the components of the standard mixture using Isolute cyano at various trap

ix

temperatures 103

3.13. Recoveries of the components of the standard mixture using Isolute diol at various trap

temperatures 104

3.14.Rinsing efficiency ofhexane using Hypersil O D S 108

3.15. Rinsing efficiency of iso-octane using Hypersil O D S 108

3.16. Rinsing efficiency ofhexane using Isolute CI 8 109

3.17. Rinsing efficiency of iso-octane using Isolute C18 109

3.18. Rinsing efficiency ofhexane using Isolute silica 110

3.19. Rinsing efficiency of iso-octane using Isolute silica - Ill

3.20.Rinsing efficiency of hexane using Isolute cyano Ill

3.21. Rinsing efficiency of iso-ocatne using Isolute cyano 112

3.22. Rinsing efficiency of hexane using Isolute diol 112

3.23. Rinsing efficiency of iso-ocatne using Isolute diol 113

3.24. Effect of hydrodistillation on m e recovery of m e standard mixture 115

3.25. Time course of the extraction of total oil from sandalwood at various densities 118

3.26. Time course of the extraction of volatile oil from sandalwood at various densities 118

3.27. Theoretical extraction profile of an analyte from a solid matrix 119

3.28. Yield and composition of volatiles extracted from sandalwood at various densities 121

3.29. Compositional variation in components of sandalwood oil extracted at various densities 122

3.30. Yield and volatile composition of oil extracted from sandalwood of various particle sizes by

SFE (Study 1) 125

3.31. Yield and volatile composition of oil extracted from sandalwood of various particle sizes by

SFE (Study 2) 127

3.32. Yield and composition of volatiles extracted by different various techniques 131

3.33. Compositional variation in sandalwood oil extracted by various extraction techniques 132

3.34. Chemical conversion of lanceol to nuciferol 132

3.35. Time course of the extraction of total oil from sandalwood using a 300 m L extraction vessel... 133

3.36. Comparison of oil yield from 7 m L and 300 m L extraction vessel 135

3.37. Oil yield and percentage volatiles of sandalwood oil along Tree 1 137



3.40. Major compositional changes along Tree 1 140



3.43. Hypothetical derivation of acyclic and monocyclic sesquiterpenes in sandalwood 144

3.44. Hypothetical derivation of bicyclic and tricyclic sesquiterpenes from the bisabolonium cation in

sandalwood 145

3.45. Variation in oil yield and percentage volatiles across the diameter of Tree 1 147



3.48. Sampling positions across the diameter of the three trees 148

x

3.49. Major compositional changes across the diameter of Tree 1 149



3.52. Variation in the oil content of sandalwood trees from various geographical locations 153

3.53. Variation in the percentage volatiles of sandalwood trees from various geographical locations... 153

3.54.Fisher's pairwise comparison for statistical differences in oil yields between locations 154

3.55. Fisher's pairwise comparison for statistical differences in percentage volatiles between location 154

3.56. Average rainfalls and temperatures for sandalwood sampling locations 156

3.57. Variation in the average percentage santalol from sandalwood trees from various geographical

locations 158

3.58. Variation in the average percentage a- bisabolol from sandalwood trees from various

geographical locations 158

3.59. Variation in the average percentage t,t- farnesol from sandalwood trees from various

geographical locations 159

3.60. Variation in the average percentage nuciferol from sandalwood trees from various geographical

locations 159

3.61. Fisher's pairwise comparison for statistical differences in a- santalol between locations

160

3.62.Fisher's pairwise comparison for statistical differences in P- santalol between locations 160

3.63. Fisher's pairwise comparison for statistical differences in t,t- farnesol between locations 161

3.64. Fisher's pairwise comparison for statistical differences in nuciferol between locations 161

3.65. Variation in oil yield from 5 locations sampled during 4 different seasons 163

3.66. Variation in percentage volatiles from 5 locations sampled during 4 different seasons 163

XI

Table of Tables

1.1. Species and world-wide distribution of commercially exploited sandalwood 2

1.2. Host species of S. spicatum 5

1.3. Oil yield and Santalol content of commercially exploited sandalwood 14

1.4. Variation in the composition of S. spicatum oil from various sections of the tree 20

1.5. Orders of magnitude of physical data of gas, supercritical fluid and liquid 25

1.6. Critical conditions for pure components 29

1.7. Relative solubility in C 0 2 of classes of compounds typically found in plant material 52

2.1. H P L C grade solvents used as rinse solvents for SFE 59

2.2. Compounds used in the standard mixture 59

2.3. Extraction and trapping conditions for exhaustive extraction of the standard mixture 61

2.4. G C conditions for the analysis of the standard mixture 62

2.5. Specifications of trapping materials 62

2.6. Extraction and trapping conditions used to examine the effect of the trapping material 64

2.7. G C conditions for the analysis of sandalwood oil 68

2.8. Extraction and trapping conditions for exhaustive extraction of sandalwood oil 69

2.9. Extraction and trapping conditions used to completely extract the sandalwood oil from the

matrix at various densities 70

2.10. Range of particle sizes used in study 1 71

2.11. Extraction and trapping conditions for exhaustive extraction of sandalwood oil in the particle

size studies 72

2.12. Extraction times used to completely extract the sandalwood oil from the matrix at various

particles sizes 73

2.13. Extraction and trapping conditions for the large-scale extraction of sandalwood (300 m L ) 76

2.14. Optimised SFE conditions for the extraction of sandalwood oil 78

2.15. G C conditions for the analysis of sandalwood oil to determine variation between trees 81

2.16.GC-MS conditions for the analysis of sandalwood oil 82

2.17. Locations and dimensions of sandalwood trees in the geographical survey 83

2.18. Locations and dates of sampling of sandalwood trees in the seasonal survey 86

2.19. Locations and dimensions of the 3 trees in the sectional survey 86

3.1. Molecular weights and boiling points of the components of the standard mixture 89

3.2. Polarity indexes of solvents used for desorption of standard mixture 105

3.3. Cumulative percentage of volatile oil extracted from sandalwood of various particle sizes by

SFE (Study 1) 124

3.4. Cumulative percentage of volatile oil extracted from sandalwood of various particle sizes by

SFE (Study 2) 126

3.5. Peak number, retention time and compounds of Western Australian sandalwood oil 128

3.6. Minor compositional changes along the three trees 142

3.7. Oil yield and santalol content of trees sampled in geographical survey 152

xii

3.8. Identification number corresponding to the geographical locations used in Fisher's pairwise

comparisons 155

1

1. Introduction

1.1. Essential Oils

By definition, essential oils are highly volatile substances isolated by a physical method

or process from an odiferous plant of single botanical species \ These oils were termed

'essential' because they were thought to represent the very essence of odour and flavour.

The term 'volatile oil' is interchangeably used with essential oil since it refers to the fact

that most of the components of the oil have low boiling points and can be recovered

from the plant tissue by steam distillation. Volatile oil is often the preferred term as it

provides a complete distinction from the higher boiling point compounds of natural

products such as triglyceride oils and fats .

Essential oils consist of a complex mixture of compounds in which the final flavour and

fragrance results from an intricate combination and proportion of the constituents. The

principle components of essential oils are mono- and sesquiterpenoids, with minor

amounts of other compounds such as aromatic and heterocyclic compounds ' . Many

essential oils contain a high proportion of mono- and sesquiterpene hydrocarbons. The

odour of these compounds is low in comparison to their oxygenated derivatives, and is

often removed from the oil by processes such as distillation .

Essential oils can be extracted from the blossoms, seeds, fruits, fruit peels, leaves,

stems, bark, wood, roots or secretions from plants. The oils are usually stored in

specialised structures within the plant tissue, such as glandular hairs on the epidermis,

oil tubes in the pericarp, or isolated cells within the tissue 4. The time taken to remove

the oil from the plant material is highly dependent on the localisation of these structures.

2

For example, the extraction of oil from the leaves or blossoms of a plant would be easier

than the extraction of oil from the wood or seed matrix.

Essential oils have many uses, ranging from flavourings, fragrances and therapeutic

agents to many industrial uses. They have been isolated from more than 1500 species

belonging to over 87 plant families. Approximately 150 different types-are commonly

traded on today's world market5.

1.2. Sandalwood

1.2.1. Sandalwood Species

The essential oil extracted from sandalwood (genus Santalum) is one of the oldest

ingredients for perfumery 6. The aromatic wood consists of a light yellow sapwood

surrounding a dark brown inner heartwood which contains the oil. The value of the

wood is determined by the amount and quality of oil contained within the heartwood.

Only those species that contain the highest quality oil are commercially viable to

harvest. Of the 29 species of sandalwood world-wide, only 7 are commercially

exploited. These 7 species along with their global distribution are shown in Table 1.1.

Sandalwood Species Distribution

Santalum album India, Indonesia, Timor and Australia Santalum austrocaledonicum N e w Caledonia and Vanuatu

Santalum ellipticum Hawaiian Islands Santalum lanceolatum Australia Santalum macgregorii N e w Guinea

Santalum spicatum Australia Santalum yasi Fiji and Tonga

Table 1.1. Species and world-wide distribution of commercially exploited sandalwood

3

S. album (East Indian sandalwood) has served as the benchmark for sandalwood quality,

a factor that has placed a high demand on the species for centuries. This, coupled with

inadequate management programs, illegal smuggling, and disease, has caused natural

stands of the trees to diminish over time 7.

The reduced availability of S. album has resulted in a greater demand for, and

acceptance of, sandalwood of lower quality. This has placed enormous stress on the

populations of other sandalwood species, particularly those found on small Pacific

islands. S. yasi, S. austrocaledonicum, S. ellipticum, and S. macgregorii have been

overexploited with drastic reductions in the sizes of the species and significant declines

in the quality of trees ' . A species of sandalwood (S. fernandezianum) in the Juan

Fernandez Islands off the coast of South America was exploited to extinction in the

early 1900s 10.

The geographic isolation and late settlement (1788) of Western Australia has meant that

natural stands of S. spicatum remained virtually untouched, while stands of sandalwood

in India and the Pacific were overexploited. The early settlers first initiated trade of S.

1117

spicatum in Western Australia in 1845 ' . Due to the widespread distribution of the

tree throughout the state and the imposition of stringent management programs, export

of S. spicatum has continued to the stage that Western Australia is now the main

supplier of internationally traded sandalwood 13'14.

4

1.2.2. S. spicatum

1.2.2.1. Description

Western Australian sandalwood (Santalum spicatum (R. Br.) A. DC.) is a small tree or

shrub which may reach heights between 3 and 8 m when fully mature. The tree consists

of irregular spreading branches with green-grey narrow leaves (Figure 1.1). It is slow

growing, taking between 50 and 90 years to fully mature depending on rainfalll . The

stem diameter can range from 100 to 300 m m and the tree is considered harvestable

when the diameter is greater than 127 m m at 150 m m above ground level.

Figure 1.1. Santalum spicatum

The tree flowers from 3 years and flowering can occur at any time of the year depending

on rainfall16. Flowering is most frequent during mid-summer (February) to autumn

(May). Between October and January, the fruit usually ripens into a leathery red-brown

epicarp with a smooth round hard endocarp (nut) up to 2 cm in diameter 1415.

5

Sandalwood seeds germinate following imbibition of rain water which cracks the nut.

Only 1 to 5% of the total seeds germinate 16. This, along with sandalwoods

susceptibility to fire and grazing by domestic and feral herbivores, mainly sheep, goats

and rabbits, has resulted in low levels of regeneration outside conservation reserves.

Establishing plantations of Western Australian sandalwood has proven difficult since it

is a root hemi-parasite, as are all species of sandalwood. While these trees do produce

their own photosynthates, they also require part of their essential nutrients to come from

a host plant(s)17. The tree uses haustoria that protrude from the roots and attach

themselves to the roots of one or more host plants. There is much conjecture as to

whether sandalwoods are facultative or obligate parasites. A list of host species recorded

for S. spicatum commonly pertaining to the Acacia and Eremophila genera, is shown in

Table 1.2. During periods of drought, the parasite is able to adapt by drawing moisture

and nutrients from the root system of the host through a direct xylem-to-xylem union.

Under extreme drought, it has been observed that the host plant may die before the

parasite 15. Little is known about the role of the host, and whether the rate of growth and

the quality of sandalwood varies with different hosts .

Host Species Recorded for Santalum spicatum

Acacia acuminata Dodonaea lobulata Acacia aneura Eremophila alternifolia

Acacia collectioides Eremophila dempsteri

Acacia hemiteles Eremophila ionantha

Acacia linophylla Eremophila oldfieldii Acacia tetragonophylla Eremophila oppositifolia Cassia chatelainiana Eucalyptus loxophleba Cassia nemophila Eucalyptus wandoo

Casuarina cristata

Table 1.2. Host species of S. spicatum 15

6

1.2.2.2. Distribution

Western Australian sandalwood is distributed over an area of 161 million hectares of

southwestern Australia (Figure 1.2) 19. Prior to clearing of the land for agriculture in the

'Wheatbelt', in the southern region of Western Australia, the total area was much larger,

ranging from a latitude of 24°S to 35°S. However the total harvestable area at present is

only 82 million hectares. The remaining areas consist of reserves and national parks 15.

Stands of S. spicatum were also widely distributed in South Australia, but have largely

disappeared due to agricultural and pastoral activities20.

Figure 1.2. Distribution of& spicatum in Western Australian

7

1.2.2.3. Climate

S. spicatum is generally found in the arid and semi-arid regions of Western Australia

where rainfall is between 200 and 600 mm annually . In rainfall areas of 200 mm,

sandalwood has a tendency to be found on lower slopes and drainage lines . The

amount of rainfall has been shown to have a marked effect on the regeneration and

growth rate of the trees 15. The time required to reach a commercial size in a 500 mm

rainfall zone can be 23 years, whereas in a 300 mm rainfall zone the tree may take up to

100 years to reach full size 21.

1.2.2.4. Soils

Western Australian sandalwood is generally found growing on red soils with a pH

91

ranging from neutral to mildly acidic (pH 4.5) . The most important aspect of the soil

is that it must be free draining. Sandalwood prefers lighter type soils such as loams and

sandy loams with a granite composition. Granite soils are characteristically high in

potassium that has been shown to be important in sandalwood nutrition ' . Heavy clay

soils, waterlogged sites and saline areas are not suitable.

1.2.2.5. History of the Sandalwood Industry in Australia

Six species of Santalum grow naturally in Australia: S. acuminatum, S. album, S.

lanceolatum, S. murrayanum, S. obtusifolium and S. spicatum . Their distributions

throughout Australia are illustrated in Figure 1.3. With the exception of S. album, all are

native to Australia. Only S. album, S. lanceolatum and S. spicatum are of the necessary

quality to be commercially acceptable.

8

Figure 1.3. Distribution of sandalwood species in Australia

Statham has comprehensively examined the history of the involvement of Australia in

the sandalwood trade. She identified three distinct stages: The Offshore Industry (1805 -

1860), Australian Sandalwood (1844 -1929) and Government Control (1929 - present)

1 \ and these are summarised below.

Australia's involvement with sandalwood began in 1805, 17 years after foundation of

the colony in 1788. Whalers and sealers from Sydney, during the off-season periods in

the Pacific Islands, brought back sandalwood to trade and thus fund future expeditions.

This was essentially a small scale trade and quantities did not exceed several hundred

tonnes. By the mid 1860s, the offshore industry ceased due to diminishing commercial

stands in the Pacific region.

In 1843, a report reached Perth on the west coast of Australia of the high prices being

obtained for a wood of a tree similar to one found growing in the region. It is thought

that the early Indian and Chinese labourers were the first to recognise the commercial

potential of the wood. Up until this time, the early settlers on the west coast had used the

wood for firewood and as a building and fencing material, and large areas were burned

during clearing of the land 24.

The first shipment of Western Australian sandalwood took place in 1845, when 4 tonnes

of wood was exported to Bombay as a trial to determine if harvesting the wood would

be a commercially viable option to help overcome a balance of trade deficit. News was

received that the wood would fetch £10 per tonne in Bombay and £20 - 30 per tonne if

shipped directly to Canton. Even though the wood was inferior to the top quality S.

album and not suitable for carving, it was in high demand as the powdered form for

incense. During this period, harvesting of sandalwood rapidly increased and by 1848, it

had become the colony's primary industry.

During the late 1800s, the demand for Western Australian sandalwood increased due to

export restrictions of S. album from India. Following the gold rush in Kalgoorlie (600

km east of Perth) came the construction of railways that gave access to vast areas of

10

uncut sandalwood and lowered the cost of transportation to the coast. Consequently the

industry trebled in size during this period, with annual export quantities reaching 9000

tonnes.

In 1913, Braddock established a plant to distil sandalwood just outside Perth. The

reported claims of the healing properties of sandalwood oil, and the outbreak of World

War I, significantly increased the demand for the oil. In 1917, over 3000 lbs of

sandalwood oil were being exported to England. The industry flourished throughout the

1920s and was not affected until the 1930s by deterioration of conditions in China and

the outbreak of World War H

By the late 1920s the need of Government regulation in the industry became clear as

stockpiles began to build up on the wharves at Fremantle and the sandalwood workers

could not be paid for their harvest. The Sandalwood Control Act, introduced in 1929, set

restrictions on the amount of sandalwood harvested. Over the following years, new

plans were negotiated mainly by altering harvesting quotas, aimed at stabilising the

market and increasing the price of sandalwood. At this point Australia held 80 % of the

international market.

During the mid 1900s the export of sandalwood remained highly profitable. The

distillers of oil through this period absorbed 10- 20 % of the wood harvested, but the

price they were prepared to pay for the wood was much less than what was paid on the

overseas market. As a consequence, sandalwood oil production in Australia ceased in

1971.

11

Throughout the 1980s, the market demand for incense continued to increase due to

rising populations in Asia and exports in Australia ranged from 1600- 2000 tonnes per

year. Concern was expressed over how long sandalwood stands would be able to

support the current demand. To reduce the amount of native wood harvested and as an

opportunistic way to make money, numerous companies established sandalwood

plantations. By far the largest project to date is the plantation of S. album in Kununurra,

in the northern tropical region of Western Australia, which began in 1997. The

plantation is situated on the Ord River which provides irrigation during the dry season.

S. album was chosen over & spicatum since & album has a faster growing rate and

provides higher return.

In 1997, sandalwood oil production was commenced once again in Western Australia by

Mt Romance Pry. Ltd, situated in Albany . The company won a government tender for

the sale of sandalwood, in which they receive up to 1000 tonnes of S. spicatum per

annum. M t Romance currently produces its own range of cosmetics using the distilled

oil as a base, and is looking to sell the oil in the international market as a base for the

perfumery industry.

1.2.2.6. Conservation

Sandalwood harvesting in Western Australia is administered by the Department of

Conservation and Land Management (CALM), according to the provisions of the Forest

Products Commission Act 2000. The harvesting and marketing of the sandalwood is

managed by the Forest Products Commission (FPC). Wescorp Pty. Ltd. is the sole

processor and marketing agent for S. spicatum.

12

Harvesting of the wood is performed by licensed sandalwood pullers under contract by

the FPC. The trees are harvested by attaching a chain around a tree connected to a four-

wheel drive vehicle, and pulling the tree out of the ground. Invariably, not all the tree is

utilised as some of the roots remain in the ground.

1.2.3. Uses

Sandalwood's highly aromatic wood has been prized for centuries, and its use in

religious and cultural ceremonies can be traced back through mythology to the 7th

century BC, and through the literature to 2300 years ago26. The wood is traditionally

used by Indians and Chinese for carvings, incense and oil.

Carvings

The highest quality wood is utilised for carvings. Logs are graded for carving and many

specifications must be met. The log must be 1 m in length, free of any cracks, rot or

blemishes, have a heartwood diameter of at least 125 mm, and an oil content of at least

i o

2 % . These logs are turned into religious ornaments, boxes, beads and other

handicrafts.

Major exporters of high grade logs are Australia, Hawaii, Fiji and Indonesia. Export is

to Hong Kong and Taiwan who in turn distribute to China, Japan and Singapore. India

who produces the highest grade of logs, due to its fine grain and oil content, have a ban

on all sandalwood export due to diminishing resources. Some sandalwood is illegally

exported and sold for exorbitant prices on the black market. Harsh penalties have been

implemented in attempts to curtail this practise.

13

Incense

Incense plays an important role in Buddhist and Hindu religious ceremonies. This,

coupled with the spread of the practice in Western countries, has led to a large and

increasing market for incense.

The incense market uses logs of all quality. Australia supplies most of-the incense world

market at present. The wood is mostly ground to a powder on site in Western Australia

and exported to Singapore and Taiwan for processing. The powdered wood is attached

to bamboo slithers with wood resin or compacted into incense sticks.

Oil

Sandalwood oil is extracted from the stem and roots and is one of the most valuable

essential oils in the world. More than 90 % of the world production of sandalwood oil is

from India 13 and represents over 50% of the country's total exports 27. The essential oil

is a pale yellow viscous liquid and is described as having a sweet, warm, persistent

woody odour.

The oil has various traditional uses. It has been used as an antiseptic, antiscabietic,

diuretic, and for the treatment of dermatitis, gonorrhoea, bronchitis and bladder

infection 13'28,29. More modern applications have used the oil in treatment against the

organisms Staphylococcus aureus, Bacillus anthracis, Bacillus subtilis and the Herpes

virus30'31. The oil has also shown to have chemopreventive action on carcinogenisis in

32

mice .

By far the major modern day use of the oil is as a base fragrance in perfumery. Much of

the sandalwood oil produced in India is exported to perfumeries in France and New

14

York. The oil blends very well with almost all types of perfumes and flavours and has

become a common fixative in countless woody floral and oriental floral bases .

Sandalwood oils are also used extensively in soaps, moisturising creams and powders. It

has been shown to act as an insect repellent in some creams34.

1.2.4. Sandalwood Oil

The amount and quality of oil contained in sandalwood differs considerably between

species (Table 1.3). The santalol content, made up of the sesquiterpene alcohols a- and

(3-santalol, is responsible for the aroma and quality of the sandalwood oil. The highest

quality oils have a santalol content of over 90%. The santalol content of the 7

commercially exploited sandalwood species is shown in Table 1.3.

Species

Santalum album

Santalum austrocaledonicum Santalum ellipticum Santalum lanceolatum Santalum macgregorii Santalum spicatum Santalum yasi

Oil Yield (%)

6-7 a

3 -5 a

n/a la

4-6 a

2 - 5 a

5a

Santalol Content (%)

>90b

>90c

n/a n/a n/a

10-70d

87c

Table 1.3. Oil yield and santalol content of commercially exploited sandalwood . . . ..... 13, 35 36 . 37-41

(n/a= not available) a b e d

1.2.4.1. Composition of S. album

The composition of East Indian sandalwood oil has been examined extensively. The oil

is a complex mixture of over ninety chemical compounds, of which at least fifty have

been suitably characterised 42_44. The santalol content consists of approximately 60 % a-

santalol and 30 % P- santalol37'45-47. Brunke has shown that p- santalol is the main

contributor to the aroma of the oil, possessing a typical sandalwood odour with powerful

15

wood, milky and urinous tones 48'49. By contrast, a- santalol possesses a much weaker

woody, cedarwood-like scent.

There is much disagreement in the literature on the compounds present in the remaining

10 % of the oil. There is general agreement that the sesquiterpene hydrocarbons a- and

p- santalene, and santalyl acetate contribute between 4- 6% to the oil 43-45>46>50>51. other

sesquiterpene alcohols include (+)-(Z)-epi-P~santalol, (-)-(E)-p-santalol, (-)-Z-lanceol

and (+)-(Z)-nuciferol45,49. Numerous other acids, aldehydes, ketones and alcohols have

also been identified 48'52"58.

1.2.4.2. Composition of S. spicatum

The composition of Western Australian sandalwood has been investigated to a much

lesser extent than the East Indian variety. An examination of the literature revealed only

seven references to Western Australian sandalwood oil38"41,59"61. Since most of these

refer to work carried out without the benefit of modern analytical techniques, a number

of discrepancies in the results reported are evident, in particular the santalol content.

Early work by Penfold estimated a santalol content of 35 %39'40, while Guenther

claimed a higher value between 40 and 45 % . In a more recent study, S. spicatum

collected near Kalgoorlie, Western Australia, afforded an oil containing 10% santalols60

and30%santalols41.

Due to the lower santalol content of S. spicatum, the other components of the oil

contribute significantly to the odour. There is general agreement that one of the major

components ofS. spicatum is trans, trans- farnesol, responsible for approximately 30 %

of the oil41'59'60'62. Other compounds previously identified and significantly contributing

16

to the oil composition include the sesquiterpenes epi-a- bisabolol, Z- nuciferol,

dendrolasin and cis- lanceol. The oil also contains a hydrocarbon fraction (~6%) that is

composed of a- santalene, epr-P-santalene, p~ santalene, and a and P- curcumene. The

structures of some of the main components of sandalwood oil are shown in Figure 1.4.

a- bisabolol OH

Nuciferol a- curcumene

Dendrolasin (Z)-a-frans-berg amotol

a- santalene p- santalene

CH2OH

(Z)-cc- santalol

,CH2OH

(Z)-epi-p-santalol

CH2OH

(Z)-p- santalol

Figure 1.4. Major components of sandalwood oil

17

1.2.5. Factors affecting Sandalwood Oil Quantity and Quality

Within sandalwood species, a great deal of variation occurs in both the oil yield and

composition from stand to stand and tree to tree. As shown in Table 1.3, the reported

santalol content for S. spicatum varied widely. Although little research has been

performed to account for these differences, some generalisations can be made and are

discussed with below.

1.2.5.1. Environmental

Environmental factors such as geographic location, climate, temperature, rainfall,

altitude and soil type may lead to differences in oil quantity and composition.

Jayappa has shown that the oil extracted from East Indian sandalwood grown in

different geographical regions throughout India differs in physical characteristics 63.

Differences were found in percentage yields, refractive indices, optical rotation and

santalol content, yet no correlation was made between the environmental conditions

prevailing at each location and the differences in the oils.

It is thought that rainfall has a profound effect on the development of heartwood, which

may account for differences in oil yield. For individual trees of similar size, the trees

with a faster growth rate may well produce a lower proportion of heartwood 13. Since it

is known that trees in high rainfall areas have faster growth rates that those in arid

conditions 14'15,645 the heartwood content of a given tree would be lower in higher

rainfall areas.

18

Much of the research on the effect of soils has concentrated on the growth of

sandalwood seedlings with various nutrition supplements. Results have shown that

chelated iron increases the growth of sandalwood seedlings65, as do calcium

supplements . Although these results show added benefits at the nursery stage, the long

term effects of these nutrients on the amount and composition of the oil are not known.

1.2.5.2. Host Species

As with the research into the effect of soil on sandalwood, studies on host species have

been limited mainly to the nursery level. Consequently, limited information on the

influences of the host on heartwood oil formation is available. The results, however, do

show quite conclusively the need for a host in seedling development64,67'69. With an

adequate host, the biomass of the seedling can increase almost 9 fold. Since host quality

is considered to be one of the most important siviculture components influencing early

growth, heartwood yield may also be dependent on the host species.

1.2.5.3. Section of Tree

One of the major reasons for the variation observed in oil yields and composition is

most likely due to the differences in oil found within the different sections of the trees.

There is general agreement that the roots and buttwood contain more oil with a higher

santalol content than the stem and branches. It was also found that oil content decreased

by 45% along the length of the tree (from root to tip). Work carried out on East Indian

sandalwood showed that the roots contained 8.43 % oil, compared to 5.79% in the trunk

and 3.52% in the branches 63'70. Little difference was found in the santalol content along

the length of the tree, decreasing by only 3 %. The santalol content found in the roots,

19

trunk and branches were 91.87, 89.09 and 88.62% respectively. This slight decrease was

accompanied by a 3% increase in the sesquiterpene hydrocarbons, santalyl acetate and

santalenes. Although this increase in santalyl acetate and santalenes may seem small, it

corresponds to an overall 35 and 45% increase for each of the sesquiterpene

hydrocarbons.

Similar studies on & spicatum have shown completely different trends in the oil and

santalol content from different sections of the tree, as shown in Table 1.4. The oil

content was found to decrease moving up the length of the tree, from a maximum of

8.38% in the buttwood to a minimum of 1.78% in the high branches62. It is interesting

to note that the buttwood has a higher oil content than the roots (7.41%), in contrast to

the results seen in S. album.

The santalol content of the oil in S. spicatum was shown to be higher in the roots and

buttwood, and decline markedly moving further up the tree (Table 1.4) 41>62'71. Relative

amounts of both trans, fr-aws-farnesol and a-bisabolol can be seen to differ in the oil

extracted from various sections of the tree. The percentage of a- bisabolol ranges from

3.1% in the buttwood to 18.8% at 30 cm above the first branch. These results clearly

indicate that the section of the tree extracted will have a great influence on both the oil

content and composition.

20

Percentage

(%)

Yield

a- bisabolol

a- santalol trans,trans-farnesol

P- santalol nuciferol

Root

7.41

3.9 6.9 3.1

2.7 2.0

Butt

8.38

3.1 10.0

5.3

3.8 2.2

Mid-trunk

4.80

12.1

1.6 6.6

0.6 2.5

Top-trunk

4.58

15.9

1.1 7.9

-

1.9

Trunk

30a

4.51

18.8

0.6 8.8

-

1.5

Trunk

70b

4.08

18.0

0.6 7.5

-

1.4

High

Branch

1.78

17.2 -

8.0

-

-

Table 1.4. Variation in the composition of S. spicatum oil from various sections of the tree 4I

* Section of tree 30 c m above Top-trunk Section of tree 70 cm above Top-trunk

1.2.5.4. Cross Section

Not only has the oil content and composition been shown to vary along the length of the

tree, changes have also been shown to occur across the diameter of the tree. In a 51

album heartwood disc taken at approximately lm above ground level, there is an

average 20% decrease in the oil content from the core to periphery . There is also a 2%

decrease in the santalol level, while the content of santalyl acetate and santalenes

increase by 60 and 30% respectively.

A similar study on a wood disc from the buttwood of 5. spicatum showed no clear trend

in the amount of oil across the diameter of the disc 62. The oil was not analysed for

composition. The different results obtained in the two studies may be due to

interspecific differences, or the positions along the tree from which the disc was taken.

1.2.5.5. A g e of Tree

As expected, it was found that the oil content in young trees (0.2 - 2.0%) was markedly

lower than that of mature 5. album (2.8 - 5.6%)72. More surprising was the difference in

the oil composition between young and mature trees. The santalol content in young trees

ranged from 72 to 83%, which was considerably lower than the 86 to 91% range seen in

21

mature trees. The level of santalyl acetate/santalenes was found to be higher in young

trees, which was thought to arise from a conversion of santalyl acetate/santalenes to

santalols as the trees develop

1.2.5.6. Ageing of Sample

The age of a sandalwood oil sample may influence the composition of the oil. It has

been shown that the colour of freshly distilled oil was generally pale yellow and turned

golden yellow on standing for 4 to 6 months 72. Analysis of the oils before and after

ageing showed an increase in the santalol content and a decrease in a- and P- santalene,

and santalyl acetate.

1.2.5.7. Extraction

Different extraction procedures can afford different oils. This can be illustrated by the

extraction of 5. album and 5. spicatum by steam distillation and supercritical fluid

extraction (SFE) 41>73'74. For both species, higher oil yields were obtained by SFE

compared to steam distillation. The composition of both oils also differed depending on

extraction techniques. This was particularly evident in the 5. spicatum sample. The more

volatile components of the oil were present in higher proportions in the steam distilled

extract, whilst the less volatile components were much less abundant. Compositional

differences in the oils between the two extraction techniques was assumed to reflect the

harsh extraction conditions associated with steam distillation, namely prolonged times at

relatively high temperatures in the presence of traces of acids. Many of the trace

compounds identified may be enzymatic or oxidative degradation products of the main

22

sesquiterpenoid compounds, particularly the more volatile components. The effect of

different extraction methods is described in more detail in Section 1.5.

1.2.5.8. Adulteration

One of the more obvious reasons for differences in sandalwood oil compositions,

particularly in the East Indian variety, is due to adulteration of the oil. This is a process

in which an adulterant is added to the oil before or after extraction to increase the

amount of oil ' . The adulterant is usually polyethylene glycol, a high boiling

compound that is difficult to detect by G C 77,78. Cheaper essential oils are also used,

such as 'Morpankhi oil' which is produced from Thuja orientalis growing in north India

79

1.2.5.9. Other factors

There are a number of other factors that are capable of producing differences in the

composition of essential oils. Although these factors have not been investigated for

sandalwood oil, they have been examined for other essential oils.

Climatic conditions have been shown to have a marked affect on the composition of

Thymus vulgaris80. At low altitudes in warm, dry mediterranean environments, the

plants contained mainly phenolic compounds (thymol and carvacrol). Moving further

from the Mediterranean coast towards more humid conditions, the plants produced

fewer phenolic compounds. The main components of these plants were cyclic non-

phenolic terpenes (thujanol-4 and a- terpineol) and acyclic monoterpenes (linalool and

geraniol).

23

Closely linked to the effect of climate are those due to season. Seasonal changes were

observed in the essential oil in the leaves of Artemisia judaica. The oil content was low

during winter (1%) and peaked in late summer at 4%. The composition of the oil was

also shown to change. Although the combined amount of camphor and piperitone in the

oil was constant (64%), the proportion of the two compounds varied with the season. In

winter, the piperitone content was reduced whilst levels of camphor increased. The

reverse was the case in summer. It is important to understand how these oils change

with the seasons as it allows the optimal time for harvesting to be determined.

Although the environmental and processing factors discussed above are capable of

producing changes in the essential oil of a plant species, the genetic makeup of the plant

is the underlying factor governing the production of oil. It has been shown that genetic

variability occurs between plants of the same species. Variability in the composition of

an essential oil of the same species can be qualitative (involving the presence or absence

of a compound), or quantitative (components present at different amounts). Qualitative

characteristics are dependent on a single or few genes whereas quantitative

characteristics are determined by several genes 80. The oil content depends on the

number and quantity of precursors formed and the capacity of oil producing structures.

As environmental factors can have a substantial influence on the composition of oil,

examination of these genetic differences can only be performed in a controlled

environment. Seedling ofArtemisia judaica grown under the same conditions contained

essentially the same oil composition as the parent plants. Therefore, by genetic

selection, it may be possible to cultivate plants of high quality using the seed stock from

those plants of superior oil content and quality.

24

1.3. Supercritical Fluid Extraction

1.3.1. What is a Supercritical Fluid?

A supercritical fluid is defined as a substance that is above its critical temperature and

critical pressure. This definition can be explained through the use of a phase diagram

(Figure 1.5). A phase diagram for a pure substance shows the temperature and pressure

regions where the substance occurs as a single phase (solid, liquid, and gas) and the

phase transition lines where two of the phases can exist in equilibria. These two phase

regions between solid- gas, solid- liquid and liquid - gas involve the phase transitions of

sublimation, melting and vapourisation respectively. The three curves intersect at the

triple point, where the solid, liquid and gaseous phases coexist in equilibrium. The gas-

liquid equilibrium curve ends at the critical point. Increasing the temperature along this

curve increases the pressure at which both phases can coexist. This, in turn increases the

density of the gas phase and decreases the density of the liquid phase. At the critical

point the density of both phases are equal and the two phases are indistinguishable. At

this point, the substance is termed supercritical and consists of a single phase. If the

temperature and pressure are above the critical parameters, the substance will always be

single phase, and no further liquefication or vapourisation of the substance will occur.

25

P r e s Pc

s u r e

Tc

Temperature

Figure 1.5. Phase (pressure- temperature) diagram of a pure substance Pc= critical pressure, Tc= critical temperature, C P = critical point, T P = triple point

1.3.1.1. Properties of Supercritical Fluids

Since a supercritical fluid exists from the merging of a liquid and gas, supercritical

fluids exhibit physical properties intermediate between the two states (Table 1.5). The

relevance of these properties in relation to applications of supercritical fluids will be

discussed below.

Density (g/mL) Dynamic Diffusion Viscosity Coefficient

(g/cm-sec) (cm2/sec)

Gas 0.0006-0.002 0.0001-0.003 0.1-0.4 Supercritical Fluid 0.2-0.5 0.0001-0.0003 0.0007 Liquid 0.6-1.6 0.002-0.03 0.000002-0.00002

Table 1.5. Orders of magnitude of physical data of gas, supercritical fluid and liquid81

Viscosity/Diffusivity

Supercritical fluids exhibit significantly lower viscosities and higher diffusivities than

liquids. These properties are dependent on both pressure and temperature. The viscosity

and diffusivity of a supercritical fluid approach those of a liquid as the pressure is

increased. As pressure increases, viscosity will increase unlike a liquid, and diffusivity

26

will decrease. As the temperature is increased the viscosity will decrease unlike a gas,

and diffusivity will increase. The increase in viscosity with pressure and diffusivity with

temperature is more pronounced in the region close to the critical point.

The low viscosity and high diffusivity of supercritical fluids means that mass transfer is

higher than in liquids alone. This, combined with their very low surface tension,

means that they can more readily penetrate porous materials and are able to transport

dissolved solutes through materials very efficiently.

Density

The solvent power of a fluid is dependent on the number and strength of interactions

between the solute and solvent, and therefore is dependent on the density of the fluid. In

general, the greater the density, the greater the solvent strength of the fluid. Since

density is dependent on pressure and temperature, changes in any of these parameters

can have an effect upon the solvent power of a fluid. The relationship between density

of a supercritical fluid and pressure at various temperatures is illustrated in Figure 1.6.

The variables are all expressed as reduced variables, which is the ratio between their

actual values and critical values. Therefore, the critical point results from the triple

intersection where all the reduced values are equal to one. The change in density with

pressure at a constant temperature can be seen to be typically non-linear. In the vicinity

of the critical point small changes in pressure or temperature result in the largest

changes in density, whereas moving further away from the critical point the change in

density is less pronounced. The unique ability to control the density of a supercritical

fluid leads to the ability to control the solvent power with small changes in pressure and

temperature.

27

2.0

11 1.0

0 0.1 1.0 10.0

P*-P'PC

Figure 1.6. Variation of the reduced density of a pure component in the vicinity of its critical point82

P R = reduced density, P R = reduced pressure, T R = reduced temperature, CP= critical point

Although liquids generally have higher densities than supercritical fluids, the density of

a liquid is essentially independent of pressure. It is the ability of a supercritical fluid to

change it density, hence solvent power, combined with the low viscosity and high

diffusivity that give supercritical fluids their unique properties that can be used in a

variety of applications. These applications include supercritical fluid extraction,

supercritical fluid chromatography and supercritical fluids as reaction solvents.

1.3.2. Supercritical Fluid Extraction

Supercritical fluid extraction (SFE) is an extraction process that uses a supercritical

fluid as the extraction solvent. This extraction technique takes advantage of the high

diffusivity, low viscosivity and minimal surface tension which allows rapid penetration

into the desired matrix, often the rate limiting step in traditional extraction techniques.

The high mass transfer rates allow rapid transport of solute leading to decreased

extraction times.

28

The most advantageous property of supercritical fluid extraction is its adjustable solvent

strength through alteration of its density. If the density of the supercritical fluid is

increased, the fluid will be able to extract compounds of a higher polarity and size. This

allows selective extraction for a particular class of compound through changes in

pressure and temperature.

Since many supercritical fluids are gases at ambient conditions, the extracted analytes

can be separated from the solvent without the need for a concentration step; often a

requirement with liquid solvent extractions.

1.3.2.1. Applications of SFE

Although supercritical fluids have been known for some time, their use in extraction is

only recent . SF E gained acceptance in the 1980s mainly due to two reasons. Firstly,

the U S Environmental Protection Agency began to discourage the use of some organic

solvents commonly used in extraction because of the adverse effect of these solvents on

the environment84. SFE was seen as a 'clean' method of sample preparation and a

superior method of extraction. Secondly, the production of commercial analytical scale

S F E equipment made this technique generally available to research laboratories.

Although SFE can be used to extract liquid samples, by addition of an inert solid matrix,

its main use has been as an alternative for the extraction of solid matrices85. Areas of

applications include environmental monitoring (pesticides, PAHs, PCBs)86"88, natural

products (flavours, fragrances, pharmaceuticals) " , foods (fats, oils) ' , and polymer

i • 95-97

analysis

29

1.3.2.2. Choice of Supercritical Fluid Solvents

The fluids that have been used for SFE are listed in Table 1.6. These supercritical fluids

cover a large range in polarity. In theory, a solvent can be chosen based on the polarity

of the target compound/s. However, practical considerations, such as the temperature

and pressure required to obtain the critical parameters, often govern the selection the

solvent. Low critical parameters are favoured since the cost of equipment increases

when high pressures and temperatures are needed.

C o m p o u n d TC(°C) Pc (Bar)

Xenon 16.6 58.4 Trifluoromethane 26.2 48.6

Carbon Dioxide 31.0 73.8 Ethane 32.2 48.8 Nitrous Oxide 36.5 72.4 Sulfur Hexafluoride 45.6 37.6 Propane 96.7 42.5 Ammonia 132.4 112.8 Methanol 239.5 80.9 Water 374.2 221.2

Table 1.6. Critical conditions for pure components Tc= critical temperature, P c= critical pressure

The use of some fluids is restricted due to problems encountered at high pressures and

temperatures. Water and ammonia are both corrosive at supercritical conditions,

ammonia is toxic, ethane is highly flammable, and nitrous oxide has been reported to

explode under pressure ".

Carbon dioxide is the most commonly used supercritical fluid for extraction for the

following reasons:

i) It has a relatively low critical temperature and pressure;

ii) It is relatively inert and therefore has a low reactivity;

iii) It is classed as non-toxic, which allows it to be used in the food industry;

30

iv) O n a small scale, it is not classed as an environmental pollutant;

v) It is widely available at low cost and in high purity.

One of the major disadvantages of supercritical C02 as a solvent is its low solvent

strength. The solvating power of supercritical C 0 2 is generally equated to that ofhexane

. This limits the application of C 0 2 to the extraction of compounds of low polarity.

For the extraction of more polar compounds, rather than using a solvent of higher

polarity, this problem can be overcome through the use of a modifier. A modifier is a

polar organic solvent, such as methanol, that is added to the supercritical carbon dioxide

to increase the polarity of the solvent. The modified C 0 2 equates to having a solvent

strength equivalent to that of chloroform and more polar compounds can be extracted

100

1.3.2.3. Instrumentation

The instrumentation of SFE is very simple. Basic instrumentation must allow the

following; an extraction fluid to be pressurised and heated to supercritical conditions,

the supercritical fluid to contact the sample and dissolve the analytes of interest,

transport of the supercritical fluid with dissolved analyte from the sample, and

collection of the analytes by removal of the supercritical fluid. A typical schematic of a

basic SFE device is illustrated in Figure 1.7.

31

Pressure/Flow Controller

1 •=•—i Extraction vessel

Collection Vessel

Fluid Pre-Heat

Extraction Fluid

Figure 1.7. Schematic diagram of basic SFE apparatus

Method of Extraction

The extraction fluid is usually withdrawn from a cylinder containing a liquid-gas

mixture. The cylinder contains a dip tube, which allows the more dense liquid phase to

be withdrawn from the cylinder. The carbon dioxide is thereby supplied to the pump as

a liquid where it is pressurised to the desired value. If the extraction fluid is supplied to

the pump as a gas, the pump will have difficulty in sustaining the necessary flow.

Once the desired pressured has been reached, the fluid passes through a preheating zone.

This heats the fluid to above the critical temperature where it becomes supercritical. The

supercritical carbon dioxide then passes through the extraction cell containing the

sample. Those analytes within the sample matrix which are soluble in supercritical

carbon dioxide are dissolved and flow out of the cell with the extraction fluid. The

conditions required to remove the analytes from the matrix will be discussed in detail in

Section 1.4.1. At this point, the temperature no longer needs to be controlled. This is

because a decrease in temperate will bring about an increase in density and the analytes

will remain soluble in the liquid carbon dioxide.

Fluid Pump

/*N

32

The fluid and dissolved analytes are passed through a restrictor where the pressure is

decreased. Restrictors are usually fixed diameter tubes or electronically controlled

variable orifices which provides the back pressure in SFE. With a fixed restrictor, flow

cannot be controlled independently of the pressure. Using a variable restrictor, flow

rates can be controlled using pressure and size of the variable orifice. A s the fluid passes

out of the restrictor the pressure is lowered to atmospheric conditions over a short

distance. Associated with this rapid expansion is Joule-Thomson cooling which can

cause plugging of the restrictor, especially by ice. To help overcome this problem, the

restrictor zone is heated. Blockages tend to occur more frequently in fixed restrictors

since variable restrictors can increase the orifice diameter if pressure build up is

detected.

As the fluid depressurises, the dissolved analytes are no longer soluble in the extraction

fluid. Gaseous carbon dioxide is released leaving behind precipitated analytes. The

analytes are collected on cryogenically controlled solid bed traps or in a liquid solvent in

which the analytes are soluble. A more detailed description of the collection methods

will be described in Section 1.4.2.

1.4. Optimisation of SFE

For successful SFE, two processes must occur efficiently. Firstly, the analytes of interest

must be completely removed from the matrix, and secondly, there must be quantitative

recovery of these analytes in the collection vessel. Therefore, optimisation of S F E can

be thought of as two separate processes, extraction and collection. Each of these

processes will be discussed in detail below.

33

1.4.1. Extraction Conditions

The ability to extract target analyte/s from a matrix is determined by101:

• Analyte solubility in the supercritical extraction fluid

• Analyte-matrix interaction

• Position of the analyte within the matrix

• Porosity of the matrix

As can be seen from the above determinants, the matrix can have a major effect upon

the extractability. Even though an analyte may be soluble in a supercritical fluid it may

be incompletely extracted. The effect of the matrix is clearly evident with polyaromatic

hydrocarbons (PAHs) spiked onto various matrices. Complete recovery was achieved

from silica wool, polyurethane foam, silica gel and ODS. Alumina and diatomaceous

earth gave low recoveries of some compounds, whereas activated charcoal exhibited

strong matrix-analyte interactions which prevented recovery of any of the PAHs

There are a number of parameters in the extraction procedure that can be optimised to

achieve complete extraction. These parameters are density (pressure), temperature,

mode of extraction (static or dynamic), flow rate, extraction time and particle size.

1.4.1.1. Density

The effect of density on solvating power has been discussed in detail in Section 1.3.1.

Density governs the solubility of an analyte in a supercritical fluid. Numerous reports

have examined the solubility of a wide range of compounds in supercritical carbon

dioxide 103,104. This has allowed correlations to be made between the size and

functionality of a compound to its solubility in supercritical C02. If the sample contains

34

low molecular weight, non-polar compounds, low densities will be sufficient to

solulibise the compounds. For higher molecular weight, non polar compounds, high

densities will be required. For high molecular weight polar compounds, high densities

with polar modifiers are required.

Since at higher densities the solubility of a compound increases, density can be utilised

to decrease extraction times. This is because less volume of solvent is required to

dissolve the same amount of solute as the density is increased. Increasing the density,

combined with dynamic extraction (Section 1.4.1.3), can be extremely useful in

decreasing extraction times if selectivity in the extraction is not required.

The density parameter has very little effect on the removal of the analytes from the

matrix. It is the physical characteristics of viscosity and diffusivity that are utilised to

interact with matrix. It has been proposed, however, that for intracellular material, the

increased pressure resulting from increasing the density can break open cellular

structures to free some analytes.

1.4.1.2. Temperature

Temperature is an important but complex parameter for controlling extraction. As the

temperature increases under isobaric conditions the density decreases. Lower

temperatures are therefore used to obtain maximum densities for maximum solvating

power. Temperature can also be used to aid in the extraction of the analytes from the

matrix. Increasing the temperature can increase diffusion and disrupt matrix associations

thereby aiding the extraction process.

35

Increasing temperature will increase diffusivity of the supercritical fluid. For samples

whose matrices are difficult to penetrate, or for analyte- matrix association that is large,

higher temperatures can be used to increase mass transfer. For many environmental

samples, it is not the solubility of the analytes that is the rate-limiting step, but it is

desorption from the matrix that determines the rate of extraction. Extracting these

samples at higher temperatures will increase diffusivity and mass transfer to improve the

desorption kinetics 105.

An example of temperature disrupting matrix association is evident in the extraction of

additives from polymers 106,107. As a matrix, polymers have very little porosity at room

temperature. If the extraction temperature is increased high enough to soften the

polymer, the matrix is disrupted. The supercritical fluid is now able to interact with the

polymer additives within the polymer matrix, and extraction efficiency is also increased

due to an increase in both diffusivity and mass transfer as discussed above.

In some cases, high temperatures can cause problems. Analytes that are thermally

unstable at high temperatures can decompose thus altering the composition of the

analytes. This is particularly important in the extraction of natural products and will be

dealt with in detail in Section 1.5.

1.4.1.3. Mode of Extraction

SFE can be performed in two modes; static or dynamic extraction. The method of

• 10R

extraction depends on the sample size to extraction cell volume ratio . In static

extraction, a fixed amount of supercritical fluid interacts with the analyte matrix. The

extraction vessel is pressurised to supercritical conditions and the sample is soaked in

36

the solvent for a given period of time. This mode of extraction utilises the supercritical

fluid properties of high diffusivity and mass transfer to access the analytes within the

matrix. Following extraction, the restrictor is opened and as the fluid depressurises, the

analytes are swept out of the extraction cell to a collection device.

In dynamic extraction, fresh solvent is continuously passed through the extraction cell

containing the sample. The restrictor is constantly open, and combined with the

pressure, delivers the extraction fluid at a desired flow rate. The collection of analytes is

continuous throughout the entire extraction time.

The static mode of extraction is limited in its solubility capability. As time increases, an

equilibrium is established between the analytes in the supercritical fluid and analytes in

the matrix. If solubility is not favourable in the supercritical fluid, exhaustive extraction

may not occur. Static extraction is often followed by a short period of dynamic

extraction to help overcome this problem.

During dynamic extraction, the dissolved analytes are constantly removed from the

matrix so analyte-matrix interactions are limited. This is generally the preferred mode of

extraction and is used for 90% of all reported applications of SFE 109.

One drawback of dynamic extraction is that it uses far more extraction fluid than static

extraction. During long periods of dynamic extraction, problems maybe encountered if

the supercritical fluid is impure. The contaminants will ultimately concentrate in the

collection device and interfere in the analysis of the extracts. This poses a particular

problem in the trace analysis of environmental samples.

37

1.4.1.4. Flow Rate

As discussed above, flow rate is only applicable in the dynamic mode of extraction.

Flow rates are limited by the capability of the pump but generally fall in the range of 0.5

mL/min to 4 mL/min for analytical applications. Faster flow rates allow for more

extraction fluid to pass through the cell per unit time, and typically, results in shorter

extraction times. Higher flow rates however, make trapping more difficult for the more

volatile compounds. This is because a greater flow rate will produce a greater expansion

once depressurised, which propels the analytes from the restrictor at greater velocities.

The lighter the compound, the more difficult it is to precipitate in the collection device

since it is able to escape with the depressurised high velocity extraction fluid. This will

be discussed in Section 1.4.3.

1.4.1.5. Extraction Time

The extraction time is dependent on sample size, density and flow rate. The larger the

sample size, and the lower the density and flow rate, the longer the extraction time will

be. When optimising SFE, the time taken to completely extract the components of

sample must be determined. This enables extraction and collection parameters to be

examined.

1.4.1.6. Particle Size

Increased permeation of the extraction fluid into the matrix increases the rate of

extraction. Therefore, for a solid sample, the extraction rate increases with a higher

surface area and smaller particle size. The greater surface area allows the extraction

fluid to permeate the matrix more efficiently and the smaller particle size leads to

38

shorter internal diffusion pathlengths over which the extraction fluid must travel to

reach the solutes.

For solid samples, where analyte extraction is restricted by diffusion through the matrix,

grinding can be used to obtain smaller particle sizes. Grinding is usually performed in a

blender followed by mechanical sieving to achieve the desired particle-size or particle

size range. Due to its importance in SFE, numerous studies have reported the effect of

particle size on extraction110"113. Studies on rosemary have shown that reducing the

particle size accelerated extraction, improved extraction efficiency and shortened

extraction time 114. Extraction was complete within 20 mins with particle sizes below

500 um, whereas for unground samples extraction was incomplete after 60 min.

The heat generated by reducing the particle size can lead to loss of the more volatile

components of the sample 114"116. To overcome this problem, cryogenic grinding can be

employed. This involves grinding in the presence of dry ice or liquid nitrogen.

Cryogenic grinding can also help in sample preparation of natural products. When plant

material is ground, cellular membranes are disrupted and this may release certain

enzymes which can decompose the components of interest114. Cryogenic grinding

inactivates these enzymes and prevents enzymatic degradation.

Problems can be encountered if the particle size is too small. Particle sizes of below 50

um should be avoided because they may become compacted in the extraction vessel

which can lead to channelling of the extraction fluid 117. This is where the supercritical

fluid passes through the small channels in the matrix leading to inefficient contact

between the supercritical fluid and parts of the sample matrix.

39

1.4.2. Collection Conditions

The collection of extracted analytes in SFE must involve quantitative transfer of the

extracted analytes from the extraction vessel to a collection device, and quantitative

collection of the analytes in a collection device. In developing an SFE method,

collection conditions must be addressed before extraction conditions, otherwise poor

recoveries may be falsely attributed to extraction conditions.

As previously discussed, the effect of the matrix can have a significant effect on the

extractability of certain compounds. To optimise the collection conditions it is important

to eliminate the effect of the sample matrix. This is usually achieved by spiking a small

amount of a standard mixture into the extraction vessel on an inert matrix. Recoveries of

compounds in the standard mixture are measured to determine if 100% of the extracted

components have been collected, indicating quantitative collection. Once this has been

determined, real samples can be extracted and the effectiveness of the extraction

conditions and matrix effects evaluated.

The collection device is situated directly after the restrictor (Section 1.3.2.3). Collection

of the extracted analytes is achieved after depressurisation of the extraction fluid to

ambient conditions. A liquid C02 flow rate of 1 mL/min equates to a gaseous C02 flow

rate of approximately 500 mL/min. Trapping of the analytes from a such a high flow

rate can prove extremely difficult, particularly for volatile analytes. There are three

common collection devices used for trapping in SFE; liquid solvent traps, inert solid bed

traps, and active solid bed traps.

40

Liquid Solvent Trapping

Liquid solvent trapping 118"121 is depicted in Figure 1.8. A narrow bore tube from the

restrictor is immersed into a liquid solvent (5-20mL) within a small container. The

depressurising extraction fluid bubbles through the solvent and vents to the atmosphere

leaving behind the analyte dissolved in the liquid solvent. The liquid solvent is often

cooled to aid in the collection of the volatile analytes. The most important parameter in

this type of trapping is the choice of solvent. It must solubilise the target analytes and

should be compatible with the analysis technique.

Heated Restrictor

Pressure Release

Transfer Tube •

Collection Solvent o

o Oo

,o &

Figure 1.8. Liquid solvent SFE trapping device

Solid Bed Traps

In solid bed trapping 119'122-1245 the depressurised extraction fluid flows through a packed

trap containing a solid support onto which the analytes adsorb (Figure 1.9). The solid

support within the trap baffles the expanding flow to aid in the precipitation of solute

molecules while allowing the expanding solvent gas to escape. The trap is often

cryogenically cooled to prevent the escape of volatile components from the trap. The

solid support consists of particles ranging from 30-100 urn in diameter. There are two

41

types of solid supports used in solid bed trapping; inert and active. An inert support,

such as silanised glass or stainless steel beads, exhibits no functionality. Active supports

comprise a chromatographic stationary phase where adsorption of the analytes on the

solid support is enhanced through interactions with the stationary phase. Commonly

used active supports include silica based supports such as octadecylsilica (ODS), cyano,

diol and amino.

Heated Restrictor ^_

Solid Bed Trap^^^ L_

Frit

To waste *-

(a) (b)

Figure 1.9. (a) Solid bed trapping device (b) cross section of solid bed trap showing the solid bed packing

Once the analytes have been collected on the solid support in the trap, they are eluted

from the adsorbent with a small volume of solvent (l-3mL). A solvent that exhibits a

high solvating power for the analytes is chosen. Alternatively the trap can be heated

during the desorption process to increase the amount of solute dissolved.

For successful collection of the analytes, both the adsorption and desorption of the

collection processes should be quantitative. There are several important parameters to

consider when optimising these processes.

Solid Bed Packing

42

1.4.3. Adsorption

Trapping material functionality, trap temperature, and extraction fluid flow rate can all

influence the adsorption process. The effect of each of these parameters is discussed

below.

1.4.3.1. Trapping Material

The trapping of compounds on solid bed traps can occur through two mechanisms;

physical trapping (often called cryotrapping) and/or chemical trapping. Trapping on

inert solid bed traps can only occur through cryotrapping since the trapping material

possesses no functionality. Active solid bed traps have the advantage that one or both

mechanisms can be used in the trapping process. The differences between inert and

active sorbent trapping mechanisms are evident in P A H extraction. The recoveries of

various P A H s were determined by collecting spiked P A H s onto cryogenically cooled

stamless steel and O D S . All P A H s were trapped quantitatively on the O D S trap,

however on the stainless steel trap the more volatile bi- and tricyclic P A H s were lost.

The retentive nature of the O D S was sufficient to prevent the compounds passing

through the trap with the gaseous extraction fluid.

The effect of the polarity of active solid supports has been studied in detail

118,119,122,124,126,127 p Q r increase(j interactions between the support and compounds, the

polarity of the trapping material must match that of the target compounds. This is

particularly important if the compounds cannot be trapped cryogenically. For example,

for a mixture of hydrocarbons (CIO to C32), the recoveries on the polar traps (cyano,

silica, diol, and amino) were lower than those on non-polar traps (C8 and O D S ) . The

effect of the trapping material is clearly evident in the trapping of the lower molecular

43

weight hydrocarbons. The polar traps yielded recoveries of < 8 0 % for CIO and CI2

hydrocarbons, whereas 1 0 0 % recovery was achieved with non-polar traps.

1.4.3.2. Trap Temperature

Cryogenic trapping is extremely important in solid support trapping, in particular when

using an inert solid support. Obviously, the more volatile the compound, the lower the

trap temperature required. At a trap temperature of-25°C, hydrocarbons below CI 1 are

not trapped on glass beads. If the temperature is lowered to -65°C, 9 5 % of CI 1

hydrocarbons can be trapped 128. L o w temperatures and the appropriate choice of active

solid support can result in increased recovery.

1.4.3.3. Extraction Fluid Flow Rate

In dynamic SFE, the extraction fluid flow rate dictates the flow of expanding gas from

the restrictor. If the velocity of the expanding gas is too great, the analytes have less

time to interact with the solid support, resulting in low recoveries. Again, this is mainly

a problem with more volatile compounds. Extraction fluid flow rates can generally be

higher with active solid supports (1-4 mL/min) than with inert solid supports due to the

multiple trapping mechanisms 129.

1.4.4. Desorption

During the desorption step, the analytes collected on the trap are eluted from the trap by

a rinse solvent. Parameters such as choice of rinse solvent, solvent flow rate, and trap

temperature can influence recovery. The effect of each of these parameters is discussed

below.

44

1.4.4.1. Rinse Solvent

The choice of rinse solvent in the desorption process depends on several considerations

130.

• The solubility of the analyte(s) in the rinse solvent;

• Compatibility of the rinse solvent for subsequent analysis or processing;

• Solvent power required to desorb the analytes from the trap packing material.

During inert solid support trapping only the first two points need be considered and

quantitative recovery is dependent on choosing an appropriate solvent. The third point

must be carefully considered when using an active solid support, since the sorbent has

the ability to retain the analytes from the trap. Selection of a rinse solvent is guided by

the same considerations involved in the selection of an appropriate elution solvent in

liquid chromatography (LC). Non-polar solvents can be used to elute analytes from a

non-polar support. As the polarity of the support increases, the polarity of the rinse

solvent must also increase since the trapped compounds will have a greater affinity for

more polar solvents. By using two rinse solvents of different polarity, fractions

containing different classes of compounds may be obtained. This aspect of selective

1 90

elution has not been extensively studied

1.4.4.2. Solvent Flow Rate

The solvent flow rate generally has an influence on the volume of solvent required to

rinse the analytes from the trap. As mentioned above, the trap acts as a LC column

during desorption. The flow rate will affect the zone broadening of the analytes as they

elute from the trap. The lower the flow rate, the lower the zone broadening, hence

minimising the volume of solvent used. Lower flow rates however increase the time

45

required to elute the analytes from the trap. Selecting the appropriate solvent flow rate

requires a balance between the volume of solvent used and time.

1.4.4.3. Trap Temperature

Depending on the application, the temperature of the rinse solvent may be varied during

the desorption process. For difficult to dissolve analytes, higher temperatures may be

required for increased solubility. Conversely, for thermally labile compounds lower trap

temperatures may be desirable.

During desorption, the temperature of the restrictor and trap should be 5-10°C lower

than the boiling point of the rinse solvent, otherwise vaporisation of the solvent occurs.

Furthermore, the temperature must also be above the melting point of the rinse solvent

to prevent freezing.

Liquid Trapping vs Solid Support Trapping

Liquid and solid support traps vary considerably in their trapping mechanisms and each

possesses various advantages and disadvantages. Liquid traps do not have the ability to

baffle the expanding extraction fluid from the restrictor. Thus, high flow rates cannot be

used as excessive bubbling will occur and the more volatile analytes will be lost. Studies

have shown that higher recoveries are obtained using solid bed traps over liquid traps

124,127

Another disadvantage of liquid trapping involves the Joule-Thomson cooling associated

with the depressurising of the extraction fluid that leads to low temperatures in the

46

collection vessel. Because the restrictor is often immersed in the solvent, freezing can

occur and small pieces of ice may clog the restrictor if it is not heated.

One major advantage of solid bed trapping is its ease of automation. Samples can

continually be extracted without the need to replace the trap, providing all the analytes

had been completely removed from the trap by the previous desorption process. An

aggressive solvent is quite commonly used after collection of the sample is complete to

ensure no analytes remain on the trap.

1.5. Extraction of Essential Oils

Since many fragrance and flavour compounds are sensitive to acids, the composition

and quality of the oil is dependant on the extraction procedure. The common methods

for the extraction of essential oils are considered below.

1.5.1. Steam and Hydrodistillation of Essential Oils

Essential oils have traditionally been extracted by steam distillation. This method

consists of passing steam through the plant material held in a suitable container

equipped with a condenser. The heat of the steam causes the volatile oils to expand,

thereby bursting open the cells containing the oil. The volatile oil and the water vapour

pass into the condenser where they return to the liquid state. The oil and water have

different densities and form two distinct layers that can be separated. In general, the

essential oil fraction is less dense than water unless it contains a significant proportion

of aromatic compounds.

47

Hydrodistillation is a similar technique in which the plant material is immersed in a

suitable quantity of water and the mixture boiled.

In both of these extraction techniques, high temperatures (~100°C) are involved. These

temperatures can often result in modifications of the original constituents in the oil by

heat-induced reactions and loss of volatile compounds can occur. The odour of the

steam distilled extract can therefore vary considerably from that of the fresh plant

material. Studies have been performed comparing the composition of the headspace

over fresh herbs to that of the essential oil recovered by steam distillation 131. It was

found that the headspace sample contained numerous compounds which make a

significant contribution to the aroma of the herb that were absent from the steam

distilled oil.

A number of studies have compared the composition of essential oils recovered by

steam, hydrodistillation and SFE 90>91>132-135. The extraction temperature used in SFE is

considerably lower since the critical temperature for CO2 is 31.3 °C. Modification of

the oils during extraction is therefore much less likely, and the odour of the SFE extracts

is thought to more closely resemble that of the natural material. Moyler136 extracted the

oil from clove buds using steam distillation and liquid C02 extraction. It was shown that

prolonged distillation caused hydrolysis of eugenyl acetate to eugenol and acetic acid.

The odour of the liquid C02 extract was sweeter, less medicinal and slightly floral and

resembled more closely the aroma of the natural clove bud than the steam distilled

extract. The liquid C02 extract had a much higher level of eugenyl acetate which was

responsible for the more floral odour. The liquid C02 extract was steam distilled and the

oil collected had a lower content of eugenyl acetate than the starting material. Traces of

48

acetic acid could be detected in the water distillate serving as evidence that degradation

was occurring during steam distillation.

Similar studies on rosemary have shown that the hydrodistilled extract contained a

higher proportion of monoterpene hydrocarbons which contribute minimally to the

aroma of the oil137. In contrast, the SFE extract contained a higher percentage of

oxygenated monoterpenes that strongly contribute to the fragrance of the oil.

Organoleptic tests were performed by a standard testing panel which judged the SFE oil

to have a strong fragrance of rosemary leaves while the hydrodistilled oil possessed a

less intense aroma considered to be slightly different from the starting material.

The composition of oils extracted by steam/hydrodistillation and SFE has also been

shown to differ for reasons other than thermal degradation. Since steam and

hydrodistillation require volatilisation of the components, collection of these

compounds require condensation. If the compound is too volatile to be condensed it may

escape through the condenser and avoid collection. This results in the loss of the top

notes of the fragrance. O n the other hand, if the compound has a low vapour pressure,

volatilisation m a y not take place, and the compound will not be found in the extract.

This typically can be detected by a loss of back notes in the fragrance.

Moyler 138 has shown that a liquid C02 extract of ginger contained the volatile

component hexan-1-al in the oil, which is completely absent from the steam distilled oil

due to its volatility. It was found that an extra 3 % of these top notes where present in

the C 0 2 extract. This extract was also found to contain more of the higher molecular

weight components (eg gingerols) which have lower vapour pressures. Since these

49

compounds are less volatile, they are only marginally responsible for the odour of the

oil, however they do posses fixative properties.

Apart from temperature effects in steam- and hydrodistillation, the pH of the liquid

phase can cause acid catalysed reactions. Most plant material in the presence of water

develops a pH between 4 and 7, and for fruits the pH may be lower139, Evidence for

these acid catalysed reactions was obtained in a study involving steam distillation of

Juniperus sabina at various pH values (2.2 to 8) 140. As the pH of the distillation water

was lowered, the concentration of sabinene decreased with concomitant increases in the

levels of its decomposition products (Figure 1.10).

sabinene

H7H20

1

^y

cc-terpinene y-terpinene terpinolene terpinen-4-ol

Figure 1.10. Conversion of sabinene in acidic conditions 140

The advantages of steam/hydrodistillation are mainly apparent on a commercial scale

because of the simplicity and low cost of design compared to SFE.

50

1.5.2. Solvent Extraction of Essential Oils

Solvent extraction employs the use of an organic solvent, or a mixture of organic

solvents, to extract the oil from plant material. The solvent used depends on the

solubility of the oils in the solvent. The plant material can be soaked in the organic

solvent at ambient temperatures, or extracted by boiling solvent in a Soxhlet-type

apparatus. Solvent extraction assisted by ultrasound, also known as sonication, is

another variation on this technique.

To recover the oils, the solvent must be removed by evaporation, which is achieved by

heating, usually under high vacuum, co distillation, or nitrogen sparging. If heat and

high vacuums are used to remove the solvent, components of the oil with a boiling point

similar to the solvent may also be removed, resulting in the loss of the more volatile

compounds.

Solvent extraction involves lower temperatures than those used in steam distillation.

Removal of the solvent can be achieved at <70 °C and modification of the composition

through heat and acid catalysed reactions is minimised.

One of the shortcomings of solvent extraction is that the organic solvents have low

selectivity. Thus, apart from the desired volatile compounds, high molecular weight,

non-volatile compounds, such as fatty oils, resins, waxes and colouring matters, are co-

extracted. This m a y lead to visually unattractive oils due to their viscous nature and dark

colouring even though oil yields are higher than those obtained by other extraction

techniques. Also, the extract may require a further clean up before analysis due to these

unwanted extracted compounds.

51

Hexane extraction of grape seed oil resulted in a yield (7.5%) comparable to that

obtained by SFE (6.9%) U1. The greater yield obtained from the solvent extraction

method was shown to be due to the presence of a higher free fatty acid concentration

and an unsaponifiable fraction due to the non-selective nature of the solvent. The

reduced high molecular weight compounds in the SFE extract simplifies the analytical

process and prolongs the GC capillary column lifetime, without affecting the aroma of

the oil.

1.5.3. Other Extraction Techniques

Less traditional methods of extraction of essential oils include:

a) Accelerated solvent extraction (ASE) which is a form of high pressure solvent

extraction used to shorten the extraction time.

b) Cold pressing in which the oils are crushed from the plant material.

c) Microwave assisted processes (MAP) in which liquid- phase or gas-phase extractions

can be performed.

1.5.4. SFE for the Extraction of Essential Oils

One of the major advantages of SFE (C02) in the recovery of essential oil relates to the

selectivity of the extraction process. Low molecular weight oxygenated compounds that

are responsible for the odour of the oil are readily soluble in the extraction fluid,

whereas the high molecular weight polar compounds are practically insoluble. It is this

selectivity that makes supercritical C02 an attractive solvent for the recovery of essential

oils. The oils obtained contain more top notes resulting from the low molecular weight

compounds that are usually lost in traditional extraction techniques. The oils also

contain a small amount of high molecular weight compounds which, although not

52

making any contribution to the fragrance, are able to act as natural fixatives and help

stabilise the oil. A list of the solubilities in C02 of classes of compounds pertaining to

natural products is shown in Table 1.7.

Very Soluble

Non-polar and slightly polar organic compounds of low molecular weight < 250

Examples include mono and

sesquiterpenes

Thiols, pyrazines and thiazoles Acetic acid, benzaldehyde, hexanol, glycerol, acetates

Sparingly Soluble Almost Insoluble

Organic compounds with higher molecular weights (up to ~ 400)

Examples include substituted

terpenes and sesquiterpenes, water, oleic acid, decanol and saturated lipids up to C12

Sugars, protein

Tannins, waxes, inorganic salts Chlorophyll, carotenoids, citric and malic acids Glycine, nitrates and many components in pesticides and insecticides

Table 1.7. Relative solubility in C 0 2 of classes of compounds typically found in plant material 142

The high diffusivity of supercritical fluids is utilised in the extraction of essential oils.

Most plant material contains the oil within cellular structures. Because of the high

diffusivity, supercritical C02 as a solvent is able to penetrate the plant material and

dissolve the essential oil more readily than in traditional methods of extraction, leading

to much shorter extraction times.

The low temperatures used with SFE, discussed previously in Section 1.4.1.2, produce

an oil with an odour that more closely resembles that of the natural plant material.

SFE does not pose the many environmental and safety concerns that surround other

extraction techniques, relating to both the consumers and producers. Since the extraction

fluid is a gas, the final product contains no solvent residue. Although C02 is a major

contributor to the greenhouse effect, any increase in the amount of C02 arising from

industrial operations would be negligible 143. C02 also has much less of an effect

53

on the environment than many traditional solvents used in large quantities in solvent

extraction.

Analytical scale SFE has the potential to be completely automated. Many of the

commercial instruments are capable of extracting numerous samples per batch,

minimising time and labour. SFE can also be directly coupled to a chromatographic

system for analysis, termed on-line S F E 126>144>145. This has the advantage of eliminating

sample handling and the possibility of sample loss and contamination, and decreases the

overall analysis time.

Because of the high pressure associated with SFE, the initial cost of equipment is high.

However, it has been found that the energy associated with the normal heat requirements

for distillation and evaporation of organic solvents is greater than that associated with

the running cost of SFE 146. This decreased cost, coupled with a fully automated system,

could produce considerable savings compared to other traditional extraction techniques.

1.5.4.1. Applications of C 0 2 S F E to Natural Products

SFE has been traditionally used for the preparative-scale isolation of compounds from

plant matrices, eg. the decaffeination of coffee, and extraction of hops and tobacco

150

In more recent times the trend has been toward analytical-scale SFE and its use as a

sample preparation method prior to analysis. Numerous essential oils have been

extracted using SFE 90'104>135>138>151-155. The extracts obtained require little clean up, so

54

analysis can be performed directly after extraction. The fast sample turnaround makes

SFE an ideal technique for the screening of components in many natural products when

combined with the appropriate analytical technique.

1.6. Analysis of Essential Oils

The analysis of essential oils primarily involves the separation and identification of their

components. This requires the use of chromatographic and spectroscopic techniques.

The advent of new analytical methods combined with the development of computers has

dramatically improved the methods of analysis over the past 50 years. These methods

are discussed below.

1.6.1. Gas Chromatography

The most important development in the field of essential oils analysis came in 1952

with the introduction of gas chromatography (GC). The transition from packed column

GC to capillary GC in 1957 increased the popularity of the technique. In more recent

times, companies have manufactured specific columns for essential oil applications.

These columns contain a polar stationary phase (polyethylene glycol) which is able to

interact with the oxygenated compounds in the oil.

GC is ideally suited for the analysis of essential oils due to the volatility of their

components. Compounds are separated according to their boiling point and affinity for

the stationary phase. The separated compounds are usually detected by a flame

ionisation detector (FID). Although FID provides high sensitivity, it does not provide

structural information. If structural identification of the separated components is

55

required GC can be coupled to mass spectrometry (GC-MS) 156,157 or Fourier transform

infrared spectroscopy (GC-FT-IR)158'159.

Other techniques such as headspace analysis and solid phase micro-extraction (SPME)

can be used to analyse the aroma of essential oils. These techniques isolate the

volatile components, which can then be analysed by GC or GC-MS with no sample

preparation required.

1.6.2. Liquid Chromatography

Liquid chromatography (LC) is used as a method of analysis when the essential oil

contains compounds that are not easily volatilised. LC is often used to analyse essential

oils extracted by solvent extraction since this extraction technique removes the higher

molecular weight components of the oil such as oleoresins. LC can be coupled to a

number of spectroscopic techniques such as ultraviolet (UV), FT-IR, MS and NMR for

detection and structural identification 161,1 2.

1.6.3. Supercritical Fluid Chromatography

Supercritical fluid chromatography (SFC) is a hybrid analytical technique between GC

and LC, in which the mobile phase is a supercritical fluid. Therefore SFC offers

chromatographic parameters between both techniques such as liquid solvating power but

with much higher resolving power than HPLC. However SFC is often not favoured as a

routine method of analysis as method development often proves more difficult. SFC

instrumentation is similar to that of LC, and separation can be performed on both

capillary and packed columns. The advantage of SFC is that it can be used to analyse a

wide range of compounds due to its hybrid chromatographic parameters. Currently the

56

major application of SFC is the separation of chiral compounds. Detection and structural

identification in SFC can be achieved with both liquid and gas phase detectors 163~165.

57

1 -7. Aims of Project

The aim of the project was to conduct a field study on Western Australian sandalwood

examining variations in the quantity and quality of the oil from trees at different

geographic locations, at different seasons and from various sections of the tree. The

results provide valuable information for the management and utilisation of S. spicatum.

To this end, the development of a fast, automated SFE method for extracting the large

number of samples was required. Optimisation of the method would involve the

examination of the effect of various extraction conditions (density and particle size), and

collection conditions for solid bed trapping (trapping material, flow rate, trap

temperature, rinse solvent, rinse temperature and rinse volume).

The effectiveness of SFE as an extraction technique was also investigated by

comparison with the traditional extraction methods of hydrodistillation and solvent

extraction.

58

2. Experimental

2.1. Equipment

Supercritical Fluid Extraction

Extractions were performed using a Hewlett- Packard 7680T supercritical fluid

extraction module. The SFE module was controlled through the SFE HP Chemstation

software (REV. A.04.04). The extraction fluid used was N45 Grade C02 (Air Liquide)

and Food Grade C02 (Air Liquide) was used for cryogenic cooling.

Gas Chromatography

Analysis was performed using a Hewlett- Packard 5890 Series II Gas Chromatograph

equipped with a flame ionisation detector (FID). The GC consisted of a HP 7673

autosampler, split/splitless inlet, and electronic pressure control (EPC) of the carrier and

detector gases. The carrier gas used was UHP He (Air Liquide) and the detector gases

were HP H2, HP N2 and Medical Air (Air Liquide).

Gas Chromatography/Mass Spectrometry

Analysis was performed using a Hewlett-Packard 5890 Series II Gas Chromatograph

equipped with a Hewlett-Packard 5972 Mass Selective Detector. The GC consisted of a

HP 7673 autosampler, split/splitless inlet, and electronic pressure control (EPC) of the

carrier gas (UHP He, Air Liquide). Identification of unknown compounds were

determined by library searches using the NIST/EPA/NIH Mass Spectral Library (NIST

•98).

59

Solvents

Reagent grade ethanol was dried by refluxing the ethanol over magnesium for a

minimum of 24 hours and then distilled. All other solvents were HPLC grade (Table

2.1).

Rinse Solvent Supplier Hexane Iso-octane Methyl-tert-butyl Ether (MTBE) Ethyl Acetate

Mallinckrodt Mallinckrodt

Fluka Mallinckrodt

Table 2.1. H P L C grade solvents used as rinse solvents for SFE

2.2. Optimisation of Trapping Conditions

2.2.1. Materials

Standard Mixture

The percentage recovery was determined by using a standard mixture (Section 2.2.3.2).

The components of the mixture are listed in Table 2.2 and their structures shown in

Figure 2.1.

Compound Supplier

Limonene Cineole Citronellal Nerol (3- Ionone trans, trans- Farnesol a- and 0- Santalol Manool

Sigma-Aldrich Fluka Fluka

Sigma-Aldrich BDH

Sigma-Aldrich Dragoco B D H

Table 2.2. Compounds used in the standard mixture

60

P- ionone

CH2OH

"OH

Citronellal Nerol

a- santalol

trans, trans- farnesol

CH2OH

P- santalol Manool

Figure 2.1. Structure of compounds used in the standard mixture

Internal Standard

AR grade octan-1-ol (Sigma-Aldrich) was used as an internal standard (IS). The IS was

diluted 1:20 with ethanol for use.

2.2.2. Time Course Extraction of Standard Mixture

2.2.2.1. Preparation of Extraction Thimbles

A 80 u L aliquot of the standard mixture (Section 2.2.1) was pipetted into a 7 m L

extraction thimble containing 1 cm of filterflocs (Machery Nagel). A further 1 cm of

filterflocs was placed into the thimble and sealed with caps at both ends.

61

2.2.2.2. Exhaustive Extraction

The prepared thimbles were extracted using the conditions set out in Table 2.3. The time

taken at each density (0.25, 0.65 and 0.95 g/mL) to completely extract the spiked sample

from the thimble was determined by using stepwise extractions over a total time of 120

minutes. Samples were collected after 10, 20, 30, 50, 70, 90 and 120 minutes in 1.5 mL

vials containing 20 pL of IS. Extraction was considered complete when no response was

detected on GC analysis.

Extraction Conditions

Density (g/mL) Pressure (bar) Extraction Temperature (°C) Flow Rate (mL/min) Equilibration Time (min) Extraction Time (min) Nozzle Temperature (°C)

0.30, 0.65, & 0.90 81,104, & 281 40 1.0 0 Up to 120 45

Trap Conditions

Nozzle Temperature (°C) 45

Trap Packing ODS Trap Temperature (°C) 20 Trap Rinsing Temperature (°C) 40 Trap Rinse Solvent Ethanol Trap Rinse Volume (mL) L5

Table 23. Extraction and trapping conditions for exhaustive extraction of the standard mixture

2.2.2.3. G C Analysis

The concentrated samples were diluted with ethanol to provide an appropriate response.

1 uL of sample was injected into the GC and analysed by the method shown in Table

2.4. The amount of each compound extracted was measured as peak area and the octanol

IS used to normalise the peak areas to adjust for small discrepancies in rinse and

injection volumes.

62

Conditions

Column HP- Innowax (30m x 0.25 m m x 0.25 um)

Injection Mode Splitless Constant Flow Rate (mL/min) 1.2 Injection Temperature (°C) 250 Detector Temperature (°C) 250

Temperature Program

Initial 50°Caforl5min R a m p Rate 1 50-220 °C at 20 °C/min Final Hold 220 °C for 10 min

Table 2.4. GC conditions for the analysis of the standard mixture

Wh e n hexane and M T B E were used as solvents the initial temperature was decreased to 35°C to achieve complete separation of limonene and cineole

2.2.3. Trapping Conditions

2.2.3.1. Materials

Trap Packing

Six types of solid sorbent trapping materials with various functionalities were examined.

The specifications of each of the solid sorbents used are shown in Table 2.5.

Trapping

Material

Stainless Steel

ODS C18 Cyano Silica

Diol

Supplier

Hypersil

Isolute Isolute

Isolute Isolute

Particle Size

(p.m)

30 30 62 61 57 65

Pore Size

(A)

n/a 120 54 54 56 54

Surface Area

(m2/g)

n/a 175 561 561 n/a 561

Carbon

Loading

(%)

n/a 9-10

19.3

8.5 n/a 6.8

Table 2.5. Specifications of trapping materials (n/a= not applicable)

Packing of Traps

The traps were packed with the aid of a purposely designed funnel which was screwed

into the top of the trap. The solid sorbent was passed through the funnel into the trap.

The trap was vibrated to ensure uniform packing. Once the trap was full, the funnel was

removed and the trap vibrated further. A stainless steel frit contained within a Teflon

63

ring was placed in the top of the trap positioned by screwing in a trap plug. The trap was

then connected to the SFE module and rinsed with approximately 20 m L of solvent and

the void volume measured before use.

2.2.3.2. Effect of Collection and Extraction Conditions on Recovery

The effects of various trapping and extraction conditions on the recovery of the

components of the standard mixture were examined. The recovery of the components

was calculated using the peak areas of the components of the extracted standard mixture

compared to a control representing 1 0 0 % recovery. The control was prepared by

pippetting 80 uL of the standard mixture directly into a vial containing 20 uL of IS, to

which 1.4 m L of solvent was added. The IS was used to normalise the peak areas to

account for small discrepancies in rinse and injection volumes. All extracts were

analysed by G C using the method in Table 2.4.

Effect of Trapping Material

The extraction thimbles were prepared with the standard mixture as described in Section

2.2.2.1. Extractions of the standard mixture were performed in triplicate for each of the

six solid sorbents as trapping materials using the conditions shown in Table 2.6.

64


Density (g/mL) Pressure (bar) Extraction Temperature (°C) Flow Rate (mL/min) Equilibration Time (min) Extraction Time (min) Nozzle Temperature (°C)

Trap Conditions

Nozzle Temperature (°C) Trap Packing

Trap Temperature (°C)

Trap Rinsing Temperature (°C) Trap Rinse Solvent Trap Rinse Volume (mL)

0.65

104 40 1.0(0.5, 1.0 & 2.0) 0 30 45

-

45 As shown in Table 2.5 20 (0,20 & 40) 40 (20,40 & 60) Ethanol

1.5

Table 2.6. Extraction and trapping conditions used to examine the effect of trapping material

Effect of Trap Temperature

Trap temperatures of 0°, 20° and 40°C for each type of trapping material were examined

using the conditions shown in Table 2.6.

Effect of C02 Flow Rate

Flow rates of 0.5, 1.0 and 2.0 mL/min for each type of trapping material were examined

using the conditions shown in Table 2.6.

Effect of Rinse Solvents

The efficiency of five different rinse solvents of various polarity to reconstitute the

adsorbed compounds from the six different types of trapping materials was examined.

The five rinse solvents used were hexane, iso-octane, methyl-tert-butyl ether (MTBE),

ethyl acetate and ethanol, as in Section 2.1 and Table 2.1.

The standard mixture was extracted using the extraction conditions described in Table

2.6. The trap conditions, however, varied slightly in that the six trapping materials used

65

were each rinsed with ethanol, hexane, iso-octane, M T B E , and ethyl acetate. To

determine whether the adsorbed components were being completely removed from the

trap by the first 1.5 m L rinse volume, a further three 1.5 m L rinses were collected in

separate vials.

Effect of Rinse Temperature

Rinse temperatures of 20°, 40° and 60°C for each type of trapping material were

examined using the conditions shown in Table 2.6.

2.2.4. Hydrodistillation of Standard

The standard mixture (250 uL) was placed into a 100 m L round bottomed flask attached

with a condenser, and hydrodistilled for 24 hours in 50 m L of distilled water. The

distillate was extracted with M T B E (3 x 50 m L ) and dried (MgS04). The solvent was

removed under a stream of nitrogen until 25 m L of solvent remained. The remaining

M T B E was left to evaporate under ambient conditions to afford the hydrodistilled

standard mixture. A volume of the standard mixture (80 uL) was transferred to a vial

containing 20 uL of IS and analysed by G C (Table 2.4). The recoveries of the

individual components of the standard mixture were calculated as described in Section

2.2.3.2.

66

2.3. Optimisation of Sandalwood Extraction

2.3.1. Materials

Sandalwood

A piece of sandalwood buttwood, approximately 1 m long, was obtained from the

Department and Conservation and Land Management (CALM). The wood was reduced

to fine wood chips using a wood carving tool fitted to an angle grinder. The chips were

reduced to a powder in a stainless steel Waring commercial blender in the presence of

dry ice. The powdered wood was dried in a dessicator for 48 hours prior to extraction.

Preparation of Extraction Thimbles

7 mL extraction thimbles with a bottom cap in place were packed with 2 cm of

filterflocs (Machery- Nagel). The weight of the empty thimble and filterflocs was

weighed on a 5 point balance (Sartorius MC 1). Approximately 1 g of powdered

sandalwood was placed into the extraction thimble and weighed to determine the exact

mass of wood to be extracted. A further 2 cm of filterflocs was positioned on top of the

wood, and the top end cap sealed in place.

For all samples of sandalwood extracted, the percentage oil yield, percentage volatiles

and composition were measured. A description of how each of these parameters were

measured is outlined below.

67

2.3.2. Calculations of Percentage Yield, Volatiles and Composition

Measurement of Percentage Yield

Each extracted sample was collected in a pre-weighed 1.5 mL amber vial. The solvent

was left to evaporate under ambient conditions and placed in a dessicator to remove any

residual water. The vial containing the oil was reweighed to determine the amount of oil

extracted. The percentage yield of oil from dry wood was calculated using the following

equation:

% Yield = weight of oil extracted / weight of dry wood x 100%

Measurement of Volatiles

The volatile component was defined as the portion of the oil that eluted from the GC

column. The non-volatile material does not elute from the column and deposits on the

inlet liner or on the analytical column. The percentage of volatiles was calculated using

the IS. To estimate the percentage volatiles a number of assumptions were made. Firstly

it was assumed that the peak area response from octanol corresponds to the mass of

octanol added prior to analysis. The second assumption was that octanol has the same

FID response as the components of the sandalwood oil. If these assumptions hold true,

the mass of sandalwood oil eluting from the column (mass volatile oil) can be calculated

using the equation below. Even if the second assumption is not true, the error would be

consistent for all samples and allow direct comparisons to be made.

Mass octanol / Peak area of octanol = Mass volatile oil / Peak area of volatile oil

68

The mass of volatiles in the extracted total oil can be calculated knowing the dilution

factor, and if required the percentage of volatiles in the oil can also be calculated using

the equation below.

% Volatiles = Mass volatile oil/Mass total oil x 1 0 0 %

G C Analysis of Sandalwood Oil


2.7.

Conditions

Column

Injection M o d e Constant Flow Rate (mL/min) Injection Temperature (°C) Detector Temperature (°C)

Initial R a m p Rate 1

Final Hold

HP- Innowax (30m x 0.25 m m x 0.25 um)

Splitless

1.3 250 250

Temperature Program

50°C 50to240atl5°C/min

240°Cforl0min

Table 2.7. G C conditions for the analysis of sandalwood oil

Compounds with a peak area of greater than 1 % of the total volatile oil components

eluting from the column were used to determine the chemical composition of the oil.

2.3.3. Exhaustive Extraction

The thimbles containing the ground sandalwood were extracted using the conditions set

out in Table 2.8. The time taken at each density (0.45, 0.55, 0.65, 0.75, 0.85, and 0.95

g/mL) to completely extract the oil from the sandalwood was determined by using

69

stepwise extractions over a total extraction time of 90 minutes. Samples were collected

after 10, 20, 30, 45, 60 and 90 minutes. All extractions were performed in triplicate.


Density (g/mL)

Pressure (bar)

Extraction Temperature (°C) Flow Rate (mL/min)

Equilibration Time (min) Extraction Time (min)

Nozzle Temperature (°C)

0.45, 0.55, 0.65, 0.75, 0.85, & 0.95 89, 93, 104, 134, 211, 281, & 383 -40 1.0 0 Up to 90 45

Trap Conditions

Nozzle Temperature (°C) Trap Packing Trap Temperature (°C)

Trap Rinsing Temperature (°C) Trap Rinse Solvent Trap Rinse Volume (mL)

45 Isolute Diol 20 40 Ethanol

1.7

Table 2.8. Extraction and trapping conditions for exhaustive extraction of sandalwood oil

2.3.4. Effect of Density on Percentage Yield, Percentage Volatiles, and

Composition

The time taken to completely remove the volatile portion of the oil for each of the six

densities was used as the extraction time. Extractions at each density were conducted

using the conditions outlined in Table 2.9. The percentage yield, percentage of volatiles

and composition of the extract for each density were measured.

70


Density (g/mL)

Pressure (bar)

Extraction Temperature (°C) Flow Rate (mL/min) Equilibration Time (min) Extraction Time (min) Nozzle Temperature (°C)

0.45

89

40 1.0 0 90 45

0.55

93

40 1.0 0 60 45

0.65

104

40 1.0 0 45 45.

0.75, 0.85

&0.95

134, 211, & 383 40 1.0 0 30 45

Trap Conditions

Nozzle Temperature (°C) 45 45 45 45 Trap Packing Isolute Diol Isolute Diol Isolute Diol Isolute Diol Trap Temperature (°C) 20 20 20 20

Trap Rinsing Temperature (°C) 40 40 40 40 Trap Rinse Solvent Ethanol Ethanol Ethanol Ethanol Trap Rinse Volume (mL) L5 L5 L5 1.5

Table 2.9. Extraction and trapping conditions used to completely extract the sandalwood oil from the matrix at various densities

2.3.5. Particle Size

Materials

Hammer-milled sandalwood powder was obtained from Mt Romance Pty. Ltd. via

Wescorp Sandalwood Pty. Ltd. The wood was dried in a dessicator for 48 hours prior to

extraction.

Preparation of Wood

Two studies, using different particle sizes were performed. In Study 1, the wood

extracted was of a particle size range, and in Study 2, the wood extracted fell below a

single particle size. The percentage yield, percentage volatiles and composition for each

extract were measured as described in Section 2.3.2.

Study 1

12 sieves of various mesh diameters were stacked vertically. The mesh sizes of the

sieves ranged from the largest at the top (1.7 mm) to smallest at the bottom (53 um). A

quantity of hammer-milled sandalwood was placed into the top sieve and the stack

shaken manually allowing the sandalwood to fall through the sieves. The sandalwood

particles collected in the sieve where the mesh size was smaller than the particle size of

the wood. The wood collected in each sieve represented a range in particle size between

the mesh size of the two sieves in which the powdered wood fell through and collected.

The ranges of particle sizes where the powdered wood was collected is shown in Table

2.10. The samples of wood of differing particle sizes were removed from the sieves and

stored in labelled glass jars.

Range of Particle Sizes Collected

Above 1700 1700-1400-1180-1000 850-710-600-500-355-250-180

•1400 •1180 •1000 -850 •710 •600 •500 •355 •250 •180 -53

(um)

Table 2.10. Range of particle sizes used in study 1

Study 2

Particle size experiments were performed in collecting 5 samples, which fell under only

one particle size. The 5 particle sizes used were <1700, <1400, <1000, <710, and <500

um. The hammer-milled sandalwood was placed on each of the sieves and the sieves

were shaken manually. The wood passing through the sieves were collected in a plastic

bucket and transferred to labelled glass jars.

72

Exhaustive Extraction

Prepared samples were extracted using the conditions illustrated in Table 2.11. The time

to completely extract the oil from the sandalwood for each particle size range was

determined by using stepwise extractions over a total extraction time of 60 minutes.

Samples were collected after 15, 30,45, and 60 minutes. The percentage yield,

composition and percentage of volatiles were measured as outlined in Section 2.3.2.


Density (g/mL)

Pressure (bar)

Extraction Temperature (°C) Flow Rate (mL/min) Equilibration Time (min) Extraction Time (min) Nozzle Temperature (°C)

0.75 134 40 1.0 0 U p to 60

45

Trap Conditions

Nozzle Temperature (°C) Trap Packing Trap Temperature (°C) Trap Rinsing Temperature (°C)

Trap Rinse Solvent Trap Rinse Volume (mL)


1.7

Table 2.11. Extraction and trapping conditions for exhaustive extraction of sandalwood oil in the particle size studies

Effect of Particle Size on PercentageYield, Percentage Volatiles and Composition

The time taken to completely remove the volatile portion of the oil (Section 3.4) was

used as the extraction time. Extractions were carried out using the conditions in Table

2.11 with the extraction times for both study 1 and 2 given in Table 2.12.

73

Extraction Conditions Extraction Time (min) 90 60 45 Particle Size (um) Above 1700, 1180-1000, 600-500,500-Study 1 1700- 1400, 1000 - 850, 355,355 - 250,

1400-1180 850-710,710 250-180,180--600 53

Particle Size (um) < 1700 < 1400,1000, Study 2 710, & 500

Table 2.12. Extraction times required to completely extract the sandalwood oil from the matrix at various particle sizes

2.4. Large Scale Extraction

2.4.1. Materials

Sandalwood

Powdered sandalwood was obtained from Wescorp Sandalwood Pty. Ltd. The wood was

dried in a dessicator for 48 hours prior to extraction.

Preparation of Extraction Cell

A 300 mL high pressure gas cylinder (Whitey) was packed with 2 cm of filterflocs

(Machery- Nagel). The empty cylinder and filterflocs was weighed on a 2-point balance

(Satorius Basic). Approximately 100 g of powdered sandalwood was placed into the

cylinder and reweighed to determine the exact mass of wood to be extracted. Enough

filterflocs was positioned into the cylinder on top of the wood to prevent the movement

of particles out of the cylinder. The extraction cell was positioned inline by attaching a

V? NPT - 1/16" union to the cylinder and 1/16" stainless steel tubing in the flow path as

described below.

74

2.4.2. Instrument Modification

Figure 2.2 shows the modifications made to the SFE to permit scale-up of the extraction

process. The existing stainless steel fluid line was disconnected at both the union before

the chamber preheat unit, and at the three-way union prior to the high pressure isolation

valve. Approximately 5 m of 1/16" coiled stainless steel tubing was connected to the

union before the chamber pre-heat. A 300 m L high pressure gas sampling cylinder

(Whitey) was connected to the end of this tubing, and served as the extraction chamber.

Another section of stainless steel tubing was connected from the end of the 300 m L high

pressure gas sampling cylinder to the three-way union prior to the high pressure

isolation valve. The solid bed trap was removed and replaced by a piece of 1/8" stainless

steel tubing that was connected to a custom made stainless steel trapping device (Figure

2.3), capable of collecting large amounts of extracted oil. The trapping device was

immersed in an ice bath during extraction to aid in the precipitation of the extraction

analytes. The 300 m L extraction vessel was placed in a thermostated water bath. The 5

m of tubing connected to the extraction vessel was submerged in the water bath, and

acted as a preheat for the fluid before entering the extraction chamber. A standard 7 m L

metal thimble was left in the SFE sample carousel to allow for normal instrument

operation by triggering the appropriate sensors during an extraction.

The SFE was operated through the program Mod 1, which allowed manual control over

the operations of the SFE. The time taken to reach pressure using the 300 m L extraction

vessel exceeded the instruments shut off time warning of underpressure conditions.

Using M o d i the pressurisation process could be restarted until the desired pressure was

attained. Following depressurisation, there was no rinse step as the oil could be

collected directly from the tapered bottom of the custom made trap.

75

High Pressure Disconnected SFE Isolation Valve Tubing Tubing to Large-Scale Trap

1 /16" Stainless Coiled Tubing Steel Tubing y for Pre-heat

Thermostat and Stirrer

SFE Thimble Pre-heat Fluid Pump High Pressure 300 m L Extraction Cell On/Off Valves

Figure 2.2. Modification of the S F E apparatus to allow scale-up of the extraction vessel to 300 m L

Pressure Release

Top of Trap

Body of Trap

Tapered Bottom

1/16" Stainless Steel Tubing from SFE

A

* •

Extension of 1/16" Stainless Steel Tubing

Figure 23. Large-scale trapping device

76

Time Course and Percentage Yields

The prepared extraction cells were extracted using the conditions set out in Table 2.13.

The time taken at each density (0.45, 0.55, 0.65, 0.75, 0.85 and 0.95 g/mL) to

completely extract the sandalwood oil was determined by using stepwise extractions

over a total extraction time of 800 minutes. Samples were collected after 100, 200, 300,

400, 500 and 800 minutes in a pre-weighed custom made large scale trapping device.

The percentage yield was calculated for each density using the total amount of oil

extracted over the 800 minute period.


Density (g/mL) 0.45, 0.55, 0.65, 0.75, 0.85 & 0.95

Pressure (bar) 89, 93, 104, 134,

211, 281, & 383 Extraction Temperature (°C) 40 Flow Rate (mL/min) 2.0 Equilibration Time (min) 0 Extraction Time (min) Up to 800 min

Nozzle Temperature (°C) 45

Trap Conditions

Nozzle Temperature (°C) 45 Trap Packing Trap Temperature (°C) 0-10°C Trap Rinsing Temperature (°C)

Trap Rinse Solvent

Trap Rinse Volume -

Table 2.13. Extraction and trapping conditions for the large-scale extraction of sandalwood (300mL)

2.5. Comparison of Extraction Techniques

2.5.1. Materials

The wood used for the comparison of the extraction techniques was from the same

source that was used for the particle size study in Section 2.3.5. The percentage yield,

77

percentage volatiles and composition for each extract was measured as described in

Section 2.3.2.

2.5.2. Methods

Hydrodistillation

Approximately 50 g of powdered sandalwood was placed into a modified Dean-Stark

apparatus and hydrodistilled for 24 hours in 500 m L of distilled water. The distillate was

extracted with M T B E (3 x 50 m L ) and dried (MgS04). The solvent was removed under

a stream of nitrogen until 25 m L of solvent remained. The remaining M T B E was left to

evaporate under ambient conditions to afford a pale yellow oil.

Ethanol Extraction

Approximately 50 g of powdered sandalwood was magnetically stirred for 24 hours in

ethanol (500 m L ) . The sandalwood powder was removed by vacuum filtration and

washed with ethanol (100 mL). The ethanol was removed under a stream of nitrogen

until 25 m L of solvent remained. The remaining ethanol was left to evaporate under

ambient conditions to afford a dark red-brown viscous oil.

Hexane Extraction

Approximately 50 g of powdered sandalwood was magnetically stirred for 24 hours in

hexane (500 m L ) . The sandalwood powder was removed by vacuum filtration and

washed with hexane (100 mL). The hexane was removed under a stream of nitrogen

until 25 m L of solvent remained. The remaining hexane was left to evaporate under

ambient conditions to afford a yellow oil.

78

SFE

Approximately 1 g of sandalwood oil was placed into extraction thimbles as described

in Section 2.3.1. The sandalwood was extracted under the optimised conditions set out

in Table 2.14.


Density (g/mL) Pressure (bar)

Extraction Temperature (°C) Flow Rate (mL/min)

Equilibration Time (min) Extraction Time (min)

Nozzle Temperature (°C)

0.75 134 40 1.0 0 30 45

Trap Conditions

Nozzle Temperature (°C) Trap Packing

Trap Temperature (°C) Trap Rinsing Temperature (°C) Trap Rinse Solvent Trap Rinse Volume (mL)


1.7

Table 2.14. Optimised SFE conditions for the extraction of sandalwood oil

2.6. Sandalwood Survey

2.6.1. Geographic

2.6.1.1. Materials

Sandalwood Samples

Core samples were taken from the buttwood of living sandalwood trees 10 cm above

ground level. The outer bark of the tree was removed and samples were taken by drilling

through the tree with a Vz inch auger drill bit attached to a Tanaka (TED-262-L) two-

stroke fence post borer (Figure 2.4). The drill bit was rotated slowly during core

sampling to limit the heat generated and minimise volatilisation of components of the

oil within the wood. The sandalwood shavings were collected by holding a plastic zip

79

lock bag directly beneath the drill bits point of entry into the tree. To prevent infection in

the tree, a small branch of dead wood was positioned inside the hole, and sealed to the

atmosphere with Selleys polyfiller (Figure 2.5).

The diameter of the trees 15 cm above ground level was measured and the approximate

height and condition of the trees noted. The GPS of the trees were recorded using a

Magellan GPS 2000XL Satellite Navigator. The trees were tagged with aluminium tags

attached to copper wire for future reference.

Figure 2.4. Coring of sandalwood trees.

The shavings were collected and the oil extracted and analysed.

80

Figure 2.5. Drill hole filled with Selly polyfiller post-core sampling

Sample Preparation

The sandalwood shavings collected from core sampling in the field were brought back to

the laboratory for preparation before extraction. Any bark in the sample was removed.

The shavings were blended in a stainless steel Waring commercial blender in the presence

of solid carbon dioxide until a powder was attained. The powder was removed from the

blender and stored in labelled glass jars. The ground samples were dried for 48 hours in a

dessicator prior to extraction.

2.6.1.2. Methods

Extraction of Sandalwood

The sandalwood samples were placed in thimbles (Section 2.3.1) and extracted using the

SFE conditions shown in Table 2.14. Percentage yield, percentage of volatiles and

81

composition of the oil was determined as previously described (Section 2.3.2). Each

sample was extracted in triplicate.

G C Analysis of Sandalwood Oil


2.15.

Conditions

Column

Injection Mode

Constant Flow Rate (mL/min)

Injection Temperature (°C) Detector Temperature (°C)

HP- Innowax (30m x 0.25 m m x 0.25 um) Splitless 1.3 250 250

Temperature Program

Initial

Ramp Rate 1 Ramp Rate 2 Final Hold

60°C for 5 mins 4°C/minfol65°C 7°C/minto240°C 10 mins

Table 2.15. G C conditions for the analysis of sandalwood oil to determine variation between trees

The chemical composition of the oil was calculated based on peak area. Only

compounds with a peak area of >1% were presented in the results. This GC analysis

method differs from the method used for the sandalwood optimisation (Table 2.7) in

that the analysis times were longer. The compositional differences were not as important

in the optimisation of the SFE methods. Greater separation was required to accurately

determine differences in composition of the sandalwood oil.

G C - M S Analysis of Sandalwood Oil


2.16. The linear velocity and temperature program were the same as those of the

previous GC method, thus allowing retention times to be matched. MS was performed

82

in Electron Ionisation (EI) mode (70eV). This allowed identification of unknown

compounds to be determined by GC-FID on a routine basis from retention times.

Conditions

Column

Injection Mode Constant Flow Rate (mL/min) Injection Temperature (°C) Detector Temperature (°C)

HP- Innowax (30m x 0.25 m m x 0.25 urn) Splitless 0.75 250 250

Temperature Program

Initial Ramp Rate 1

Ramp Rate 2 Final Hold

60°C for 5 mins 4°C/mintol65°C 7°C/minto240°C 10 mins

Table 2.16. GC-MS conditions for the analysis of sandalwood oil

2.6.1.3. Sandalwood Locations

The 87 sandalwood core samples were taken from 12 regions throughout Western

Australia (WA). Figure 2.6 shows the geographic location of the 12 regions in WA.

Table 2.15 lists the region where each sample was taken, the height and diameter of the

tree, and the GPS location. A number of regions were further subdivided into locations

(26 in total). Sampling of locations within a region was only performed when

sandalwood trees were found in different landtypes within the same region. Sandalwood

trees from a location were found within a 200 m radius.

Sample

Wanjarrie 1.1.1

1.1.2

1.1.3

1.1.4

1.2.1

1.2.2

1.2.3

1.3.1

1.3.2

1.3.3

Midgut

1.4.1

1.4.2

1.4.3

1.5.1

1.5.2

1.5.3

Gascoyne

1.6.1

1.6.2

1.6.3

1.6.4

1.6.5

Approx Height (m)

3.0

n/a

2.5

3.0

4.0

3.5

4.0

2.5

3.5

3.0

4.0

3.0

4.0

4.0

6.0

5.5

Junction

3.0

5.5

4.5

3.5

n/a

Diameter (mm)

129

88

111

154

155

138

151

161

170

132

167

135

139

140

150

189

166

247

146

147

83

GPS(deg, min)

27 26 S 120 34 E 27 26S 120 34 E 27 26E 120 34 E 27 26E 120 34 E 27 25S 120 35 E 27 25S 120 35 E 27 25S 120 35 E 27 20S 120 37 E 27 20S 120 37 E 27 20S 120 37 E

24 39S 11820E 24 39S 11820E 24 39S 11820E 24 52S 118 24 E 24 52S 118 24 E 24 52S 11824E

25 24S 116 06 E 25 24S 116 09 E 25 24S 116 06 E 25 24S 116 06 E 22 24S 116 06 E

Sample Approx Height (m)

Shark Bay 1.7.1 2.5

1.7.2 5.0

1.7.3 4.5

1.7.4 4.0

1.8.1 2.0

1.8.2 2.0

1.8.3 2.0

Murchison

1.9.1 2.5

1.9.2 4.0

1.9.3 3.0

1.10.1 2.0

1.10.2 2.0

1.10.3 3.0

Burnerbinmah

1.11.1 2.5

1.11.2 2.5

1.11.3 2.5

1.11.4 n/a

1.12.1 3.0

1.12.2 5.5

1.12.3 2.0

1.13.1 3.0

1.13.2 2.5

1.13.3 3.0

Diameter GPS (deg, (mm)

176

202

231

178

147

155

229

194

130

120

113

140

166

138

144

165

77

180

274

113

129

117

163

min)

25 50 S 113 40 E 25 50S 113 40 E 25 50S 113 40 E 25 50 S 113 40 E 25 51S 113 40 E 25 51S 113 40E 25 51S 113 40 E

26 52S 115 56E 26 52S 115 56E 26 52S 115 56E 27 00S 11603E 27 00S 116 03 E 27 00S 116 03 E

28 47S 11715E 28 47S 117 15E 28 47S 11715E 28 47S 117 15E 28 47S 117 17E 28 47S 117 17E 28 47S 11717E 28 44S 117 21 E 28 43S 11723E 28 43S 117 23 E

Table 2.17. Locations and dimensions of sandalwood trees in the geographic survey Diameter of tree was measured 150 m m above ground level

Sample

MtElvire 1.14.1

1.14.2

1.14.3

1.14.4

1.15.1

1.15.2

1.15.3

1.16.1

1.16.2

1.16.3

Approx Height (m)

2.0

n/a

2.5

3.5

4.0

4.5

2.5

3.0

4.0

2.5

Goongarrie

1.17.1

1.17.2

1.17.3

1.18.1

1.18.2

1.18.3

1.19.1

1.19.2

1.19.3

2.0

4.5

4.0

3.5

4.0

3.5

3.0

2.5

2.5

Bullock Holes

1.20.1

1.20.2

1.20.3

3.0

3.0

3.0

Diameter (mm)

106

89

200

137

190

187

149

159

165

135

121

174

204

140

182

171

124

130

139

143

143

151

GPS (deg, min)

29 15S

119 38 E

29 15S

119 38E

29 15S

119 38 E

29 15S

119 38E

29 19S

119 36E

29 19S

119 36E

29 19S

119 36E

29 24S

119 35 E

29 24S

119 35 E

29 24S

119 35 E

30 01S

121 02 E

30 01S

121 02 E

30 01S

121 02 E

30 01S

121 07 E

30 01S

121 07 E

30 01S

121 07 E

30 04S

121 08 E

30 01S

121 08 E

30 01S

121 08 E

30 32S

121 45 E

30 32S

121 45 E

30 32S

121 45 E

Sample Approx Height (m)

Bullock Holes

1.21.1

1.21.2

1.21.3

1.22.1

1.22.2

1.22.3

3.5

4.0

4.0

4.0

5.0

4.5

Katanning

1.23.1

1.23.2

1.23.3

1.23.4

1.23.5

1.23.6

Ninghan

1.24.1

1.24.2

1.24.3

1.24.4

1.24.5

Jaurdie

1.25.1

1.25.2

1.25.3

1.26.1

1.26.2

1.26.3

3.0

3.5

2.5

3.5

2.5

4.0

2.0

3.0

2.0

3.5

2.5

2.5

2.0

4.5

5.0

3.0

2.5

Diameter GPS (deg, (mm)

141

135

143

180

122

126

80

200

160

205

125

130

122

102

160

182

150

150

152

170

178

138

115

min)

30 32S

121 46 E

30 32S

121 46 E

30 32S

121 46 E

-30 32 S

121 45 E

30 32S

121 45 E

30 32S

121 45 E

33 26S

117 40E

33 26S

11740E

33 26S

11740E

33 26S

117 40E

33 26S

11740E

33 26S

117 40 E

29 29S

117 10E 29 29S 11710E 29 29S 117 10E 29 29S 117 10E 29 29S 11710E

3107S 120 18 E

3107S

120 18 E

3107S

120 18 E

30 51S

121 09 E

30 51S 121 09 E

30 51S

121 09 E

Table 2.17 \cont.... Locations and dimensions of sandalwood trees in the geographic survey Diameter of tree was measured 150 m m above ground level

85

Figure 2.6. Sampling locations throughout W A

2.6.2. Seasonal Variation

Sandalwood trees from Wanjarrie, M t Elvier, Goongarrie, Bullock Holes and Katanning

were sampled over an 18 month period to determine if differences existed in the oil due

to seasonal variation. Samples were collected in spring, summer, autumn and winter. The

dates each sample was taken from the regions are shown in Table 2.18.

86

Date of Core Sampling Region

Wanjarrie Mt Elvire

Goongarrie Bullock Holes Katanning

Spring

21/09/98 25/09/98 25/09/98 28/09/98 30/09/98

Summer

12/01/00 13/01/00 14/01/00 14/01/00 21/01/00

Autumn

07/03/99 08/03/99 09/03/99 09/03/99 15/03/99

Winter

04/08/99 05/08/99 06/08/99 06/08/99 23/08/99

Table 2.18. Locations and dates of sampling of sandalwood trees in the seasonal survey

2.6.3. Sectional Examination

2.6.3.1. Materials

Three sandalwood trees were harvested from Lakeside Reserve approximately 20 km

east of Kalgoorlie. The size, diameter and GPS location of the trees are given in Table

2.19.

156

159

144

30 49 85 E 121 36 72 S 30 49 96 E 121 36 69 S 30 49 94 E 12136 71 S

Sample Approximate Height Diameter (mm) GPS (m) (deg, min, sec)

Treel 2.5

Tree 2 3.0

Tree 3 3.0

Table 2.19. Locations and dimensions of the 3 trees in the sectional surveys Diameter of tree was measured 150 m m above ground level

Harvesting of Sandalwood Trees

The trees harvested were situated on the edge of washed out river beds and were chosen

for ease of removal. The roots were exposed (Figure 2.7) by washing away the soil on

the edge of the river bank with water from a fire-fighting water hose supplied by a water

tanker. The tree was loosened from the soil by a combination of digging and washing

the soil from the tree with water. The trees were removed whole with the roots intact

and cut 50 cm above ground level with a chainsaw for ease of transport.

Figure 2.7. Exposed roots of a sandalwood tree. Harvesting of the tree required the roots system to remain intact. The earth was removed by washing

away the soil with water from a fire hose.

2.6.3.2. Longitudinal Sampling

Core samples were taken along a length of each tree from the roots to the branches at

5cm intervals. The method used for sampling was a variation of that described in Section

2.6.1.1. In between taking core samples, the auger drill bit was cooled in a dry

ice/acetone mixture. Instead of using a fence post borer, the drill bit was rotated by an

electric hammer drill. The shavings collected were reduced to powdered wood as

described in Section 2.6.1.1 and dried for 48 hours in a dessicator prior to extraction. All

extractions were performed in triplicate.

2.6.3.3. Cross Sectional sampling

The trees were cut using a bandsaw at 10 and 5 cm above ground level. A section of the

buttwood 5 cm in width was obtained. Core samples were taken across the diameter of

the tree with sampling method used in Section 2.6.3.2.

88

3. Results and Discussion

3.1. Optimisation of Trapping Conditions

Optimisation of the trapping conditions was performed using a standard mixture. The

standard mixture contained compounds representative of those found in real sandalwood

oil, and other compounds of varying size, volatility, and functionality that are commonly

found in essential oils (Table 2.2, Figure 2.1). These included the major sesquiterpene

(C15) alcohols of Western Australian sandalwood (trans, trans-famesol, and a- and f3-

santalol), limonene, cineole, citronellal, nerol and P-ionone, which represented the more

volatile CIO terpenes of alkene, ether, aldehyde, alcohol and ketone functionality

respectively. Manool, a diterpene alcohol (C20) was included to examine the

effectiveness of the collection procedure on higher molecular weight alcohols. The

boiling points and molecular weight of the components are shown in Table 3.1.

3.1.1. Extraction Time Course of Components of Standard Mixture

Exhaustive extraction was performed on the standard mixture to determine the time

required to completely extract and transfer the compounds to the solid bed trap for three

C02 densities (0.30,0.65, and 0.95 g/mL). The recoveries of each of the components

were measured after 10,20, 30, 40, 60, 80 and 120 minutes of extraction at each density.

An extraction time course showing the cumulative percentage recovery of each

component of the standard mixture recovered over time at densities of 0.30, 0.65, and

0.90 g/mL is given in Figures 3.1- 3.3 (Appendix Al). The compounds along the x-axis

represent their elution order from the GC column, hence, order of decreasing volatility

and increasing size.

89

It can be seen that the rate of extraction of each compound increased as the density of

the extraction fluid also increased. This was because as the density of the extraction

fluid was increased, the solvent strength also increased, allowing more compound to be

dissolved per unit time. An exception to this occurs where the rate of extraction of

limonene and cineole at a density of 0.30 g/mL is greater than at 0.65 g/mL, which was

an unexpected result.

Compound Limonene Cineole Citronellal Nerol P- ionone t,t- farnesol a- santalol p- santalol Manool

Molecular Weight 136.2 154.3 154.3 154.3 192.3 222.4 220.4 220.4 290.5

Boiling Point (°C)

176 176 208 225 229

263, 137@ 3 m m 148@ 5mm 158@ 5mm

144-145® 0.2mm

Table 3.1. Molecular weights and boiling points of the components of the standard mixture

At a density of 0.90 g/mL, all of the compounds in the standard mixture were extracted

at similar rates, indicating that each compound had a similar solubility in the C02

solvent. This was also evident at 0.65 g/mL, although the initial rate of extraction was

slower. Extraction with C02 at a density of 0.30 g/mL showed significantly different

rates of extraction for the various classes of compounds in the standard mixture even

though the cumulative percentage of the CI 5 and C20 compounds extracted at 0.30

g/mL may be an underestimate as not all of these compounds were extracted after 120

minutes (Figure 3.3) as the recoveries of these compounds after 80,100 and 120

minutes had not begun to decrease. After 30 minutes, over 60% of the monoterpenes

(limonene, cineole, citronellal, nerol and p-ionone) were extracted, but only 3% of the

sesquiterpenes {trans, fra/w-farnesol, and a- and p-santalol) and the diterpene manool.

90

This shows the larger CI5 and C20 compounds are far less soluble in C 0 2 at this density

than the CIO compounds.

120

Limonene Cineole Citronellal Nerol P-ionone a-santalol f,f-farnesoI P-santalol Manool

Figure 3.1. Cumulative percentage of individual components of the standard mixture extracted

over 80 minutes at a C 0 2 density of 0.90 g/mL

i2o

100

2

Warn Limonene Cineole Citronellal Nerol P-ionone a-santalol f.f-farnesol P-santalol Manool



°80

a 60

•40

°30

•20

• 10

91

• 1 2 0

• 100

• 8 0

• 6 0

• 4 0

• 3 0

• 2 0

• 1 0

Limonene Cineole Citronellal Nerol P-ionone "-santalol r,?-farnesol P-santalol Manool



A density of 0.65 g/mL was chosen to examine the effect of the various trapping

conditions on recovery since the compounds were completely recovered (>95%

recovery) after 30 minute of extraction.

3.1.2. Trapping

SFE can be can be divided into two distinct steps, extraction and collection. Extraction

involves the removal and transfer of the analytes from the matrix to the collection device,

while collection involves the recovery of the extracted analytes by the collection device.

For successful SFE, both these steps must be quantitative. The development of efficient

collection methods can prove difficult, due to the high gaseous C02 flow rates at the

restrictor after depressurisation. It is therefore important to develop a quantitative

collection method before addressing the extraction step.

Collection in SFE using a solid bed trap as the collection device relies on the adsorption

of the analytes onto the solid support (trapping). This can occur through two

mechanisms; physical and chemical adsorption. In physical adsorption on both inert and

92

active solid supports, the solid surface onto which the analytes precipitate is

cryogenically cooled. For efficient trapping of volatile components, sub-ambient trap

temperatures are required (cryotrapping). In chemical adsorption on active sorbents,

trapping occurs through interactions between the analytes and the active sorbent. There

is not one universal trap that will be effective for all classes of compounds. Active solid

supports have the advantage over inert solid supports because trapping, can occur

through both physical and chemical adsorption, and the analyst can choose the sorbent

that is most compatible with the target analytes.

A number of solid supports at three trap temperatures (0, 20 and 40°C) were examined

in this study. The solid supports were chosen to cover a wide range of polarities; inert

(stainless steel beads), non-polar (Hypersil O D S , Isolute CI8) and polar (Isolute cyano,

silica, and diol). The five active solid supports are silica and silica based sorbents,

bonded to chromatographic stationary phases of CI 8 (from two manufacturers), cyano,

or diol functionality (Figure 3.4).

The effectiveness of the collection procedure was assessed from the percent recovery of

the analytes, where a recovery over 95 % indicated quantitative collection. In a real

sample, however, the recovery cannot be measured, since the initial amounts of analytes

to be extracted are not known. To calculate the percent recovery in this study, the

mixture of known standards was used (Table 2.2, Figure 2.1). A measured amount (80

uL) of the standard mixture was extracted from an inert matrix and the percent recovery

of each component was measured.

C18- Octadecyl CN- Cyanopropyl Si- Silica 20H- Diol

93

-Si-

NC

Isolute Cyano

Si

I OH

Isolute Silica

HO

-OH

Isolute Diol

Hypersil O D S Isolute C18

Figure 3.4. Phases bonded to solid silica supports used in the study

The recoveries of the components of the standard mixture on the six trapping materials

at trap temperatures of 0,20 and 40°C at a flow rate of 1.0 mL/min are graphed in the

following sections. The results show the effect of trapping material, trap temperature

and compound volatility on the recovery of the individual compounds. For simplicity,

the results from each category of trapping material (inert, non-polar, and polar) will be

discussed separately.

3.1.3. Inert Trapping Material

The recovery of the components of the standard mixture using stainless steel beads

(Figure 3.5, Appendix A2) varied considerably. Quantitative recovery was not achieved

for any of the compounds at any of the three trap temperatures. This indicated that

physical adsorption alone did not efficiently trap the compounds.

94

Due to the lack of chemical functionality associated with the stainless steel beads, the

poor recoveries were a result solely of trap temperature and compound volatility. The

influence of trap temperature on recovery was much less for the less volatile compounds

(i.e higher boiling points). The recoveries of all compounds less volatile than citronellal

differ only minimally at the trap temperatures examined. Recoveries of over 90% were

achieved at a trap temperature of 0°C for all compounds. Increasing the temperature to

20°C, resulted in a slight loss of these compounds, with recoveries between 63 and 79%

being achieved. A further increase in the trap temperature from 20 to 40°C did not lead

to further loss and recoveries ranged between 61 and 79%. These results show that

physical adsorption is less efficient at a trap temperature of 20°C compared to 0°C. At

the higher temperature more of each compound can escape from the trap with the

expanding gas.

The recoveries of the more volatile compounds (limonene, cineole, and citronellal) were

much lower. The differences in volatility of these compounds appeared to have a strong

influence on their recoveries. At a trap temperature of 0°C, the recovery of citronellal,

the least volatile of the three compounds (b.p 208°C), was 77%, while the more volatile

limonene and cineole, which have the same boiling point (176°C), were recovered at 7

and 14% respectively. These recoveries are considerably lower than the recoveries seen

for those compounds less volatile than citronellal (90.1 -92.5 %).

The loss in recovery as the trap temperature increased was more evident for these more

volatile compounds (particularly evident for citronellal) than those compounds less

volatile than citronellal. An increase in the trap temperature from 0 to 40°C resulted in a

67% decrease in recovery of citronellal, compared to only a 25% decrease in nerol.

Complete loss of limonene and cineole was observed at trap temperatures of 20 and

95

40 C. The different losses in recoveries of these compounds were again attributed to

differences in the compounds volatihty. Greater physical adsorption occured between the

nerol molecules and the stainless steel beads as the trap temperature increased compared

to more volatile compounds.

In summary, it appears that those compounds with a boiling point greater than citronellal

(>225°C) exhibit similar recoveries and similar losses as the trap temperatures were

increased. The recoveries of these compounds were on the most part influenced by trap

temperature. Compounds with a boiling point similar to or below that of citronellal

<225°C show much poorer recoveries, and were much more dependent on the volatility

of the compound. The more volatile the compound, the lower the recovery. Greater

losses in recoveries were also experienced as the trap temperatures increased.

120

Limonene Cineole Citronellal Nerol B-ionone a-santalol t.t -farnesol B-santalol Manool

Figure 3.5. Recoveries of the components of the standard mixture using stainless steel balls as the

trapping material (inert trap) at trap temperatures of 0, 20 and 40°C and flow rate of 1 mL/min.

The non-quantitative recoveries on the stainless steel bead trap were due to the escape of

the compounds with the expanding gas, by inefficient physical adsorption. Decreasing the

velocity of the expanding gas by decreasing the extraction fluid flow rate might

96

improve recoveries. Flow rates of 0.5,1.0 and 2.0mL/min were examined at a trap

temperature of 20°C (Figure 3.6, Appendix A2). As the flow rate increased, the recovery

of all compounds in the standard mixture decreased. This clearly demonstrates the

limited ability of the inert trapping material to retain the components of the standard

mixture by physical absorption processes at the higher flow rates.

The recoveries of those compounds with a volatility less than citronellal were improved

by decreasing the flow rate to 0.5 mL/min. The lower flow rate enables more efficient

physical interactions with the stainless steel beads, due to the decreased velocity of the

expanding gas through the restrictor. At a trap temperature of 20°C the recoveries of

these compounds improved approximately 35- 40% by decreasing the flow from 2.0 to

0.5 mL/min.

The effect on the recoveries of the more volatile compounds (limonene, cineole, and

cirtronellal) as the flow rate decreases was much less apparent. The recovery of

citronellal was increased 19.7% by decreasing the flow rate from 2.0 to 0.5 mL/min.

Limonene and cineole were not trapped at any flow rate. Although these compounds had

a longer time to interact with the stainless steel beads at the lower flow rates,

presumably lower trap temperatures were required to aid in precipitation due to their

high volatility.

97

• 0.5

• 1

• :

limonene Cineole Citronellal Nerol B-ionone a-santalol //-farnesol B-santalol Manool

Figure 3.6. Recoveries of the components of the standard mixture on stainless steel beads (inert

trap) at extraction fluid flow rates of 0.5,1.0 and 2.0 mL/min at a trap temperature of 20°C.

3.1.4. Non-polar traps

Chemical adsorption along with physical adsorption can occur on the CI 8 sorbents

(Hypersil O D S and Isolute CI8, Figure 3.4). This can occur by interactions between the

compounds with the CI8 phase (dispersive forces), and the compounds with residual

silanol groups on the silica sorbent (hydrogen bonding).

Figures 3.7 and 3.8 (Appendices A 3 and A4) shows the recoveries obtained using the

two CI 8 traps at 0, 20 and 40°C at an extraction fluid flow rate of 1 mL/min.

Quantitative recoveries of all compounds, except limonene and citronellal were achieved.

Limonene, containing only alkene functionality, interacts with the CI 8 phase through

dispersive forces and physical adsorption. The other compounds have functional groups

that can form strong hydrogen bonds with the residual silanol groups on the surface of

the base silica along with the dispersive forces. The weaker interactions between

limonene and the CI 8 sorbent may result in the lower recoveries with the Hypersil

ODS trap. It appears however, that these interactions can be greatly enhanced

98

from a combination of chemical and physical adsorption, as the recovery improved from

17 to 9 4 % when the temperature of the trap was decreased from 20 to 0°C.

Interestingly, Isolute CI 8 and Hypersil ODS traps differ in their interaction with

limonene. Quantitative trapping was seen at all 3 trap temperatures using the Isolute CI8

trap, but only at 0°C on the Hypersil ODS trap. These different trapping abilities can be

rationalised by consideration of the different manufacturer specifications (Table 2.5),

such as average particle size, average pore size, specific surface area, and in particular

carbon loading. Carbon loading is a measure of the percent of phase (CI 8) bonded to the

base silica. Isolute CI 8 has a higher carbon loading (19.3%) than Hypersil ODS (9-10%)

and the potential for interactions between limonene and the CI 8 bonded phase is greater

with Isolute CI 8.

10

120

140

limonene Cineole Citronellal Nerol B-ionone o-santalol //-farnesol B-santaloI Manool

Figure 3.7. Recoveries of the components of the standard mixture using Hypersil ODS as the

trapping material (non-polar trap) at trap temperatures of 0, 20 and 40°C at a flow rate of 1.0 mL/min

99

Limonene Cineole Citronellal Nerol (J-ionone a-santalol /./-farnesol P-santalol Manool

Figure 3.8. Recoveries of the components of the standard mixture using Isolute C18 as the trapping

material (non-polar trap) at trap temperatures of 0, 20 and 40°C at a flow rate of 1.0 mL/min

Quantitative trapping of citronellal was not observed on either of the non-polar traps at

any of the three trap temperatures. This result was unexpected, since citronellal is

capable of forming hydrogen bonds with the residual silanol groups on the sorbent in the

same manner as the other polar compounds. It is tempting to suggest that the aldehyde

group, at least in part, may lead to the formation of a hemiacetal, which would covalently

link the aldehyde to the silanol group, resulting in partial retention on the trapping

material. This, however, is not tenable in view of the quantitative recoveries obtained

with Isolute silica and diol sorbents described below (see Figures 3.11 and 3.13).

Although low recoveries of both limonene and citronellal were observed on the Hypersil

ODS trap, and citronellal on the Isolute CI 8 trap, the recoveries were greater than those

using the inert trap (Figure 3.5). This shows that some form of chemical adsorption is

occurring. On the inert trap, the trapping of these compounds was very much dependent

on their volatility, the more volatile compounds experiencing lower recoveries.

However on both CI8 traps, cineole, which has the same boiling point as limonene, was

trapped quantitatively at all three trap temperatures, while limonene and citronellal

(which has boiling point higher than cineole) were not, showing the importance and

selectivity of chemical adsorption in the trapping process.

The effectiveness of the non-polar sorbents in trapping the components of the standard

mixture was seen by varying the flow rate (Figures 3.9 and 3.10, Appendices A3 and

A4). For the compounds trapped quantitatively on both traps, the flow rate was shown

to have no effect on recovery, illustrating the strength of the chemical adsorption in

retaining the compounds. The strength of the hydrogen bonding was sufficient to retain

these compounds even at a higher velocity of expanding gas. Limonene was poorly

retained on Hypersil ODS but quantitatively recovered on Isolute CI8 at all three flow

rates. This can be assumed to be due to the superior carbon loading of the Isolute CI 8.

* m

D0.5

• 1

• 2

-i r

Limonene Cineole Citronellal Nerol B-ionone a-santalol f,i-farnesol P-santalol Manool

Figure 3.9 Recoveries of the components of the standard mixture using Hypersil ODS as the trapping material (non-polar trap) at extraction fluid flow rates of 0.5,1.0 and 2.0 mL/min at a

trap temperature of 20°C.

120

100

80 --

> 60 o

40 -

20

0 -

l*Pj ±ri

no.5 m • 2

Limonene Cineole Citronellal Nerol P-ionone a-santalol/,/-farnesol P-santalol Manool

Figure 3.10 Recoveries of the components of the standard mixture using Isolute C18 as the trapping material (non-polar trap) at extraction fluid flow rates of 0.5,1.0 and 2.0 mL/min at a

trap temperature of 20°C.

3.1.5. Polar Trapping Material

Chemical adsorption on the polar traps occurs mainly through interactions with the polar

groups of the bonded phases and hydrogen bonding with the residual silanol groups.

Isolute silica contains a hydroxyl group as its main functionality (Figure 3.4). It would be

expected that hydrogen bonding might be the dominant interaction. On the other hand,

Isolute cyano and diol posses a short non-polar hydrocarbon chain in their functionality

of 4 and 8 carbons respectively (Figure 3.4), that could also enable interactions with

compounds through dispersive forces together with hydrogen bonding. This has the

advantage that a wider range of compounds can be trapped.

The recoveries of the compounds at 0, 20 and 40°C on the three polar traps (Isolute

silica, cyano, and diol) are shown in Figures 3.11-3.13 (Appendices A5- A7). All

compounds, except limonene and citronellal, were quantitatively recovered on the three

polar traps, at all three trap temperatures. These were able to hydrogen bond with the

polar bonded phases and residual silanol groups. The recoveries of limonene and

citronellal on the three polar traps varied, but were above 8 5 % at all trap temperatures,

showing far superior recoveries over the non-polar traps.

The lowest recoveries of limonene and citronellal on the three polar traps were seen

with the silica sorbent, affording recoveries of 85.2 and 86.0% at 40°C respectively

(Figure 3.11). These lower recoveries were due to the inefficient ability of Isolute silica

to interact with these less polar compounds through dispersive forces. As was seen with

the non-polar traps, by decreasing the trap temperature to 0°C, both compounds were

quantitatively recovered.

Citronellal was quantitatively trapped on the cyano trap at all temperatures, but the

recovery of limonene at 40°C was slightly lower (90.5%). The non-polar portion of the

bonded phase was unable to sufficiently prevent the escape of limonene at 40°C. A

decrease in the trap temperature to 0°C was sufficient to completely retain limonene on

the trap.

Isolute diol showed quantitative recoveries of all compounds irrespective of the trap

temperature. In this case, the longer non-polar portion of the bonded phase was able to

interact more strongly with limonene and citronellal.

103

• 0

• 20

• 40

Limonene Cineole Citronellal Nerol p-ionone a-santalol/,/-farnesol B-santalol Manool

Figure 3.11. Recoveries of the components of the standard mixture using Isolute silica as the trapping material (polar trap) at trap temperatures of 0,20 and 40°C at a flow rate of 1.0 mL/min.

120

100

P 60 o o

tltir ft H~

10

120

140

Limonene Cineole Citronellal Nerol P-ionone a-santalol /./-farnesol p-santalol \fenool

Figure 3.12. Recoveries of the components of the standard mixture using Isolute cyano as the

trapping material (polar trap) at trap temperatures of 0, 20 and 40°C at a flow rate of 1.0 mL/min.

104

10

120

140

I I I I I I Limonene Cineole Citronellal Nerol P-ionone a-santalol/./-farnesol p-santalol Manool

Figure 3.13. Recoveries of the components of the standard mixture using Isolute diol as the

trapping material (polar trap) at trap temperatures of 0,20 and 40°C at a flow rate of 1.0 mL/min.

Isolute diol was chosen as the trapping material to be used for sandalwood extractions as

quantitiative recoveries were shown at all trap temperatures. The trap temperature

chosen was 20°C, since this temperature was closest to ambient. A trap temperature of

20°C was favoured over 0°C as it minimised the amount of cryogenic carbon dioxide

required to cool the trap during the extraction process, and favoured over 40°C since the

time required to heat the trap would increase pre-run time.

There was no significant difference between the recoveries of the compounds between

flow rates of 0.5 and 2.0 mL/min at a trap temperature of 20°C on the three polar traps

(Appendices A5- A7). This showed that the interactions between the compounds and

polar sorbents were much greater than those with the non-polar sorbents, which showed

a loss in recoveries of limonene and citronellal as the flow rate was increased.

105

3.2. Desorption

The recoveries of components of the standard mixture are dependent on both the

adsorption and desorption processes. Even though compounds are trapped efficiently,

inefficient desorption from the trap will result in low recoveries. It is therefore important

to optimise the desorption conditions, such as rinse solvent, rinse volume and rinse

temperature.

The most influential parameter on the desorption of compounds from the trap is the

rinse solvent. Non-polar interactions between the analyte and the sorbent are best

disrupted by non-polar solvents and polar interactions are more effectively disrupted by

polar solvents. To examine the effect of the rinse solvent on desorption of the

components of the standard mixture from each of the six solid sorbents, five organic

solvents of various polarity were chosen. The solvents were, in order of increasing

polarity, hexane, iso-octane, methyl-tert-butyl ether (MTBE), ethyl acetate and ethanol.

The polarity index for each of these solvents is shown in Table 3.2.

Solvent

Hexane Iso-octane MTBE Ethyl Acetate Ethanol

Polarity Index

0.0 0.1 2.5 4.4 5.2

Table 3.2. Polarity indexes of solvents used for desorption of standard mixture

The standard mixture was extracted using the optimal conditions established previously

(Table 2.6), and the traps were rinsed with four successive 1.5 mL rinse volumes. The

recovery of each component was measured in each of the rinse volumes to determine the

effectiveness of each solvent on the desorption of the compounds. Optimal desorption

occurred when the compounds were desorbed from the trap by the first 1.5 mL rinse

volume. The results from the recovery of the last rinse volume were not included, as

prior to the final rinse, the SFE depressurised which invariably resulted in higher

recoveries measured in the final rinse compared to the rinse previous. The results are

again discussed in relation to the categories of trapping material (inert, non-polar, and

polar).

3.2.1. Inert Trapping Material

Using the stainless steel beads as trapping material, over 99% of the total amount of

each compound recovered was rinsed from the trap by the first 1.5mL rinse volume

(Appendix A2). The recoveries of limonene and cineole could not be determined, as

they were not trapped at 20°C.

With an inert trap, the compounds do not interact with the stationary phase, and

recoveries are determined by their solubility in the rinse solvent. The results showed that

all the compounds were soluble in each of the rinse solvents.

3.2.2. Non-Polar Trapping Material

Figures 3.14 to 3.17 (Appendices A3 and A4) show the rinsing efficiency of the non-

polar solvents (hexane and iso-octane) with the non-polar traps (Hypersil ODS and

Isolute CI8). Limonene, cineole, and citronellal were most efficiently desorbed from

both non-polar traps by the non-polar rinse solvents due to their limited ability to

interact with the silanol groups. Limonene only experiences dispersive forces with the

CI8 bonded phase, while cineole and citronellal have limited H-bonding ability

compared to alcohols. These interactions can easily be disrupted by the non-polar

solvents and these compounds are more efficiently eluted from the trap. Iso-octane was

less efficient in desorbing the compounds from the trap than hexane. This can be

107

explained to be due to more inefficient disruption of the polar interaction (H-bonds)

using iso-octane compared to hexane since the same inefficient desorption was also seen

for the more polar compounds (alcohols).

The CIO alcohol (nerol), and the CI5 alcohols (t,t-farnesol, and a- and P- santalol),

were the least efficiently desorbed compounds from both non-polar traps. These

alcohols bond more strongly to the residual silanol groups on the base silica, and

consequently, the non-polar rinse solvents did not have the ability to disrupt these polar

interactions. Again, iso-octane was less efficient at eluting these compounds from the

traps compared to hexane.

The results clearly show that p-ionone and manool, which potentially can form

hydrogen bonds with the residual silanols, were desorped more efficiently from the non-

polar traps, using the non-polar solvents, than the CIO and CI5 alcohols. The

differences in rinsing efficiencies compared to the CIO and CI5 alcohols can be

explained by structural differences, p-ionone is a ketone and forms weaker hydrogen

bonds with the silanol groups than the alcohols and are more easily disrupted by the

non-polar solvents. Manool has a large non-polar portion with a strong affinity for the

non-polar solvents and can be desorped more efficiently than the smaller alcohols.

Lower recoveries were obtained for all polar compounds from the Hypersil ODS trap

compared to the Isolute CI8 trap. This was again attributed to the carbon loading

differences between the two sorbents. The higher carbon loading percentage of the

Isolute CI8 sorbent compared to the Hypersil ODS results in a lower percentage of

residual silanols that can interact with the alcohols. The non-polar solvents therefore can

more easily disrupt the polar interactions with the Isolute C18 sorbent.

108

120

100

80

60

£

Ba. - rrtfl M*i P if fti

D 1st rinse

• 2nd rinse

D 3rd rinse

Limonene Cineole Citronellal Nerol P-ionone a-santalol /,/-farnesol P-santalol Manool

Figure 3.14. Rinsing efficiency ofhexane using Hypersil ODS

120

100

80

60

40

j

Ii I

Ei

j

I - " L , ffi*i*i, -

L rfl T_

1, «*•*, , ffl1!, ffl5!, Limonene Cineole Citronellal Nerol P-ionone a-santalolf,f-fernesol P-santalol Manool

Figure 3.15. Rinsing efficiency of iso-octane using Hypersil O D S

109

120 i

100

,-, 80 -

u 60

40

20

ifi

a i

m

fL h

O 1st rinse

Q 2nd rinse

03rd rinse

Limonene Cineole Citronellal Nerol P-ionone "-santalol U-farnesol P-santalol Manool

Figure 3.16. Rinsing efficiency ofhexane using Isolute C18

120

100

80

I 6 0 | a

40 +

20 -

*

m

m *

G 1st rinse

E2nd rinse

•J 3rd rinse

Limonene Cineole Citronellal Nerol P-ionone a-santalol t,t -farnesol P-santalol Manool

Figure 3.17. Rinsing efficiency of iso-octane using Isolute C18

The polar solvents (ethyl acetate, M T B E and ethanol) were much more efficient in

rinsing the compounds from the trap than the non-polar solvents, desorbing over 9 6 % of

the compounds in the first rinse volume (Appendices A 3 and A4). These polar solvents

were capable of disrupting the polar interactions of the compounds with the residual

silanol groups.

110

3.2.3. Polar Trapping Material

A s expected, poor recoveries from the polar traps were achieved using non-polar

solvents (Figures 3.18- 3.23, Appendices A5- A7). The less polar compounds (limonene,

cineole and citronellal) were rinsed most efficiently from the traps (>90% in the first 1.5

m L rinse volume), due to their high affinity for the solvent. Much lower rinsing

efficiencies were found for the less volatile compounds, in particular the CIO and CI5

alcohols, where three 1.5 m L rinse volumes ofhexane or iso-octane were not sufficient

to completely desorb these compounds from the trap. This is due to the inability of the

non-polar solvent to disrupt the polar interactions with both the residual silanols and

polar bonded phase. As was observed for the non-polar traps, P-ionone and manool were

more effectively desorbed from the polar traps by the non-polar solvents than the CIO

and CI5 alcohols due to weaker interactions with the polar sorbents. Again, hexane was

more efficient at eluting the compounds from the trap than iso-ocatane.

120

100

80

g 60 o o P4 40

20--

(H

fl—F • r 1

.Hi , —1*1 ,

-,-

1 m

— , — n , rl 1, rfl,

D 1st rinse

D 2nd rinse

D 3rd rinse

Limonene Cineole Citronellal Nerol p-ionone a-santalol f-farnesol P-santalol Manool

Figure 3.18. Rising efficiency ofhexane using Isolute silica

11

" 1st rinse

^ 2nd rinse

^3rd rinse

Limonene Cineole Citronellal Nerol P-ionone "-santalol t,t -farnesol P-santalol Manool

Figure 3.19. Rinsing efficiency of iso-octane using Isolute silica

120

100

80

60

40 -

20 -

- ' * ~ — 1-*-

p

J* i _ r

ri u ft J — — ) — —' 1 ' *1

• 1st rinse

• 2nd rinse

• 3rd rinse

Limonene Cineole Citronellal Nerol P-ionone a-santalol t,t -farnesol P-santalol Manool

Figure 3.20. Rinsing efficiency of hexane using Isolute cyano

112

120-.

100

80 6s-

» 60 o o Pi

40

20

0- a ifl i

5

+i A g -h Tl

Limonene Cineole Citronellal Nerol p-ionone a-santalol t,t-farnesol p-santalol Manool

Figure 3.21. Rinsing efficiency of iso-octane using Isolute cyano

D 1st rinse

• 2nd nnse

• 3rd rinse

120 -m

100

£ 80

t* 60

40

20

0 4

ii A A

i

^L

fl

L -ff JL^L Limonene Cineole Citronellal Nerol P-ionone a-santalol t,t-farnesolP-santalol Manool

• 1st rinse

• 2nd rinse

• 3rd rinse

Figure 3.22. Rinsing efficiency of hexane using Isolute diol

113

D lstnase

• 2nd rinse

D 3rd rinse

Limonene Cineole Citronellal Nerol B-ionone a-santalol t.t-farnesol B-santalol Manool

Figure 3.23. Rinsing efficiency of iso-octane using Isolute diol

Similar rinsing efficiencies with the non-polar solvents were observed between Isolute

silica and diol, both of which have - O H functionalities. Isolute cyano, which has a -CN

functionality, showed better rinsing efficiencies than Isolute silica and diol for the more

polar compounds. This reflects the weaker interactions between the cyano functional

group and polar compounds that can be more easily disrupted by a non-polar solvent.

All compounds of the standard mixture were rinsed efficiently from the three polar traps

with the polar solvents (ethyl acetate, M T B E , ethanol) (Appendices A5- A7). A recovery

of over 9 7 % the total amount recovered was rinsed from the trap in the first 1.5mL rinse

volume.

Ethanol was chosen as the rinse solvent for the extraction of sandalwood samples due to

its ability to efficiently desorb all the components of the standard mixture, low cost, and

ease of purification.

114

3.2.4. Rinse Temperatures

The desorbtion of analytes from a trap may be improved by increasing the temperature

of the trap during the rinse step. This, presumably, is due to increased solubility of the

analyte and weaker H-bonding interactions. The recovery of the components of the

standard mixture, at trap rinse temperatures of 20,40 and 60°C with ethanol as the rinse

solvent were examined (Appendices A2- A7). No significant differences in the

recoveries were seen with any of the 6 traps. It would be interesting to determine

whether rinsing efficiencies could be improved by using the non-polar solvents with the

active solid bed traps by increasing the trap temperature during the rinsing process. The

conclusion could be made however, that for rinse trap temperatures between 20 and

60°C, no loss in recovery was observed during the desorption step. A trap rinse

temperature of 40°C was chosen for the sandalwood extraction since this temperature

was closest to the nozzle temperature of 45°C.

3.3. Hydrodistillation of Standard Mixture

To examine the effect of hydrodistillation on these compounds, the standard mixture

was hydrodistilled as described in Section 2.2.4. The distillate was extracted with

MTBE since this solvent was found to recover 99% of all components of the standard

mixture in the first 50 mL extraction volume. The MTBE was removed under nitrogen.

The final recovery is dependent on the efficiency of the hydrodistillation, extraction and

evaporation procedures. To determine the effect that the extraction and evaporation

steps may have on the recoveries during the hydrodistillation methodology, a control

was used, in which the mixture was extracted from water.

115

The recoveries of the components of the standard mixture in the control and

hydrodistilled extract are shown in Figure 3.24 (Appendix A8). The control showed a

recovery of 66.6 and 74.3% for limonene and cineole respectively, indicating a loss of

these compounds during the extraction or evaporation steps. The low recoveries were

mainly attributed to losses during the evaporation of the solvent, due to the high

volatility of both limonene and cineole, since it was already known the extraction step

removed 9 9 % of all compounds in the first 50 m L extraction volume. Compounds with

boiling points greater than limonene and cineole (>174°C) in the control were recovered

quantitatively in the control.

Limonene Cineole Citronellal Nerol P-ionone a-santalol t,t-farnesol P-santalol Manool

Figure 3.24. Effect of hydrodistillation on the recovery of the standard mixture

The low recoveries of limonene and cineole observed for the control were also evident in

the hydrodistilled standard. This indicates that the losses of these compounds did not

occur during the hydrodistillation process. The other compounds, with the exception

of P-ionone and manooL afforded much lower recoveries after hydrodistillation,

presumably due to thermal degradation of the compounds at the high temperatures

116

involved in hydrodistillation. It is unlikely that the low recoveries were due to

volatilisation during hydrodistillation, since the more volatile compounds (limonene and

cineole) were relatively unaffected by hydrodistillation. Unexpectably, citronellal was

not recovered in the hydrodistilled sample. This may be due to solubility effects or

oxidation of the compound to citrobellic acid that may then react further with other

alcohols. There is also a reduction in the recovery of nerol, a- and P-santalol and t,t-

farnesol.

3.4. SFE Extraction Conditions for Sandalwood Samples

The effect of extraction conditions on sandalwood samples was examined using the

optimised collection conditions determined previously (Table 2.14). The aim was to

selectively extract the volatile components of the oil from the wood, leaving behind the

non-volatile material that has little impact on the aroma of the oil. To extract the volatile

components of the oil, the compounds must be dissolved by the supercritical CO2,

removed from the matrix, and transported to the solid bed trap for collection. These

processes are affected by a number of extraction parameters; density, temperature, mode

of extraction, flow rate, extraction time and particle size.

As the method was to be used for the extraction of a large number of samples, some of

these extraction parameters were set constant during the optimisation procedure.

Dynamic extraction was used since static extraction would substantially increase the

time involved. A C02 flow rate of 1 mL/min, which had previously been shown not to

affect the recovery of the volatile analytes with Isolute diol as the trapping material

(Section 3.1.5), was selected. An extraction temperature of 40°C was used for all

117

extractions, to minimise degradation of thermally labile compounds. Therefore only

density, extraction time, density and particle size needed to be optimised for extraction.

3.4.1. Extraction Time

Exhaustive extraction was performed on sandalwood samples to determine the time

required to completely remove the volatile oil for each density examined (0.45,0.55,

0.65, 0.75, 0.85, and 0.95 g/mL). Percentage yields, percentage volatiles, and oil

composition were measured after 10,20, 30,45, 60, and 90 minutes of extraction at

each density.

The extraction time course, graphed in Figure 3.25, shows the cumulative percentage of

the total oil extracted from sandalwood at each density (Appendix A9). The rate of

extraction of the oil increases with density. This extraction profile includes many of the

non-volatile compounds that contribute minimally to the odour of the oil and are not

eluted from the GC column during analysis. Many of these non-volatile compounds

require longer extraction times because of their lower solubility in supercritical CO2.

Since the aim was to selectively extract the volatile components of the oil, a more

appropriate illustration of the extraction time course is shown in Figure 3.26 (Appendix

A9). This shows the cumulative percentage of the total volatile components of the oil

extracted from sandalwood at each density, based on the peak area of compounds

eluting from the GC (Section 2.3.2). As can be seen, the rates of extraction were greater

than for the total oil extraction (Figure 3.25), thereby resulting in shorter extraction

times.

118

S

g> 80

u

20 40 60 80

Extraction Time (min)

100 120 140

Figure 3.25. Time course of the extraction of total oil from sandalwood at various densities

20 40 60 80

Extraction Time (min)

100 120 140

Figure 3.26. Time course of the extraction of volatile oil from sandalwood at various densities

Stahl has used a theoretical model to profile the extraction of an analyte from a solid

matrix 166 (Figure 3.27). This can be used to help explain the results obtained in the

extraction of volatile oil from sandalwood (Figure 3.26). Region I shows the initial

extraction of the analytes, which is highly dependent on the solubility of the analytes in

the extraction fluid. The more soluble the analytes are in the extraction fluid, the higher

119

the initial rate of extraction. The extraction profile for sandalwood shows that the initial

rate of extraction of the volatile compounds during Region I increased with density.

After 10 min extraction at a density of 0.45 g/mL, only 22.2% of the volatile compounds

have been extracted, compared to 84.5% at a density of 0.95g/mL. The initial extraction

of the volatile compounds during Region I at the lower densities required longer times,

due to the decreased solvent power. There appears to be very little difference however,

in the rates of extraction between densities of 0.75 and 0.95 g/mL, suggesting that the

volatile components of sandalwood oil are equally soluble in supercritical CO2 at

densities in this range.

Y (amount of analyte recovered)

Y max

I I

I 'I I '" • '

Amount of Solvent ^ Amount of Time

Figure 3.27. Theoretical extraction profile of an analyte from a solid matrix166

Region II represents the transition to diffusion controlled kinetics where the analyte-

matrix interactions must be disrupted. The rate of extraction in this region is lower than

in region I since it now is dependent on diffusion rather than solubility. In the extraction

profile for sandalwood, this region is absent at the high densities. The effectiveness of

the supercritical fluid in dissolving the compounds at high densities, may cause Region I

and II to occur almost simultaneously. Region II becomes more evident at the lower

densities where the conditions for extraction are less favourable, due to lower pressures

> i

Y

and lower solubilities. The reduced rate of extraction due to the diffusivity of the

supercritical fluid can be seen between 30 and 60 minutes at densities of 0.45 and 0.55

g/mL.

Region in represents an extremely slow rate of extraction which is diffusion limited and

reflects the limited mobility of the analyte in the matrix or difficulty in-the extraction

fluid penetrating the matrix. In this region a significant amount of time is required for

little increase in the amount of analyte recovered. In many extractions, it is not feasible

to continue the extraction into Region HI due to the high energy costs involved for

minimal increase in yield. For this reason, the extraction of sandalwood was deemed to

be quantitative when >96% of the volatile portion of the oil was removed. This occurred

after 30 minutes for densities of 0.95, 0.85, and 0.75 g/mL, 45 minutes for 0.65 g/mL,

60 minutes for 0.55 g/mL and 90 minutes for 0.45 g/mL. These conditions were used to

examine the effect of density on percentage yield, mass of volatiles and composition of

the oil.

3.4.2. Density

It is well known that solubility of a compound in CO2 is closely correlated with its

vapour pressure (or volatility)167. In a complicated model, such as sandalwood, the

relative proportion of volatile and non-volatile compounds extracted would differ with

density. It was possible to determine the relative amount of the volatile and non-volatile

compounds in each extract (volatile is defined as those compounds which would elute

from the GC column during analysis, non-volatile as those that were retained on the

column). The percentage of volatiles and non-volatiles in the extracted oil was

calculated through using an internal standard previously described in Section 2.3.2.

121

Different stationary phases of the GC column can influence the elution or retention of

many compounds. Since sandalwood is a complex mixture of compounds, the results of

the percentage volatile calculation were verified using a non-polar column (HP-5MS).

The percentage of volatiles using the polar analytical column was 29.4 ±1.7%, which

agreed within experimental error to the results from the non-polar column (26.7 ±1.9%).

The percentage yield and mass of volatiles of the oil extracted from sandalwood at the

six densities is shown in Figure 3.28 (Appendix A10). The percentage yield of oil ranged

from 1.8%, extracted at a density of 0.45 g/mL, to 4.8% at a density of 0.95 g/mL and

increased almost linearly with increasing extraction densities between 0.55 and 0.95

g/mL. The amount of volatile compounds remained constant as the density was

increased, indicating that the solubility of these compounds was similar between 0.45 and

0.95 g/mL. The overall increase in yield was due to the increase in non-volatile material

extracted as the density increased the solvation power of the supercritical C02.

3 3

I mass non-volatiles

I mass volatiles

0.95 0.85 0.75 0.65 0.55 0.45

Density (g/mL)

Figure 3.28. Yield and composition of volatiles extracted from sandalwood at various densities Mass of volatiles and non-volatiles was the mass of oil extracted assuming the mass of total oil extracted

was the percentage yield (i.e. amount extracted from 100 g of dry wood)

122

Composition

The compositions of the volatile oils extracted at each density differed primarily in the

amount of the two least volatile compounds eluting from the GC (Figure 3.29, Appendix

A10). Figure 3.29 shows the 3 most common components of Western Australian

sandalwood oil and the last two compounds eluting from the GC column identified by

their retention times. The relative amounts of the compounds were similar for extraction

densities between 0.95 and 0.65 g/mL. However, at a density below 0.65 g/mL, the

solvating power of the supercritical fluid was not sufficient to completely extract the two

late eluting compounds. A decrease in the amount of these compounds from 10.5 to

4.7%, and 4.2 to 1.2% was observed as the density decreased from 0.65 to 0.45 g/mL.

To ensure that the maximum amounts of volatile compounds were extracted, a density of

0.75 g/mL was used as the optimal density for the extraction of sandalwood volatile oil

for a time of 30 minutes.

«-bisabolol a-santalol a-farnesol 26.67

Compound or Retention Time (min)

28.35

Figure 3.29 Compositional variation in components of sandalwood oil extracted at various densities

123

3.4.3. Particle Size

The size of the matrix can have a profound influence on efficiency of extraction.

Smaller particle sizes generally leads to more rapid and complete extractions by

increasing the surface area and decreasing the diffusion path length. Grinding of the

samples can be employed to decrease the particle size of solids. Sandalwood samples

were ground in a blender in the presence of solid carbon dioxide to prevent volatilisation

and degradation of compounds by the heat generated in the grinding process.

To examine the effect of the size of the matrix on extraction, two particle size studies

were conducted. The first examined the effect of various particle size ranges (Table

2.10) on percentage yield, mass of volatiles and oil composition. Although this study

provided valuable information towards the understanding of the effect of the matrix size

on extraction it would be of no commercial benefit. Obtaining the optimal particle size

range would be labour intensive and produce wastage. A second particle size study was

therefore conducted examining the effect on percentage yield, mass of volatiles and oil

composition from wood extracted below a single particle size (Section 2.3.5). This

process required the use of only one sieve rather than a series of sieves, and much less of

the wood was wasted. This study has direct commercial benefit.

3.4.3.1. Particle Size Study 1

Extraction Time

Exhaustive extraction (Table 2.11) was performed on each particle size range, to

determine the time required to completely remove the volatile oil. The cumulative

percentage of the volatile oil extracted over a 60 minute period for each particle size

range is shown in Table 3.3. It can be seen that as the particle size decreased the rate of

extraction increased. The total amount of oil extracted in the first 15 minutes ranged

from 62.1% for a particle size >1700 um to 84.7% for a particle size range of 355-250

um. The greater amount of volatile oil initially extracted from the smaller particle size

range is due to the fact that the intraparticle diffusion resistance is less for smaller

particles because of the smaller diffusion pathways.

Extraction was considered complete when >96% of the total volatile fraction was

extracted. This occurred after 60 minutes extraction for particle size ranges between

>1700 and 850-710 urn, and 45 minutes for size ranges between 600-500 and 180-53

um. These extraction times were used for extraction of the wood, to determine the effect

of the particle size ranges on percentage yield, mass of volatiles and oil composition.

Time (min)

15

30

45

60

CTJMMULATTVE PERCENTAGE OF VOLATILE OIL EXTRACTED (%)

Particle Size Ranges (um)

>1700

62.1

83.4

93.7

100.0

1700-

1400

61.8

83.7

93.9

100.0

1400-

1180

65.6

85.8

94.6

100.0

1180-

1000

66.4

86.9

95.1

100.0

1000-

850

73.2

89.7

96.4

100.0

850-

710

73.0

89.9

96.3

100.0

710-

600

76.7

91.4

97.1

100.0

600-

500

79.3

92.6

97.5

100.0

500-

355

79.1

92.0

97.2

100.0

355-

250

84.7

94.8

98.2

100.0

250-

180

83.3

93.3

98.5

100.0

180-

53

83.8

93.7

98.2

100.0

Table 3.3. Cumulative percentage of volatile oil extracted from sandalwood of various particle sizes by SFE (Study 1)

Effect of Particle Size Distribution Range on Oil Yield and Percentage Volatiles

The percentage yield and amount of volatiles extracted using the optimal extraction

conditions (Tables 2.11 and 2.12) for each of the particle size ranges are depicted in

Figure 3.30 (Appendix Al 1). The yield increased from 1.6 to 3.7%, as the particle size

range decreased from >1700 um to 250-180 um. The increase can be seen to be the

result of an increase in both the amount of volatile and non-volatile compounds (43%

125

and 57% increase respectively). Presumably, as the particle size decreased, the extraction

fluid can more efficiently extract both a higher amount of volatile and non-volatile

compounds from the matrix due to both increased surface area and smaller internal

diffusion pathlengths.

Interestingly, decreasing the particle size range to 180-53 um, yielded lower amounts of

volatiles and non-volatiles, resulting in a lower percentage yield (3.3%). This might arise

from channelling effects due to the small particle size 117. This leads to inefficient contact

between the supercritical fluid and parts of the sample matrix, thus reducing the

percentage yield.

o o r i—i

A

o o " * •

*7 o o r--*

o 00 . — i

*7 o o •* w—t

o o o *7* o 00 .—1

—*

o W~l

00 o o o .-H

o i—i

r-o «n 00

o o NO ©

r-

o o <r> O O NO

Particle Size Range (mm)

V% «0 m o o *Ti

o «rt c-< •rt «r» d

O 00

.--, o v~t CS

o *r\ o 00 ^H

Figure 3.30. Yield and volatile composition of oil extracted from sandalwood of various particle

sizes by SFE (Study 1). Mass of volatiles and non-volatiles was the mass of oil extracted assuming the mass of total oil extracted

Composition

The percentage of each component of the oil at the particle size ranges studied is given

in Appendix Al 1. Even though the amount of volatiles extracted at the various particle

size ranges differed, the relative amounts of each compound in the extracted volatile oils

were similar.

3.4.3.2. Particle Size Study 2

Extraction Time

The cumulative percentage of the volatile oil extracted over a 60 minute period (Table

2.11) from the wood below a single particle size is shown in Table 3.4. As observed in

Study 1, the rate of extraction was higher for samples of smaller particle sizes.

Extraction was deemed complete when over 96% of the volatile oil was recovered. This

was 60 minutes for a particle size <1700 um, and 45 minutes for particle sizes <1400,

<1000, <710, and <500 um.

Time (min)

15

30

45

60

CUMMULATIVE PERCENTAGE OF VOLATILE

OIL EXTRACTED (%)

Particle Size Range (um)

<1700

67.5

88.7

96.0

100.0

<1400

78.5

92.0

97.0

100.0

<1000

80.0

91.9

97.5

100.0

<710

82.3

94.4

98.0

100.0

<500

85.0

94.9

98.0

100.0

Table 3.4. Cumulative percentage of volatile oil extracted from sandalwood of various particle sizes by SFE (Study 2)

Effect of Particle Size Distribution Range on Oil Yield and Percentage Volatiles

Each particle size range was extracted using the optimised extraction condition (Tables

2.11 and 2.12). The effect of the particle sizes below a single size on the percentage

127

yield and composition of volatiles, was less than that found in Study 1 (Figure 3.31,

Appendix A12). This was because the samples in Study 2 consisted of a much broader

range of particle sizes, with many common to each sample. The percentage yield ranged

from 2.6% for wood of particle sizes <1700pm, to a maximum of 3.9% for particles

sizes <710um (Figure 3.31). The increase in amount of oil extracted was mainly due to a

67% increase in the amount of non-volatile material. No further increase in percentage

yield was observed as the particle sizes decreased from <710 to <500um which may

again be attributed to channelling 117.

4.5

4

3.5

3

£ 2-5 2 15 ? 2

1.5

1

0.5

0

" m a s s non-volatiles

^ m a s s volatiles

T

i I •

T 1

— i — •

gta

— i — — i —

J_

— E E -

<1700 <1400 <1000 <710

Particle Size Range (Mm)

<500

Figure 3.31. Yield and volatile composition of oil extracted from sandalwood of various particle

sizes by SFE (Study 2).

Mass of volatiles and non-volatiles was the mass of oil extracted assuming the mass of total oil extracted


Composition

The percentage of each component of the oil at the particles size ranges studied are given

in Appendix A12. The relative amounts of each compound in the extracted volatile oils

were similar.

3.5. G C - M S Identification of Components in Volatile Sandalwood Oil

Sections 3.7-3.10 required the identification of as many of the components as possible

of volatile sandalwood oil, in order to examine compositional differences. Each

compound that corresponded to >1% of the total composition of the volatile oil by GC-

FID was assigned a number. The compound giving rise to the peak was identified by

GC-MS and the corresponding retention time was used for routine identification using

GC-FID. A list of the compounds identified is shown in Table 3.5. The peak number

corresponds to the peak numbers in Appendices A13- A20.

Peak N o

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

Retention Time (min)

20.79

22.66

23.57

27.82 30.52 33.09

33.42

35.21

39.74 40.03

40.41

40.59 40.84 41.89

42.24

42.63 43.12

43.21

43.38 43.61 43.71

44.28 44.52

44.80

47.02 49.13 50.35 51.51 53.32 54.11

57.12 58.22

Identified Compound

p-santalene

a-curcumene

P-farnesene dendrolasin

nerolidol

P-bisabolol

y-curcumene

a-bisabolol n.i a-santalol

Z-a-frans-bergomotol

trans, fr-a/w-farnesol n.i E-cis, epi-P-santalol

cw-P-santalol

n.i £ra«.s-P-santalol

n.i n.i n.i c&y-lanceol nuciferol

n. n. n. n. n. n.i n. n.i n.i

i

n.i

Table 3.5. Peak number, retention time and compounds of Western Australian sandalwood oil The peak number corresponds to the peak number found in Apendices A13-A20

The compounds identified are consistent with those previously identified m S. spicatum,

S. album and S. austrocaledonicum 36.37,3^1,43,45,47,48,50,52,53,57-60,62,70,168,169 Compound

13 is only found in small amounts in a few of the sandalwood trees sampled.

Compounds 18,22, 24, and 25 are all minor compounds of sandalwood oil and are

prevalent in many of the trees sampled. Interestingly, many of these compounds had not

been shown to exist in previous studies 36>37>57>58'60 The most likely structure of these

compounds would be isomeric forms of C15H26O. An important unidentified compound

was compound 26, which was present at up to 20% in some extracted oils. Brophy

had also found a compound that was tentatively identified as a C15H26O alcohol

composing 6.4% of Western Australian sandalwood oil eluting after *ran,s-P-santalol

and prior to cw-lanceol.

The late eluting compounds were not identified (compounds 29-40). These compounds

are not included in the published chemical composition of numerous West Australian

sandalwood oils 41,6°. This is important to note as this work has shown that compounds

35 and 36 often consist of up to 20% of the oils (Appendices A15 -A24). These late

eluting compounds may be isomeric forms of santalyl, bergamotyl, lancyl, or nuciferyl

acetates, previously noted to exist in S. album and S. austrocaledonicum ' . The

exclusion of these compounds in the volatile oil calculations will result in an

overestimate of the other components, and be a possible source for the variation

amongst previous studies.

3.6. Comparison of Extraction Techniques

Figure 3.32 (Appendix A13) shows the percentage yield and amount of oil extracted

from Western Australian sandalwood by hydrodistillation, SFE (CO2 density of 0.75

g/mL), and solvent extractions (hexane and ethanol). Extraction conditions are given in

130

Section 2.5. The variations in the percentage yield are mainly due to the different

amounts of non-volatile compounds extracted by each method. In comparison, the

different amounts of volatile compounds contributed minimally to the differences in the

percentage yields.

Ethanol was by far the most efficient solvent for extracting the oil from the wood, with a

percentage yield of 8.6%. Its high polarity and hydrogen-bonding properties facilitated

the extraction of a large amount of non-volatile compounds, which made up 79.5% of

the total amount of oil extracted. This resulted in the extract appearing as a heavily

viscous oil with an unattractive dark brown colour. In contrast, extraction of the wood

with the less polar solvent hexane afforded a more visually attractive, yellow flowing

oil, which contained a lower amount of non-volatile compounds (62.8%), at a

considerably lower yield (2.9%). SFE (CO2 density of 0.75 g/mL) produced a visually

similar oil, at lower percentage yield (2.1%), containing a slightly lower percentage of

non-volatile compounds (57%). This yield could be improved by extracting at a higher

density, thereby increasing the strength of the solvent, enabling more of the non-volatile

compounds to be extracted. Hydrodistillation extracted the least amount of oil (1.4%).

This extraction technique only provides those compounds that are steam volatile and the

extract consists predominately of a volatile fraction, with only 39.5% due to non-volatile

compounds. Consequently the oil is much less coloured than the samples obtained by

hexane extraction and SFE.

131

T3

9

8

7

6

5

4

3 +

2

I mass non-volatiles

I mass volatiles

1

Hvdrodistillation SFE Hexane Ethanol

Figure 3.32. Yield and composition of volatiles extraction by various extraction techniques Mass of volatiles and non-volatiles was the mass of oil extracted assuming the mass of total oil extracted


3.6.1.1. Effect o n Composition

The effect of the extraction techniques on the percentage composition of the five major

components is illustrated in Figure 3.33. The complete composition of the oil extracted

by each technique is given in Appendix A13. The most noticeable feature is the

difference observed in the percentages of a-santalol and nuciferol. A lower percentage of

a-santalol (-8%) is extracted by ethanol, and a higher percentage of nuciferol (-7%) is

extracted by hydrodistillation.

The low value of a-santalol using ethanol extraction may be partially explained by the

solvent's selectivity. As has already been shown in the study with different SFE

extraction densities, higher densities will extract greater amounts of late eluting

compounds from the GC (Figure 3.29). Since ethanol extracts more non-volatile

compounds than the other extraction techniques the relative percentage of the individual

compounds, in particular the highly volatile compounds may decrease as a result. In

Figure 3.33, four of the five major compounds with the exception of t,t-farneso\ are

132

present in lower amounts compared to the other extraction techniques. The decrease in

the amount of a-santalol however is considerably larger than for the other compounds,

which suggests other process may cause the lower percentage of a-santalol.

Interestingly, the amount of nuciferol is considerably higher in the hydrodistilled oil,

compared to the other compounds. This may arise from the conversion of enols or dienes

to the aromatic nuciferol catalysed by acid and oxygen. For example, lanceol or double

bond isomers of it could be converted to nuciferol as shown in Figure 3.34.

25 #

I 20 % "3 £ 15 o u HO

1 10 o SB a,

»1 1 1 1 , 1 J 1 |_uJ 1 —1—L-^-MS

i

J—|—L^J—1—1—1—L_

' Hydrodistillation

' SFE

' Hexane

' Ethanol

a-bisabolol a- santalol /./-farnesol P-santalol nuciferol

Figure 3.33. Compositional variation in sandalwood oil extracted by various extraction techniques

Lanceol Nuciferol

Figure 3.34. Chemical conversion of lanceol to nuciferol

133

3.7. Scale up of Extraction

The extraction of sandalwood was scaled up from a 7 m L extraction vessel (~1 g) to 300

mL (~ 100 g) to achieve a larger volume of oil. The 300 mL extraction vessel was

attached externally in a water bath for temperature control, and the SFE module

modified as described in Section 2.4.

Extraction Time Course

Exhaustive extraction was conducted on sandalwood samples to determine the time

required to completely remove the oil at various densities (0.45, 0.55, 0.65, 0.75, 0.85,

and 0.95 g/mL). The extraction method was paused without depressurisation of the

system after 100, 200, 300, 400, 500, and 800 mins and the oil collected was weighed,

and the cumulative percentage yield calculated (Figure 3.35, Appendix A14).

2.5

3 1.5

1 -

0.5 *

• — t — 100

A • •

X X

X X

200 300 400 500

Time (mins)

•

X X

A

•

• 0.45

•0.55

±0.65

X0.75

*0.85

•0.95

•+•

600 700 800 900

Figure 3.35. Time course of the extraction of total oil from sandalwood using a

300 m L extraction vessel

The amount of wood extracted increased 100 fold compared to the normal mode of

operation. As expected the time required to completely extract the oil from the wood

134

also increased. The extraction was stopped after 800 minutes. In the last 300 minutes,

25.6, 21.1, 20.7, 16.4, 14.4, and 11.4% of the total amount of oil were extracted at

densities of 0.45, 0.55, 0.65, 0.75, 0.85 and 0.95 g/mL respectively. From these results it

appears that extraction is not complete, but it must be remembered that samples

collected during the first 500 minutes did not involve depressurisation, which is required

to expel the majority of the oil from the extraction vessel and tubing.

The effectiveness of the 300 mL extraction procedure in amount of oil extracted was

measured by comparing the percentage yields at each density after 800 mins extraction

to the previously optimised extraction conditions using the 7 m L extraction vessel

(Table 2.9) illustrated in Figure 3.36. Although the results were comparable, at

densities <0.75 g/mL the percentage yield is slightly greater with the larger scale

extraction, while slightly lower at densities >0.75 g/mL. This can be explained in terms

of the different trapping devices used in each method. The custom made large scale

trapping device relies on purely physical adsorption for trapping, and can not baffle the

expanding gas flow from the restrictor. Therefore, at the higher densities, the trap may

be limited in its ability to completely precipitate the extracted analytes due to the greater

volume of expanding gas.

135

J -

2.5 •

2 -

Yield (%)

i

0.5

0

• 1 • — 1 —

1 1 | 1300 mL

l7mL

0.45 0.55 0.65 0.75 0.85 0.95

Density (g/mL)

Figure 3.36. Comparison of oil yield from 7 m L and 300 m L extraction vessel

3.8. Section of Tree

It has already been shown that the percentage yield and composition of the oil extracted

varied in different sections of the tree (roots, buttwood, and branches) ' ' ' . To

examine this in more detail, core samples were taken every 5 c m along the length of the

tree from the roots to branches. Samples were extracted using the conditions described

in Table 2.14.

The percentage yield and volatiles of the oil extracted from samples taken along the

length of three trees is shown in Figures 3.37- 3.39 (Appendices A15- A17). The results

varied from tree to tree, even though the three trees were situated within a radius of each

other of 50 m, however similar trends in oil yield were observed over the length of the

trees. In all three trees the highest amount of oil was found in the roots below ground

level (Tree 1, 5.9%; Tree 2, 6.9%; Tree 3, 5.0%). Tree 3 showed an exceptionally low

percentage yield between 15 and 10 c m below ground level, decreasing to a minimum of

0.59. The reason for this is that a grub, which infects the heartwood had infected part of

the root system.

The amount of oil extracted from the wood above ground level decreased slightly along

the length of each tree. The buttwood, which is the region of the tree from just below

ground level to 20 cm above ground level, contained a slightly higher oil content in

Trees 2 and 3, than the upper sections of the tree. This confirms previous studies 41,62'71.

The higher oil content in the roots and buttwood reflects the greater proportion of

heartwood in these sections of the tree. The roots are composed mainly of heartwood,

whereas in the stem and branches the heartwood content is variable, ranging from 90%

to negligible amounts 68. Since the heartwood contains the majority of the oil, the

sections of the tree containing the highest proportion of heartwood would contain the

highest percentage of oil. It has also been shown that, along the length of the tree, the

o

ratio of heartwood/sapwood decreases from the roots to the branches . This can explain

the decrease in percentage yield of oil in samples taken along the length of the tree.

The percentage of volatiles in the oil fluctuates considerably between 40 and 65%. No

clear conclusions could be made about the effect of the different sections of the tree on

the percentage of volatile compounds in the oil.

137

co c\2 -^ | • < ^ c \ 2 c o - ^ L r 3 c o c ^ c o a ^ c 3 ' - - ' C \ 2 c o - ^ ' L O c o r ^ c o I I I _ _ ^ _ r - . ^ _ _ _

Distance of Core Sample from Ground Level (cm)

Figure 3.37. Oil yield and percentage volatiles of sandalwood oil along Tree 1

T3

LO CO 1

o> C\2 1

lO 1

C3 lO CV2

O ^h

o CO

lO Z>-

C3

OT>

138

• l ° o yield

• — ° o volatiles

70



3.8.1.1. Effect on Composition

Figures 3.40- 3.42 (Appendices A15- A17) shows the major compositional changes in

the oil extracted from the core samples taken along the length of the three trees.

Although the composition of the oils varied between the trees, similar trends in the

changes of compounds along the trees were observed.

The most noticeable trend was the change in the percentage composition of a- and P-

santalol. In all cases the combined santalol content was greatest in the roots, with a-

santalol being the major constituent of the oil. Moving above ground level from the roots

a dramatic decrease in the amount of a- and 0- santalol was observed. The amount of

each compound decreased by more than 5 fold to below 5 % over a distance of 25 cm,

from the roots (-15 cm) to the buttwood (10 cm) in all three trees. These results

differed from those observed by Piggott , who found a-santalol to be the major

constituent (17.7%) in the buttwood. Moving further up the tree, the amount of a- and

P- santalol decreased to below 1%, in agreement with the results found by Piggott.

This sharp decrease in the amount of a- and P- santalol from the roots to the buttwood

was accompanied by subsequent increases in the amount of a- bisabolol and/or t,t-

farnesol. The relative increase in these compounds was very much dependent on the tree

examined, and for this reason the results for each tree will be discussed separately.

Tree 1 (Figure 3.40) showed that the sharp decrease in the santalol content between -10

and 10cm was accompanied by an increase in the amount of a-bisabolol. A decrease in

a- and P- santalol of 26.1% between -10 and 10 cm, corresponded to an increase in a-

bisabolol of 18.7%. The increase in t,t- farnesol (2.0%) was much less over this region.

Above ground level, a-bisabolol was the major component (~30%) of the extracted oil.

This value remained unaffected by further small decreases in a- and p- santalol,

however the amount oft,t- farnesol increased.

Results from Tree 2 (Figure 3.41) show a similar changes in the composition of the oil,

however t,t- farnesol was the major component of the oil above ground level (-35%).

The 14.7 % decrease in santalol content between -10 and 10 cm had a more pronounced

effect on the change in the percentage of t,t- farnesol than seen in Tree 1. The

percentage of t,t- farnesol increased 8.3%, while a- bisabolol increased 10.6%. As seen

in Tree 1 these compounds fluctuated along the length of the tree.

140

Tree 3 (Figure 3.42) had a very low amount of a- bisabolol compared to the other two

trees. Consequently there was little increase in the percentage of a- bisabolol moving

from the roots to above ground level where the santalol content decreased. The major

constituent of the oil above ground level, t,t- farnesol, increased by 21.8% between -15

and 10 cm and the percentage of santalols decreased by 37.1%. As the santalol content

declined below 1%, the amount of t,t- farnesol increased further and fluctuated along the

length of the tree.

There are also numerous consistent changes occurring in the composition in the minor

components of the oil along the length of the tree. Minor compositional changes

occurring in samples taken from the trees between the roots and branches are shown in

Table 3.6.

a- bisabolol

a- santalol

tt- farnesol

P- santalol

o o cs

v> '

o v~t e»

o T

v> >o

O t-

i/-> o «o O v-j O "~> 00 O —< r»> •* VO t-

Distance from ground level (cm)

Figure 3.40. Major compositional changes along Tree 1

141

451 40



-X-

a- bisabolol

a- santalol

tt- lamesol

P- santalol



Compound

Dendrolasin

a- trans- bergamotol No 20

cis- lanceol Nuciferol No 27 No 28

No 30

Dendrolasin

a- trans- bergamotol cis- lanceol No 28

No 30

Dendrolasin

a- trans- bergamotol No 20 No 30

Percentage of Compound in Roots

Percentage of

Compound in Branches

Treel

<1 3.3

11.5 4.8 4.3 8.2

3.5 3.8

4.1

<1

5.4 7.4 8.0 4.3 6.6

<1

Tree 2

<1

7.3

<1 <1 6.5

4.1

<1

6.7 6.4

<1

Tree 3

<1 6.7

6.5

7.1

10.2 <1

2.1 <1

Table 3.6. Minor compositional changes along the three trees

In summary, the following was observed for all three trees;

• A sharp decrease in the amount of santalols from the roots to the butwood

• Increases in the amount of a- bisabolol and t,t- farnesol as the santalol content

decreased

• A decrease in a- trans- bergamotol, No 20, 27, and 30 along the length of the tree

• An increase in dendrolasin, cis- lanceol and No 28 along the length of the tree

These results are of some interest when considered together with the biosynthesis of

sandalwood sesquiterpenes. It is worthwhile noting that the majority of the known

sesquiterpenes in sandalwood are isoprenologues of widely occurring monoterpenes. In

other words, the third isopreniod unit does not take part in the elaboration of the carbon

skeleton (Figure 3.43). The acyclic (t,t- farnesol, nerolidol, p- farnesene) and

143

monocyclic (a- and P- bisabolol, a- and y- curcumene, cis- lanceol, nuciferol) can be

considered to arise by simple processes from farnesyl diphosphate. The formation of the

bicyclic (P-santalene, epi-P- santalol, cw-P-santalol, Z-a-frvms-bergamotoi) and tricyclic

(a- santalol) sesquiterpene requires rearrangements of carbonium ions (Figure 3.44).

This second pathway can be considered to reflect a more evolved process.

In view of this, it is not surprising that the composition of oil obtained from the higher

reaches of the trees (younger portion of the plant) is characterised by the presence of the

simpler acyclic and monocyclic sesquiterpenes. The roots and more mature hardwood

are capable of more elaborate biosynthetic processes which results in sesquiterepenes

that are unique to sandalwood.

Presuming these hypothetical pathways are correct, the precursors to some of the

unidentified compounds in Table 3.6 can be assumed. As compounds No 20,27 and 30

decrease along the length of the tree, it is assumed that the biosynthesis of these

compounds follow the pathway shown in Figure 3.44 and would be closely related to the

structure of the santalols. Compound No 28 however, since it decreases along the length

of the tree would presumably be synthesised using the pathway described in Figure 3.43,

its structure would be acyclic or monocyclic.

144

PPO

PPO

a-curcumene Z-nuciferol

a-farnesene

epi-a-bisabolol

p-bisabolol

cis-lanceol

y-curcumene

Figure 3.43. Hypothetical derivation of acyclic and monocyclic sesquiterpenes in sandalwood

145

R1 = H;p-santalene R1 = OHp-santalol

a-santalol

Figure 3.44. Hypothetical derivation of bicyclic and tricyclic sesquiterpenes from the bisabolonium cation in sandalwood

146

Cross Sectional Variation

Variation in Oil Content and Percentage Volatiles

To examine the variation in oil content and composition across the diameter of the three

trees, a 5 cm section of the tree was removed between 5 and 10 cm above ground level.

The cross section was sampled as described in Section 2.6.3.3. The results showing the

changes in percentage yield and volatiles across the tree are shown in Figures 3.45- 3.47

(Appendices A18- A20). The sampling positions across each of the trees are shown in

Figure 3.48.

Figure 3.48 also shows the light coloured sapwood surrounding the darker inner

heartwood. Generally, the outer positions of the cross section contained the least amount

of oil. This is best seen in tree 3, which has a greater proportion of sapwood compared

to the other trees. The percentage yield of oil from the sapwood at positions 1 and 6 was

below 0.4%. An exception to this can be seen in tree 2, where the oil content at position

7 was greater than at position 4. The lower percentage yield found at position 4 was due

to heartwood rot, caused by infection of a grub. This can also be seen by the split across

position 4 in tree 3 (Figure 3.48). Areas of heartwood rot are evident in the centre of the

heartwood in tree 1 and tree 3, which made sampling difficult.

Apart from the areas of heartwood affected by heartwood rot, the percentage yield of oil

across the heartwood remained essentially similar. Values ranged from 5.1-6.9% (tree

1), 5.5-7.6% (tree 2), and 4.8-5.3% (tree 3). This shows that the heartwood oil content is

unaffected by the distance across the tree. Further evidence to support this can be seen

from Figure 3.44, where the percentage yield of oil from position 7 for tree 2 was 6.3%.

Figure 3.46 shows that only a thin layer of sapwood surrounds the heartwood, and the

sample from position 7 consisted predominately of heartwood. Even though this

147

position was on the outer of the tree, the yield was comparable to that from the inner

heartwood.

I°o yield

•°o volatiles

1 2 3 4 5 6

Sampling Position

Figure 3.45. Variation in oil yield and percentage volatiles across the diameter of Tree 1

~ 5

a 4 -

O 3

• • " o vie Id

- • — ° o volatiles

3 4 5

Sampling Position


148

3 4 5

Sampling Position

I °o yield

•°o volatiles


Tree 1

Tree 2

Heartwood Rot

Heartwood Rot

/l 2 3 4 5 6 )m

Heartwood Rot

Tree 3

Figure 3.48. Sampling positions across the diameter of the three trees

149

Composition

Figures 3.49- 3.51 (Appendices A18- A20) shows the variation on the 4 major

components of sandalwood across the diameter of the three trees. Trees 1 and 3 show

little variation (<7%) in a-bisabolol, and a- and P-santalol across the diameter of the

tree. Greater variation was noted for in the amounts of ?,/-farnesol (10.9%, treel; 14.2%,

tree 3). It appears that the relative percentage of t, /-farnesol was greater on the outside

of the tree in the sapwood regions than the inner heartwood.

The results from tree 2 are interesting. At position 4, where the oil yield was significantly

lower due to heartwood rot, the amount of a-bisabolol increased 6 fold and was devoid

of a- and P-santalol. This suggests the operation of a 'phytoalexin' response. It is well

known that plants challenged by biotic or abiotic factors, can respond by diverting their

metabolism to the formation of defence compounds 170. If this is so, it indicates that a-

bisabolol might have some biological activity towards the grub infection.

3 4

Sampling Position

-•— u- bisabolol

- • — a- santalol

tt- farnesol

- X — P- santalol

Figure 3.49. Major compositional changes across the diameter of Tree 1

150

a- bisabolol

a- santalol

tt-farnesol

P- santalol

3 4 5

Sampling Position


— K -

" a- bisabolol

i i

tt- farnesol

_ P- santalol

1 2 3 4 5 6 7

Sampling Position


3.9. Geographic Variation

S. spicatum is distributed over an area of 161 million hectares throughout Western

Australia ( W A ) 19. Brand has found genetic differences to exist between S. spicatum

ecotypes 171172. It is possible that some populations of sandalwood contain better sources

of genes for heartwood development, oil production, and oil composition arising from

these genetic differences. Considering the importance of both oil yield and

composition in relation to commercial viability, it is surprising that no extensive studies

have been conducted examining variations in oil yield and composition from different

geographic locations in Western Australia.

Geographic Variation in Oil Yield and Volatile Composition

Core samples were taken from numerous sandalwood trees from 12 geographic

locations throughout Western Australia and extracted by SFE as previously described

(Section 2.6.1.3). The percentage yield and santalol content of all the trees samples are

given in Table 3.7 (the total composition of the extracted volatile oil from each tree and

percentage volatiles is given in Appendix A21). There was found to be a great deal of

tree to tree variation. The oil yields and santalol contents ranged from 0.82 to 6.05 %,

and 0 to 84.6 % respectively. The average oil yield from the 87 tree sampled was 2.86 ±

1.14 % % which is significantly greater than the reported 2% average of S. spicatum 13.

The average santalol content was 24.89 ± 23.24 %. As can be seen from the large

standard deviation, much more variation occurred between the santalol contents than the

oil yields. Although the average was low compared to the reported values of santalol in

S. album, it was encouraging to find that 10 of the 87 trees sampled contained santalol

contents above 60 %.

Differences in the oil yield and percentage volatiles due to the geographic location were

determined through measuring the average oil yield and percentage volatiles from each

of the 12 geographical locations (Figures 3.52 and 3.53). Trees from Katanning contain

the highest average amount of oil (4.6%). This value was similar to the 4.8% yield

reported from the SFE of East Indian Sandalwood 73'74. Trees from Wanjarrie, Shark

Bay and Mulgul, afforded the next highest average yields (3.8, 3.5, and 3.4%). The

lowest average yields (2.0%) were found in trees from Jaurdie and Burnerbinmah.

152

Sample

Wan

jarrie 1.1.1

1.1.2

1.1.3

1.1.4

1.2.1

1.2.2

1.23 1.3.1 1.3.2

1.33 Mulgul

1.4.1

1.4.2

1.43 1.5.1 1.5.2

1.53 Gas-coyne Junction 1.6.1 1.6.2

1.63 1.6.4

1.6.5

Shark Bay

1.7.1

1.7.2

1.73 1.7.4

1.8.1

1.8.2

1.83 Murch-ison

1.9.1

1.9.2

1.9.3

1.10.1

1.10.2

1.103

Yield (%)

2.42

1.12

3.74

3.26

3.81

3.91

5.59

3.54

2.56

5.77

2.67

5.07

3.09

1.58

4.69

3.12

1.84

1.70

2.54

2.60

1.81

3.42

3.54

3.07

3.75

2.66

3.32

4.76

3.40

2.21

1.15

2.38

3.62

2.92

Santalol

(%)

15.71

33.60

46.64

60.94

39.58

37.57

11.40

31.31

48.53

55.35

73.31

64.77

65.38

54.96

50.91

68.98

40.08

28.55

10.56

30.24

53.18

62.43

84.62

70.64

76.72

55.43

43.02

73.51

6.00

38.16

7.54

2.19

6.96

15.66

Sample

Burner-binmah

1.11.1

1.11.2

1.11.3

1.11.4

1.12.1

1.12.2

1.12.3

1.13.1

1.13.2

1.133 Mt Elvire

1.14.1

1.14.2

1.143 1.14.4

1.15.1

1.15.2

1.153

1.16.1 1.16.2

1.16.3

Goongarrie

1.17.1

1.17.2

1.17.3

1.18.1

1.18.2

1.18.3

1.19.1

1.19.2

1.19.3

Yield

(%)

2.30

0.82

1.22

1.60

3.07

2.30

1.38

1.82

2.73

2.62

2.07

2.79

3.75

2.46

4.19

4.51

2.34

1.63

1.83

1.30

2.77

4.42

2.00

2.78

2.94

2.05

3.23

3.77

4.00

Santalol

(%)

2.82

0 0 2.03

2.00

10.90

12.12

2.07

0 0

3.39

9.97

10.56

21.61

11.34

10.61

21.00

23.46

17.58

31.73

6.36

24.20

26.07

21.74

41.12

16.34

3.75

10.68

17.67

Sample

Bullock

Holes

1.20.1

1.20.2

1.20.3

1.21.1

1.21.2

1.213

1.22.1

1.22.2

1.22.3

Katanning

1.23.1

1.23.2

1.23.3

1.23.4

1.23.6

Ning-han

1.24.1

1.24.2

1.24.3

1.24.4

1.24.5

Jaurdie

1.25.1

1.25.2

1.25.3

1.26.1

1.26.2

1.26.3

Yield

(%)

1.98

2.50

2.71

2.31

2.27

1.98

4.32

2.69

"2.16

4.83

4.52

6.05

3.22

4.50

1.14

2.25

3.64

3.53

1.93

2.48

1.03

2.97

2.08

1.96

1.49

Santalol

(%)

4.83

20.41

36.21

2.41

9.49

7.25

17.39

69.22

55.37

14.65

9.66

5.39

16.06

29.60

2.30

3.70

11.94

8.70

3.56

1.36

1.36

0.81

5.02

24.90

2.89

Table 3.7. Oil yield and santalol content of trees sampled in geographical survey Dimensions and locations of trees are given in Table 2.17

153

The trees with the greatest yields also contained the highest percentage volatiles. The

sandalwood oil from MulguL Shark Bay, Katanning and Wanjarrie contained average

volatile percentage compositions of 66.5, 65.9, 63.4, and 60.4% respectively. In fact, a

strong linear correlation was found between the percentage yield and percentage volatiles

(p=0.000, rM).538).

1 3 bo f-s

" 2 xn S3

2 3

o

s e? PQ

S3 O

J3

.g <s fi g

a 3

m

2 » 00

s o g o

X/l

o

o o "3

00

a 'S £

S St .13 00

c

Figure 3.52. Variation in the oil content of sandalwood trees from various geographic locations

St

3 _op

2

S a >>.2 en S3 si 3 03

a o .13 a

•3 a

e 3 03

">

3 St 00 S3

o o O

OS

O

J4

o 3 CO

00 S3 S3

1 W

E en J3 00 S3

3 St

Figure 3.53. Variation in the percentage volatiles of sandalwood trees from various geographic locations

154

One-way analysis of variance (ANOVA) revealed significant differences for the oil yield

and percentage volatiles (p=0.000) between geographic locations. It can be seen from

the large standard deviations in Figures 3.52 and 3.53 that there was large variation

within locations. Similar variability was seen in the genotype, measured by allele

frequencies at 3 polymorphic loci in S. spicatum from 8 locations 171. The variation

however maybe random, and due to the small sample sets from each location. Fisher's

pairwise comparisons were performed on the data and locations to determine which

locations differed significantly. The results from these tests are shown in Figures 3.54

and 3.55. The identification numbers corresponding to each location used in the Figures

3.54 and 3.55 are shown in Table 3.8.

2 3 4 5 6 7 8 9 10 11 12

1 n X

0

X

X

X

0

X

0

X

X

?.

0

0

0

X

0

0

0

X

0

X

3

X

0

0

0

0

0

X

0

0

4

0

X

0

0

X

X

0

X

5

0

0

0

0

X

0

0

a

0

X

0

X

0

0

7

0

0

X

0

0

8

0

X

0

X

Q

X

0

0

10

X

X

11

0

Figure 3.54. Fisher's pairwise comparison for statistical differences in oil yields between locations (o= no significant difference: x= significant difference)

1 3 4 5 6 7 8 9 10 11 12

1 n 0

0

X

X

X

X

0

0

X

X

?

X

0

X

X

X

X

X

0

X

X

1

X

0

0

0

0

0

X

0

0

4

X

X

X

X

X

o X

X

5

0

0

o 0

X

0

0

a

0

0

0

X

0

0

7

0

0

X

0

0

8

0

X

0

X

q

0

X

X

m

X

X

11

0

Figure 3.55. Fisher's pairwise comparison for statistical differences in percentage volatiles between locations

(o= no significant difference: x= significant difference)

Identification Geographic Location Number

1 Wanjarrie 2 Mulgul

3 Gascoyne Junction 4 Shark Bay 5 Murchison

6 Burnerbinmah 7 M t Elvire

8 Goongarrie 9 Bullock Holes 10 Katanning 11 Ninghan 12 Jaurdie

Table 3.8. Identification numbers corresponding to the geographic locations used in Fisher's pairwise comparisons (Figures 3.54 and 3.55, and 3.61 to 3.64)

Brand found there was genotypic and phenotypic variation in sandalwood trees from

different locations, and the genetic distance between ecotypes increased linearly with

increased geographical distance 171,172. No such linear relationship was found between

percentage yield and percentage volatiles with geographic distance. Therefore, rather

than geographic location being the cause of the observed differences, other factors such

as environmental conditions may influence the amount of oil and volatiles found within

a location.

Differences in climatic conditions have been shown to cause variation amongst the

171

phenotypic characteristics of sandalwood such as nut size, leaf size and growth rate

The rainfall and temperature from each location are given in Figure 3.56. Trees with the

highest percentage yields from Katanning also have the highest average annual rainfall

(395 mm) and coolest climate (22.7°C). However, there does not appear to be a

correlation between the percentage yields and rainfall or temperature for the other

locations that experience an average annual rainfall and temperature between 213 and

294 mm, and 25.2 and 32.0°C. There also appears to be no correlation between

percentage volatiles and these environmental conditions.

156

450

100

50

0

•Rainfall

35

30

25 3

1 15 ha

20 | H

15

10

3 8 a CO 3

<U CU C« tlQ

IS

s-~ CO tan c o o o

o 3C ^£ o

3 D3

Figure 3.56. Average rainfalls and temperatures for sandalwood sampling locations

A number of other factors may contribute to the variation observed between locations.

Although care was taken to choose trees of similar size, the maturity of a tree is

determined by the formation of heartwood rather than size72. It is generally considered

that heartwood content tends to be higher for a given size tree in lower rainfall

conditions 13'35. It is therefore possible that trees from arid regions may reach maturity at

sizes far smaller than in areas of higher rainfall, which may bias the results from these

regions as trees of similar sizes were chosen for sampling. The proportion of heartwood,

and hence, the maturity of the trees could not be determined by the sampling method

used.

Another factor that may contribute to the variation is disease. Heartwood rot is caused

by the infection of the heartwood with a grub. This results in hollow pockets within the

heartwood, as was noticed during the core sampling of some trees. The effect this has on

the yield is not known, but it seems reasonable to suggest that it would decrease the

heartwood/sapwood ratio, and decrease the yield. Also as has previously been shown in

157

Figure 3.50, heartwood rot can cause considerable variation in the composition of the oil

extracted from the infection area.

Other factors such as soil type, host species and genetics may be responsible for

variations yet are outside the scope of this thesis.

3.9.1.1. Geographic Variation in Oil Composition

The average percentage composition of the 5 major compounds of the volatile oil of S.

spicatum from each of the 12 locations is shown in Figures 3.57- 3.60. The amounts of

a- and p-santalol were added to give the total santalol content. One-way A N O V A

showed significant differences to exist between locations for a- and P-santalol, t,t-

farnesol and nuciferol (p=0.00). Fisher's pairwise comparisons were performed on the

data and locations to determine which locations differed significantly. The results from

these tests are shown in Figures 3.61 and 3.64. The identification numbers

corresponding to each location used in the Figures 3.61 and 3.64 are shown in Table 3.8.

N o significant difference was found between a-bisabolol from different locations

(p=0.062), which was due to the large standard deviations relative to the means.

158

a- santalol

(3- santalol

in 11 m 60

^

J3 00

•S

Figure 3.57. Variation in the percentage santalol from sandalwood trees from various geographic

locations

30 -,

Figure 3.58. Variation in the percentage a-bisabolol from sandalwood trees from various

geographic locations

159

Figure 3.59. Variation in the percentage /,f-farnesol from sandalwood trees from various

geographic locations

2

Figure 3.60. Variation in the percentage nuciferol from sandalwood trees from various geographic locations

Of most interest is the santalol content, since this determines the quality and value of the

oil. Previous studies have shown that the santalol content of S. spicatum ranges between

7 and 82%38'41. These large variations may be due to a number of environmental factors

as well as different sampling, extraction and analytical techniques between studies. In

160

this study, the same sampling, extraction and analytical techniques on a large number of

trees were used, with the advantage that direct comparisons between results could be

made.

The average santalol content from each location was shown to vary considerably (3.3%

to 66.6%). The trees with the highest santalol content were found in Shark Bay, closely

followed by trees from Mulgul (63.0%). There was no relationship between the oil yield

and santalol content. The high oil yielding trees from Katarming and Wanjarrie had

santalol contents of 15.1 and 38.6% respectively. There was also no evidence of a

relationship between the santalol content and climatic data (temperature and rainfall).

This suggests that other factors such as genetic differences may cause the variation

amongst trees from different locations.

2 3 4 5 6 7 8 9 10 11 12

1 T

o X

X

X

X

X

X

X

X

X

2

X

0

X

X

X

X

X

X

X

X

7,

X

0

X

o 0

o 0

X

X

4

X

X

X

X

X

X

X

X

5

o o 0

0

0

0

0

a

X

X

X

0

0

o

7

0

o o o o

8

o o 0

0

Q

0

X

X

in

o o

11

o

Figure 3.61. Fisher's pairwise comparison for statistical differences in a- santalol between locations (o= no significant difference: x= significant difference)

2 3 4 5 6 7 8 9 10 11 12

1 •x

o X

X

X

X

X

X

X

X

X

2

X

0

X

X

X

X

X

X

X

X

3

X

0

X

0

0

0

0

0

0

4

X

X

X

X

X

X

X

X

5

0

o 0

0

0

0

o

6

X

X

X

o 0

o

7

0

o 0

0

o

8

0

0

0

0

Q

0

X

X

in

0

o

11

0

Figure 3.62. Fisher's pairwise comparison for statistical differences in P- santalol between locations (o= no significant difference: x = significant difference)

2 3 4 5 6 7 8 9 10 11 12

i n o 0

0

0

0

0

X

X

0

X

2

0

0

0

0

0

0

X

X

X

X

1

0

0

0

0

0

X

X

0

X

4

o 0

X

X

X

X

X

X

5

0

0

0

X

X

X

X

a

X

X

X

X

X

X

7

0

0

0

0

o

8

0

X

0

o

0

0

0

0

in

0

o

11

o

Figure 3.63. Fisher's pairwise comparison for statistical differences in t,t- farnesol between locations

(o= no significant difference: x= significant difference)

2 3 4 5 6 7 8 9 10 11 12

1 n O

0

X

X

X

0

0

X

X

X

2

X

0

X

X

X

X

o X

X

X

3

X

o X

o o 0

0

X

0

4

X

X

X

X

0

X

X

X

5

o 0

X

X

0

X

o

a

0

X

X

X

X

X

7

0

X

0

X

0

8

0

0

X

0

0

X

X

X

in

X

o

11

X

Figure 3.64. Fisher's pairwise comparison for statistical differences in nuciferol between locations (o= no significant difference: x= significant difference)

Results in Section 3.8 showed that along the length of the tree, in particular just above

and below ground level, the santalol content decreased significantly, accompanied by an

increase in the amount of a-bisabolol, ^-farnesol and nuciferol. Since core sampling of

the trees was performed 10 cm above ground level, many of these compositional

changes may be occurring at this position within the tree. Moreover, locations that are

subjected to greater erosive forces may have a ground level much closer to the roots

than in other locations, which may result in higher santalol contents. This may explain

the higher santalol content found at Shark Bay. These trees were the only ones in the

study situated on the coast in sand dunes. Since sand dunes themselves are a product of

erosion, the topsoil in this location is likely to be more prone to erosion than the more

compact loamy soil found in more typical locations.

162

A strong linear relationship was found between the amount of a- and P- santalol in each

tree sampled (p=0.000,1^=0.988), showing that the ratio of a- to p- santalol is consistent

in the oil extracted from all 87 trees.

Of particular interest, were the relationships between the amounts of the five major

compounds found in the extracted oil from each tree. Regression analysis showed strong

negative correlation between the santalols and a- bisabolol, U-farnesol and nuciferol

(pO.OO), as those trees with highest santalol contents typically contained the lowest

amounts of a- bisabolol, £f-farnesol and nuciferol and vice versa. This supports the

presence of two biosynthetic pathways (Figures 3.43 and 3.44), which may be regulated

by certain feedback mechanisms.

3.10. Seasonal Variation in Oil Yield and Volatile Composition

Variations in percentage yields of oils have been measured in the leaves from eucalypts

1 TX 1 *7A.

and geranium during different seasons ' . The percentage yields were lowest in the

dryer warmer months and highest in the wetter cooler months. No such variation was

found in the amount of oil and volatiles from sandalwood from 5 locations (Figures 3.64

and 3.65, Appendices A22- 24) using two-way ANOVA (p>0.05). This may be due to

the fact that essential oil in the sandalwood is situated in the heartwood, whereas the oil

of eucalypts and geranium is present in the leaves. Since the leaves are found at the

extremities of the plant, and are the structures of the plant used in photosynthesis,

environmental conditions such as temperature and sunlight, which vary with season,

will have a greater impact on the formation and loss of oil.

163

D Spring

• Summer

•Autumn

•Winter

Wanjarrie Mt Elvire Goongarrie Bullock Holes Katanning

Figure 3.64. Variation in oil yield from 5 locations sampled during 4 different seasons

D Spring

• Summer

• Autumn

• Winter

Wanjarrie Mt Elvire Goongarrie Bullock Holes Katanning

Figure 3.65. Variation in percentage volatiles from 5 locations sampled during 4 different seasons

It is important to bear in mind that the effect of the core sampling procedure is not

known. Since the same trees were sampled four times over the period of 18 months, the

damage to the tree may have affected results. Moreover, the core samples for each

season were taken from different regions of the tree. Samples were taken 5 cm above the

previous sample point, which after four samples spans a length of 20 cm. As discussed in

Section 3.7, the percentage yield and percentage volatiles can vary considerably over

such a distance.

3.10.1.1.Seasonal Variation in Oil Composition

Two-way ANOVA showed no significant differences in the amount of a- bisabolol, a-

and p-santalol, t,t- farnesol, or nuciferol between the seasons.

4. Conclusion

Optimal trapping and extraction conditions were established for the extraction of

sandalwood oil from Western Australian sandalwood. The trapping conditions, were

optimised by measuring the recovery of a standard mixture representative of

sandalwood oil. Results showed that the polar trapping materials more effectively

trapped these compounds. Poorer recoveries of the more volatile compounds (limonene

and citronellal) were observed on the inert and non-polar traps, however decreasing the

trap temperature and flow rate in some instances was able to improve the recoveries of

these compounds.

Once trapped the compounds must be eluted from the trapping material by a rinse

solvent. The ability of numerous solvents of various polarity to desorb the components

of the standard mixture from the traps was determined. It was found that the polar

solvents (ethyl acetate, MTBE, and ethanol), were capable of desorbing each compound

from all the traps by the first 1.5 mL rinse volume. This was because the polar solvents

were more effective in disrupting the interactions between the compounds and the

sorbents. The non-polar solvents (hexane and iso-octane) were less efficient at

desorbing the more polar compounds from the active traps. Poor rinsing efficiencies

were observed from the non-polar traps due to the ability of the analytes to hydrogen

bond to the residual silanol groups of the solid supports.

Optimal trapping conditions were with Isolute diol as the trapping material at a

temperature of 20°C and CO2 flow rate of 1 mL/min. Ethanol provided an easily

available, cheap rinse solvent. These conditions were used to examine the effect of the

extraction parameters on the percentage yield, percentage volatiles and oil composition

of the oil extracted by SFE from sandalwood.

An increase in the extraction density was shown to increase the percentage yield. This

increase was mainly due to an increase in the amount of non-volatile material extracted,

as the amount of volatile oil extracted remained constant over the density range

examined (0.45 to 0.95 g/mL). Over this density range, the composition of the volatile

oil was found to differ in only the two least volatile compounds, which are currently not

identified, whose amounts decreased as the density fell below 0.65 g/mL. Hence 0.75

g/mL was chosen as the optimal density to completely extract the volatile oil from

sandalwood. The identification of the compounds present in the non-volatile oil would

be a focus of future work.

The particle size of the wood matrix was shown to have an effect on the percentage

yield and percentage volatiles, but it had no effect on the composition of the extracted

volatile oil. Decreasing the particle size increased in part the amount of non-volatile

compounds extracted, and also to a lesser extent the volatile compounds, thereby

increasing the overall percentage yield. This was thought to be due to the increased

surface area and smaller internal diffusion pathlengths as the particle sizes decreased.

The sandalwood oil extracted using the optimised SFE conditions was compared to the

oil extracted using hydrodistillation and solvent extraction. The percentage yield of total

oil extracted by SFE was comparable to that extracted by hydrodistillation and hexane

extraction. A much greater percentage yield was extracted using ethanol, however the

oil was visually unattractive. The composition of the volatile oils varied slightly with

hydrodistillation extracting a greater amount of nuciferol and ethanol a lower amount of

santalols.

The SFE and GC methods developed served as fast, robust, routine techniques for the

extraction of large numbers of sandalwood samples. These methods were used to

examine variation in the oil extracted every 5 cm along the length of 3-trees from the

roots to the branches. The oil content was highest in the roots below ground level and

decreased along the length of the tree. A greater santalol content was also found in the

roots, which decreased 5 fold in the wood above ground level. This sharp decrease was

accompanied by a significant increase in the amount of a- bisabolol and t,t- farnesol.

These results are thought to be due to differences in the biosynthetic pathways occurring

in the more mature roots and less mature stems of the trees. Further studies are required

to validate the biosynthetic pathways occurring within sandalwood.

The major commercial focus of the work was to examine variations in the sandalwood

oil extracted from trees found in 12 different geographic locations throughout Western

Australia. Oil content, percentage volatiles, and oil composition were all found to vary

between locations. An attempt was made to correlate these differences with temperature

and rainfall variations between locations without success. No correlation could be

established. These results can serve as a starting point to further examine variables that

may cause variation in the oil amongst sandalwood trees, such as soil type, host species

and ecotypes.

168

Recommendations for Future Work

Many of the results from the trapping studies were influenced by the residual silanol

groups on the solid supports, in particular the non-polar supports. A greater

understanding of the interactions of the non-polar bonded phase could be achieved by

using end-capped supports, where the residual silanol groups are replaced by non-polar

functionalities.

With the current core sampling technique used, no differentiation could be made

between the heartwood and sapwood. The results therefore reflect what is found in both

the heartwood and sapwood. For instance, the results showing the decrease in oil yield

along the length of the tree maybe due to the decreasing proportion of heartwood. A

sampling technique that can distinguish heartwood from sapwood would be able to

assess changes only occurring on the heartwood.

Advantages would also be gained from the development of a completely non-evasive

technique for the sampling of volatile oil composition. As it is unknown as to the effect

of core sampling on the tree, this would better serve as a comparison in the volatile oil

composition between seasons on a longer term. Such techniques could include solid

phase microextraction (SPME), where the volatile compounds of the oil could be

adsorbed onto a fibre positioned within close proximity to the tree in the field, and

brought back to the lab for analysis by GC. Another more expensive technique could be

through the use of a portable micro-GC that could be used in the field which samples the

volatile oil directly from the tree.

The results showing the variation in oil content, percentage volatiles and oil

composition from different geographical locations can serve as a starting point to

169

examine variables that may cause these variations. The focus of further work would

involve elemental analysis of the soil, to find if any correlations exist. This could be

closely linked to the elemental analysis of the wood, which may also have an influence

on oil variation.

Another possible source of variation could be due to differences in the-genetic make up

of the trees. These studies would examine any relationships between differences in DNA

sequence or DNA expression and oil variation. If a particular genotype was found to

produce a better quality oil, this may lead to the cloning of trees. Along the same lines, it

would be interesting to see if the seed collected from the better trees would also produce

a similar quality oil.

5. References

(1) Linskens, H. F.; Jackson, J. F. In Essential Oils and Waxes; Linskens, H.

F., Jackson, J. F., Eds.; Springer- Verlag, 1991; p VII.

(2) Hay, R. K. M.; Svoboda, K. P. In Volatile Oil Crops; Hay, R. K. M.,

Waterman, P. G., Eds.; Longman Scientific and Technical, 1993; pp 1-4.

(3) Bauer, K.; Garbe, D. In Common Fragrance and Flavor Materials;

VCH, 1985; pp 113-115.

(4) Koedam, A. In Capillary Gas Chromatography in Essential Oil Analysis;

Bertch, W., Jennings, W. G., Kaiser, R. E, Eds.; Huethig, 1987; pp 13- 27.

(5) Deer, T. In Australian Essential Oils Industry Seminar; 1993;

pp 59-66.

(6) Ohloff, G. In Scent and Fragrances : The Fascination of Odors and

Their Chemical Perspectives; Springer-Verlag, 1994.

(7) Radomiljac, A. M.; Borough, C. J. Australian Forest Grower

1995,19, 1.

(8) Jiko, L. R. In Sandalwood in the Pacific; Hamilton, L., Conrad, C. E.,

Eds.; Pacific Southwest Research Station: 1991; USDA Forest Service General

Technical Report PSW-122, pp 13-18.

(9) Daruhi, G. In Sandalwood in the Pacific Region; McKinnell, F. H., Ed.;

ACIAR, 1991; pp 26-29.

(10) Brennan, P.; Merlin, M. In Sandalwood in the Pacific Region;

McKinnell, F. H., Ed.; ACIAR, 1991; pp 30-37.

(11) Statham, P. "The Australian sandalwood trade- Small but significant,"

Department of Economics, The University of Western Australia, 1987.

(12) Statham, P. In Sandalwood in the Pacific; Hamilton, L., Conrad, C. E.,

Eds.; 1990; USDA Forest Service General Technical Report PSW-122, pp 26-38.

(13) Applegate, G. B.; Chamberlain, J.; Dahuri, G.; Feigelson, J. L.;

Hamilton, L; McKinnell, F. H.; Neil, P. E.; Rai, S. N; Rodehn, B.; Statham, P. C;

Stemmermann, L. In Sandalwood in the Pacific; Hamilton, L., Conrad, C. E., Eds.;

1990; USDA Forest Service General Technical Report PSW-122, pp 1-11.

(14) Kealley, I. G. Wildlife Management Program 1991, 8.

(15) Loneragan, O. W. Historical Review of Sandalwood (Santalum

Spicatum) Research in Western Australia; Department of Conservation and Land

Management: Perth, 1990; Vol. Research Bulletin No 4.

(16) Kealley, I. G. Landscope 1989,4, 35.

(17) Merlin, M.; VanRavenswaay, D. In Sandalwood in the Pacific; Hamilton,

L., Conrad, C. E., Eds.; 1990; USDA Forest Service General Technical Report PSW-

122, pp 46-59.

(18) Applegate, G. B.; Davis, A. G. W.; Annable, P. A. In Sandalwood in the

Pacific; Hamilton, L., Conrad, C. E., Eds.; 1990; USDA Forest Service General

Technical Report PSW-122, pp 12-18.

(19) Sawyer, B.; Jones, P. "Western Australian Sandalwood Resource

Statement 2000," Department of Conservation and Land Management, 2000.

(20) Applegate, G. B.; McKinnell, F. H. In Sandalwood in the Pacific Region;

McKinnell, F. H., Ed.; ACIAR; 1991; pp 5-12.

(21) www. agric. wa.gov. au/progserv/natural/trees/treecorp/sandalw2.

(22) Fox, J. E. D. Journal of the Royal Society of Western Australia 1997, 80,

209.

(23) Struthers, R.; Lamont, B. B.; Fox, J. E. D.; Wijesuriya, S.; Crossland, T.

Journal of Experimental Botany 1986, 37, 121 A.

http://wa.gov

(24) Talbot, L. Forest Focus 1983, 30, 21.

(25) www.mtromance.com.au.

(26) Srinivasan, V. V.; Sivaramakrishnan, V. R.; Rangaswamy, C. R.;

Ananthapadmanabha, H. S.; Shankaranarayana, K. S. In Sandal; Indian Council of

Forestry Research and Education, 1992; pp 1-2.

(27) Valluri, J. V. Biotechnology in Agriculture and Forestry 1994, 28, 400.

(28) Chourasia, O. P.; Nigam, S. S. Indian Perfumer 1978, 22, 206.

(29) Dikshit, A.; Husain, A. Fitoterapia 1984, LV, 171.

(30) Garia, A. K.; Varma, R. G. Acta Ciencia Acta 1990,16, 327.

(31) Benencia, F.; Courreges, M. C. Phytomedicine 1999, 6, 119.

(32) Banerjee, S.; Ecavade, A.; Rao, A. R. Cancer Letters 1993, 68, 103.

(33) Varshney, S. C. Indian Perfumer 1992, 36, 147.

(34) Osmani, Z.; Anees, L; Naidu, M. B. Pesticides 1972, 6, 19.

(35) Srinivasan, V. V.; Sivaramakrishnan, V. R.; Rangaswamy, C. R.;

Ananthapadmanabha, H. S.; Shankaranarayana, K. S. In Sandal; Indian Council of

Forestry Research and Education:, 1992.

(36) Ehrhart, Y. "Oil Composition of the Sandalwood (Santalum

austrocaledonicum) from Erromango and Aniwa Islands, Vanuatu," CIRAD, 1998.

(37) Brunke, E.-J.; Rojahn, W. DRAGOCO Report 1980, 5,127.

(38) Guenther, E. S. The Drug and Cosmetic Industry 1943, 53, 270.

(39) Penfold, A. R. Journal and Proceedings of the Royal Society of New

South Wales 1928, 62, 60.

(40) Penfold, A. R. Journal and Proceedings of the Royal Society of New

South Wales 1932, 66.

(41) Piggott, M. J.; Ghisalberti, E. L.; Trengove, R. D. Flavour and

Fragrance Journal 1997,12, 43.

http://www.mtromance.com.au

(42) Pande, B. S. Chemical Industry Digest 1996,19, 83.

(43) Demole, E.; Demole, C; Enggist, P. Helvetica Chimica Acta 1976,59,

737.

(44) Yadav, V. G. Pafai Journal 1993, 21.

(45) Brunke, E.-J. DRAGOCO Report 1981, 4/5, 91.

(46) Verghese, J.; Sunny, T. P.; Balakrishnan, K. V. Flavour and Fragrance

Journal 1990, 5.

(47) Christenson, P. A.; Secord, N.; Willis, B. J. Phytochemistry 1981,20,

1139.

(48) Brunke, E.-J.; Schatkowski, D.; Struwe, H.; Tumbrink, L. Bergamotol

and Spirosantalol- New constituents of East Indian Sandalwood oil; Elsevier: New

York, 1986; Vol. 18.

(49) Brunke, E., -J; Fahlbusch, H.-G.; Schmaus, G.; Volhardt, J. Riv. Ital.

EPPOS1997,15,48.

(50) Adams, D. R.; Bhatnagar, S. P.; Cookson, R. C. Phytochemistry 1975,

14,1459.

(51) Shankaranarayana, K. H. Indian Perfumer 1979,23,65.

(52) Anonis, D. P. Perfumer and Flavorist 1998, 23, 19.

(53) Brunke, E.-J.; Vollhardt, J.; Schmaus, G. Flavour and Fragrance

Journal 1995,10,211.

(54) Kar, P. C. Aromi Saponi Cosmetici Aerosol 1973, 55, 847.

(55) Kretschmar, H. C; Barneis, Z. J.; F, E. W. Tetrahedron Letters 1970, 37.

(56) Nikiforov, A.; Jirovetz, L.; Buchbauer, G.; Raverdino, V. Mikrochimica

Acta 1988, //, 193.

(57) Brunke, E.-J.; Schmaus, G. DRAGOCO Report 1995, 6,245.

(58) Brunke, E.-J.; Schmaus, G. DRAGOCO Report 1995, 5, 197.

(59) Birch, A. J.; Mostyn, K. M. C; Penfold, A. R. Australian Journal of

Chemistry 1953, 6, 391.

(60) Brophy, J. J.; Fookes, C. J. R.; Lassak, E. V. Journal of Essential Oil

Research 1991, 3, 3U.

(61) Rao, B. S.; Sudborough, J. J. Journal of the Indian Institute of Science

1923, 5, 60.

(62) Piggot, M. J.; West Australian sandalwood (Santalum Spicatum): Part

(I): Comparison of essential oil extraction techniques Part (II): Isolation and

identification of some polar compounds; Thesis; University of Western Australia: Perth,

1995.

(63) Jayappa, V.; Nataraj, B. M.; Shanbhag, P. K.; Patil, K. B.; Srinvas, A.

Pafai Journal 1981, 3, 27.

(64) Radomiljac, A.; Clews, M. Landscope 1996,11,23.

(65) Hirano, R. T. In Sandalwood in the Pacific; Hamilton, L., Conrad, C. E.,


(66) Radomiljac, A. Sandalwood Research Newsletter 2000, 9, 4.

(67) McKinnell, F. H. In Sandalwood in the Pacific; Hamilton, L., Conrad, C.

E, Eds.; 1990; USDA Forest Service General Technical Report PSW-122.

(68) Rai, S. N. In Sandalwood in the Pacific; Hamilton, L., Conrad, C. E.,


(69) Radomiljac, A. M.; McComb, J. A. In Sandal and Its Products;

Radomilijac, A. M., Ananthanapadmanabho, H. S., Welbourn, R. M., Satyanarayana

Rao, K., Eds.; ACIAR; 1997, pp 54-59.

(70) Shankaranarayana, K. H.; Parthasarathi, K. Indian Perfumer 1987, 31,

211.

(71) Brand, J. E.; Shea, S. R.; Spadek, T. Sandalwood Research Newsletter

1999, 8, 1.

(72) Shankaranarayana, K. H.; Parthasarathi, K. Indian Perfumer 1984, 28,

138.

(73) Naik, S. N.; Maheshwari, R. C; Maheswari, M. L. Indian Perfumer

1988, 32, 74.

(74) Naik, S. N.; Lentz, H.; Maheshwari, R. C. Fluid Phase Equilibria 1989,

49,115.

(75) Naqvi, A. A.; Mandal, S. Indian Perfumer 1995, 39, 62.

(76) Rao, R. V.; Hemavathi, T. R.; Sujatha, M.; Chauhan, L; Raturi, R. In

Sandal and Its Products; Radomilijac, A. M., Ananthanapadmanabho, H. S., Welbourn,

R. M., Satyanarayana Rao, K., Eds.; ACIAR; 1997, pp 93-102.

(77) John, M. D.; Paul, T. M.; Jaiswai, P. K. Indian Perfumer 1991, 35, 186.

(78) Shankaranarayana, K. H.; Parthasarathi, K. Research and Industry 1984,

29, 204.

(79) Baiswara, R. B.; Nair, K. N. G.; Mathew, T. V. Research and Industry

1915,20,213.

(80) Franz, C. In Volatile Oil Crops; Hay, R. K. M., Waterman, P. G., Eds.;

Longman Scientific & Tehnical, 1993; pp 63-96.

(81) Stahl, E.; Quirin, K., -W; Gerard, D. In Dense Gases for Extracting and

Refining; Springer-Verlag, 1988; p 11.

(82) Luque de Castro, M. D.; Valcarcel, M.; Tena, M. T. In Analytical

Supercritical Fluid Extraction; Springer-Verlag, 1994; p 43.

(83) Zosel, K. German Patent 1,493,190,1964.

(84) Taylor, L. T. In Supercritical Fluid Extraction; John Wiley and Sons,

1996; pp 1-6.

(85) Gere, D. R.; Derrico, E. M. LC-GC1994,12, 353.

(86) Chester, T. L.; Pinkston, J. D.; Raynie, D. E. Analytical Chemistry 1994,

66, 106R.

(87) Bowart, S.; Hawthorn, S. B. Journal of Chromatography 1995, 703, 549.

(88) Liu, Y.; Lopez-Avila, V.; Alcaraz, M.; Beckert, W. F. Journal of High

Resolution Chromatography 1993,16, 106.

(89) Moyler, D. A. In Developments in Food Flavours; Birch, G. G.; Lindley,

M. G; Elsvier Applied Science, 1986, pp 119-129.

(90) Reverchon, E.; Ambruosi, A.; Senatore, F. Flavour and Fragrance

Journal 1994, 9, 19.

(91) Terauchi, F.; Ohira, T.; Yatagai, M.; Ohgama, T.; Aoki, H.; Suzuki, T.

Mokuzai Gakkaishi 1993, 39, 1421.

(92) Bevan, C. D.; Marshal, P. S. Natural Products Research 1994, 451.

(93) Levy, J. M.; Danielson, V.; Ravey, R.; Dolata, L. LC-GC 1994,12, 920.

(94) Lembke, P.; Engelhardt, H. Chromatographia 1993, 34, 509.

(95) Braybrook, J., H; MacKay, G. A. Polymer Interantional 1992, 27, 157.

(96) Kueppers, S. Chromatographia 1992, 33, 434.

(97) Via, J.; Braue, C; Taylor, L. T. Journal of High Resolution

Chromatography 1993,16, 279.

(98) Brunner, G. In Gas Extraction: An Introduction to Fundamentals of

Supercritical Fluids and the Application to Separation Processes; Springer, 1994; p 5.

(99) Stahl, E; Quirin, K., -W; Gerard, D. In Dense Gases for Extracting and

Refining; Springer-Verlag, 1988; pp 12-13.

(100) Knipe, C. R.; Miles, W. S.; Rowland, F.; Randall, L. G. In Designing a

Sample Preparation Method That Employs Supercritical Fluid Extraction; Hewlett-

Packard Company, 1993; pp 25-28.


1996; p 103.

(102) Vannoort, R. W.; Chervet, J. P.; Lingeman, H.; deJong, G. J.; Brinkman,

V. A. T. Journal of Chromatography 1990, 505, 45.

(103) Hawthorne, S. B.; Miller, D. J.; Nivens, D. E.; White, D. C. Analytical

Chemistry 1992, 64,405.

(104) Hawthorne, S. B.; Krieger, M. S.; Miller, D. J. Analytical Chemistry

1988, 60,472.

(105) Dionex, C. "SFE method development guidebook," Dionex, 1993.

(106) Lou, X. W.; Janssen, H. G.; Cramers, C. A. Journal of Microcolumn

Separations 1995, 7, 303.

(107) Marin, M. L; Jimenez, A.; Lopez, J.; Vilaplana, J. Journal of

Chromatography 1996, 750, 183.

(108) Luque de Castro, M. D.; Valcarcel, M.; Tena, M. T. In Analytical

supercritical Fluid Extraction; Springer-Verlag, 1994; pp 123-124.


1996; p 102.

(110) Reverchon, E.; Donsi, G.; Osseo, S. Industrial Engineering and

Chemical Research 1993, 32, 2721.

(111) Smith, R. D.; Udseth, H. R. Separation Science and Technology 1983,

18,245.

(112) Jenkins, T. J.; Kaplan, M.; Simmonds, M. R.; Davidson, G.; Healy, M.

A.; Poliakoff, M. The Analyst 1991,116,1305.

(113) Mira, B.; Blasco, M.; Bema, A.; Subirats, S. Journal of Supercritical

Fluids 1999,14, 95.

(114) Tena, M. T.; Valcarcel, M.; Hidalgo, P. J.; Ubera, J. L. Analytical

Chemistry 1997, 69, 521.


1996; p 115.

(116) Gere, D. R.; Derrico, E. M. LC-GC 1994,12, 432.


1996; p 116.

(118) Dionex Dionex 1992, Technical Note 202.

(119) Husers, N.; Kleibobmer, W. Journal of Chromatography 1995, 697, 107.

(120) Thompson, P. G.; Taylor, L. T. Journal of High Resolution

Chromatography 1994,17, 759.

(121) Thompson, P. G.; Taylor, L. T.; Richter, B. E.; Porter, N. L.; Ezzell, J. L.

Journal of High Resolution Chromatography 1993,16, 713.

(122) Eckard, P. R.; Taylor, L. T. Journal of High Resolution Chromatography

1996,19,111.

(123) Mulcahey, L. J.; Hedrick, J. L.; Taylor, L. T. Analytical Chemistry 1991,

63, 2225.

(124) Mulcahey, L. J.; Taylor, L. T. Analytical Chemistry 1992, 64,2352.

(125) Li, K; Fingas, M. F.; Balanger, J. M. R.; Pare, J. R. J. In 12th Technical

Seminar on Chemical Spills, 1995; pp 173-189.

(126) Daimon, H.; Hirata, Y. Journal of Microcolumn Separations 1993, 5,

531.

(127) Yang, Y.; Hawthorne, S. B.; Miller, D. J. Journal of Chromatography

1995, 699, 265.

(128) Anderson, M. R.; Swanson, J. T.; Porter, N. L.; Richter, B. E. Journal of

Chromatographic Science 1989, 27, 371.


1996; p 68.

(130) Knipe, C. R.; Miles, W. S.; Rowland, F.; Randall, L. G. In Designing a

Sample Preparation Method That Employs Supercritical Fluid Extraction; Hewlett-

Packard Company, 1993; pp 87-88.

(131) Chialva, F.; Gabri, G.; Liddle, P. A. P.; Ulian, F. Journal of High

Resolution and Chromatography Communications 1982, 5, 182.

(132) Hawthorne, S. B.; Riekkola, M.-L; Serenius, K.; Holm, Y; Hiltunen, R.;

Hartonen, K. Journal of Chromatography 1993, 634, 297.

(133) Chyau, C. C; Mau, J. L.; Wu, C. M. Journal of Agricultural and Food

Chemistry 1996, 44, 1096.

(134) Boelens, M. N.; Boelens, H. Perfumer andFlavorist 1997, 22, 31.

(135) Adasoglu, N.; Dincer, S.; Bolat, E. The Journal of Supercritical Fluids

1994, 7, 93.

(136) Moyler, D. A. Flavour and Fragrance Journal 1993, 8, 235.

(137) Reverchon, E.; Senatore, F. Flavour and Fragrance Journal 1992, 7,

227.

(138) Moyler, D. A.; Heath, H. B. Liquid Carbon Dioxide Extraction of

Essential Oils; Elsevier, 1986; Vol. 18.

(139) Pickett, J. A.; Coates, J.; Sharpe, F. R. Chemistry and Industry 1975, 5

July, 571.

(140) Koedam, A.; Looman, A. Journal of Medicinal Plant Research 1980,

Supplement, 22.

(141) Molero Gomez, A.; Pereyra Lopez, C; Martinez de la Ossa, E. The

Chemical Engineering Journal 1996, 61, 227.

(142) Moyler, D. A. In Extraction of Natural Products Using Near-Critical

Solvents; King, M. B., Bott, T. R., Eds.; Blackie Academic & Professional, 1993; pp

140-183.

(143) Sanders, N. In Extraction of Natural Products Using Near-Critical

Solvents; King, M. B., Bott, T. R., Eds.; Blackie Academic and Professional, 1993; pp

34-49.

(144) Modey, W. K.; Mulholland, D. A.; Raynor, M. W. Phytochemical

Analysis 1996, 7,1.

(145) Hawthorne, S. B.; Miller, D. J.; Krieger, M. S. Journal of

Chromatographic Science 1989, 27, 347.

(146) Caragay, A. B.; Little, A. D. Perfumer andFlavorist 1981, 6, 43.

(147) Fischer, M.; Jefferies, T. M. Journal of Agricultural and Food Chemistry

1996, 44,1258.

(148) Song, S. Q.; Ashley, D. L. Analytical Chemistry 1999, 71, 1303.

(149) Gardner, D. S. In Extraction of Natural Products Using Near-Critical

Solvents; King, M. B., Bott, T. R., Eds.; Blackie Academic & Professional, 1993; pp 84-

100.

(150) Lack, E.; Seidlitz, H. In Extraction of Natural Products Using Near-

Critical Solvents; King, M. B., Bott, T. R., Eds.; Blackie Academic & Professional,

1993; pp 101-139.

(151) Amnion, D. G.; Barton, A. F. M.; Clarke, D. A.; Tjandra, J. Analyst

1985,110,921.

(152) Birtigh, A.; Johannsen, M.; Brunner, G. The Journal of Supercritical

Fluids 1995, 8,46.

(153) Morita, S.; Yamada, T.; Hidaka, T. Mokuzai Gakkaishi 1995, 41,237.

(154) Smith, R. M. LC-GC 1995,12, 930.

(155) Reverchon, E.; Delia Porta, G.; Senatore, F. Journal of Agricultural and

Food Chemistry 1995, 43, 1654.

(156) Masada, Y. Analysis of Essential Oils by Gas Chromatography and

Mass Spectrometry; John Wiley & Sons, Inc., 1976.

(157) McMaster, M.; McMaster, C. GC/MSA Practical User's Guide; Wiley-

Vch:, 1998.

(158) Ragunathan, N.; Sasaki, T. A.; Krock, K. A.; Wilkins, C. L. Analytical

Chemistry 1994, 66, 3751.

(159) Mundina, M.; Vila, R.; Tomi, F.; Ciccio, J. F.; Ibanez, C; Adzet, T.;

Casanova, J.; Canigueral, S. Flavour and Fragrance Journal2000,15, 201.

(160) Hay, R. K. M.; Svoboda, K. P. Volatile oil crops; Longman Scientific

and Technical:, 1993; pp 57-60.

(161) Wolfender, J. L.; Ndjoko, K.; Hostettmann, K. Current Organic

Chemistry 1998, 2, 575.

(162) He, X. G. Journal of Chromatography 2000, 880, 203.

(163) Funada, Y; Hirata, Y. Journal of Chromatography A 1998, 800, 317.

(164) Dugo, P.; Mondello, L.; Dogo, G.; Heaton, D. M.; Bartle, K. D.; Clifford,

A. A.; Myers, P. Jounal of Agricultural Food Chemistry 1996, 44, 3900.

(165) Blum, C; Kubeczka, K.; Becker, K. Journal of Chromatography A 1997,

773, 377.

(166) Stahl, E.; Quirin, K., -W; Gerard, D. In Dense Gases for Extracting and

Refining; Springer-Verlag, 1988; p 6.

(167) Stahl, E.; Quirin, K, -W; Gerard, D. In Dense Gases for Extracting and

Refining; Springer-Verlag, 1988; pp 1-3.

(168) Alpha, T.; Raharivelomanana, P.; Bianchini, J.-P.; Faure, R.; Cambon, A.

Phytochemistry 1997, 46, 1237.

182

(169) Alpha, T.; Raharivelomanana, P.; Bianchini, J.-P.; Faure, R.; Cambon, A.

Phytochemistry 1997, 44, 1519.

(170) Stipanovic, R. D.; Bell, A. A.; Benedict, C. R. In Biologically Active

Natural Products; Cullen, H. G., Cutler, S. J., Eds.; CRC Press, 1999; pp 211-220.

(171) Brand, J. E. Mulga Research Centre Journal 1993,11, 1.

(172) Brand, J. E. Mulga Research Centre Journal 1993,11,13.

(173) Rajeswara, B. R.; Kaul, P. N.; Mallavarapu, G. R.; Ramesh, S.

Biochemical Systematics and Ecology 1996, 24, 627.

(174) Rafique, M.; Chaundry, F. M. Pakistan Journal of Science and Idustrial

Research 1999, 42, 282.

6. Appendix

Table of Appendix

Al Extraction time course ofstandardniixture at 0.30, 0.65, and 0.95 g/mL 184

A 2 Recoveries of standard mixture from stainless steel beads trap 185

A 3 Recoveries of standard mixture from Hypersil O D S trap 186

A 4 Recoveries of standard mixture from Isolute CI 8 trap : 187

A 5 Recoveries of standard mixture from Isolute silica trap 188

A 6 Recoveries of standard mixture from Isolute cyano trap 189

A 7 Recoveries of standard mixture from Isolute diol trap 190

A 8 Recoveries of standard mixture following hydrodistillation 191

A 9 Extraction time course of total and volatile sandalwood oil at various densities 191

A 1 0 Yield, volatiles and composition of sandalwood oil extracted at various densities 192

A l 1 Yield, volatiles and composition of sandalwood oil extracted using various particle size ranges

(Study 1) 193

A 1 2 Yield, volatiles and composition of sandalwood oil extracted using various particle size ranges

(Study 2) 195

A13 Yield, volatiles and composition of sandalwood oil extracted using different extraction

techniques 196

A14 Cumulative oil yield extracted at various densities over 800 minutes using large-scale SFE

apparatus 197

A15 Yield, volatiles and composition of sandalwood oil extracted every 5 cm along length of tree 1.. 198

A16 Yield, volatiles and composition of sandalwood oil extracted every 5 c m along length of tree 2.. 203

A 1 7 Yield, volatiles and composition of sandalwood oil extracted every 5 c m along length of tree 3.. 208

A18 Yield, volatiles and composition of sandalwood oil extracted across diameter of tree 1 213



A21 Yield, volatiles and composition of sandalwood oil extracted from tress from various geographic

locations (samp 1 ed during spring) 216


locations (sampled during autumn) 226


locations (sampled during winter) 230


locations (sampled during summer) 235

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extraction and variation of the essential oil from western

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