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AN EVALUATION OF SUSTAINABLE SUB– AND
SUPERCRITICAL FLUID EXTRACTION TECHNOLOGIES
AND TRADITIONAL SOXHLET FOR THE ISOLATION OF
HIGH QUALITY, NATURAL LAVANDULA spp. FLAVOURS
Amanda Edda Daluiso
Degree of Master of Science
Department of Food Science and Agricultural Chemistry
McGill University
Montreal, Quebec, Canada
April, 2015
A thesis submitted to McGill University in the partial fulfillment of the
requirements for the Degree of Master of Science
© Amanda Edda Daluiso 2015
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Abstract
Growing interest in high quality, natural foods and beverages by consumers who
are increasingly more concerned with their health and well-being has driven the
food industry to explore natural flavours in product development and
reformulations. Recognized for its valuable aromatic oil by the fragrance industry,
lavender continues to garner great interest for its use as a natural flavouring
substance in beverages, confectionery, baked goods, and dairy products by the
food industry. In this study, critical fluid technology and classical extraction
techniques were assessed for their capacity to generate high quality, natural
Lavandula extracts. The quality of these extracts was evaluated by investigating
the chemical composition of each isolate and the mobilization of select
Lavandula quality related compounds, linalyl acetate, linalool and camphor, using
gas chromatography- mass spectrometry (GC-MS). Over thirty compounds of
North American lavender Lavandula Angustifolia ‘Royal Velvet’ and lavandin
Lavandula x Intermedia ‘Grosso’ cultivated in Sequim, Washington, USA were
characterized by GC-MS from extracts obtained by means of Soxhlet,
supercritical carbon dioxide extraction (SFE-CO2) and subcritical water extraction
(SWE). Linalool and linalyl acetate were determined as the main quality
indicators of the SFE-CO2 extracts while linalool was the key character impact
compound of SWE within the Lavandula spp. cultivars. The highest yield was
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determined from Soxhlet due to the co-extraction of high molecular weight
compounds such as waxes. SFE-CO2 (1-3% w/w) and SWE (2-4% w/w) yields
agreed well with those found in literature for essential oils. SWE delivered
extracts rich in isolated oxygenated compounds. These extracts may have useful
applications in other industries. In terms of high Lavandula flavour quality, the
best extracts were those of lavender obtained with SFE-CO2. SFE performed at
low pressure of 2000 psi was recommended as optimal operational conditions.
SFE-CO2 is a promising sustainable alternative for the generation of high quality
lavender extracts for application in the flavour industry as a natural ingredient.
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Résumé
L'intérêt croissant porté aux aliments et boissons naturels et de haute qualité par
les consommateurs qui sont de plus en plus préoccupés par leur santé et bien-
être a poussé l'industrie alimentaire à explorer les saveurs naturelles dans le
développement et reformulation de produits. Reconnu pour sa précieuse huile
aromatique par l'industrie de la parfumerie, la lavande continue d'attirer un grand
intérêt pour son utilisation comme substance aromatisante naturelle dans les
boissons, la confiserie, les produits de boulangerie et les produits laitiers par
l'industrie alimentaire. Dans cette étude, la technologie de fluide critique et les
procédés d'extraction classiques ont été évalués pour leur capacité à générer
des extraits naturels et de haute qualité de Lavandula spp.. La qualité de ces
extraits a été évaluée en étudiant la composition chimique de chaque isolat et la
mobilisation des composés de qualité de Lavandula sélectionnés, l'acétate de
linalyle, linalol et le camphre, par spectrométrie de masse-chromatographie en
phase gazeuse (GC-MS). Plus de trente composés de lavande Lavandula
angustifolia 'Velvet Royale' et lavandin Lavandula x intermedia
'Grosso' d'Amérique du Nord cultivé à Sequim, Washington, Etats-Unis ont été
caractérisés par GC-MS à partir d'extraits obtenus au moyen de Soxhlet,
extraction avec dioxyde de carbone supercritique (SFE-CO2) et extraction par
eau sous-critique (SWE). Linalol et acétate de linalyle ont été déterminés les
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principaux indicateurs de qualité dans les extraits SFE-CO2 bien que linalol était
le composé à caractère d'impact clé du SWE dans les espèces de Lavandula. Le
rendement le plus élevé a été déterminé par Soxhlet en raison de la co-
extraction de composés de poids moléculaire élevé tels que des cires. Les
rendements par SFE-CO2 (1-3% p/p) et SWE (2-4% p/p) sont en bon accord
avec ceux trouvés dans la littérature pour les huiles essentielles. SWE a livré des
extraits riches en composés oxygénés isolés. Ces extraits peuvent avoir des
applications utiles dans d'autres industries. En termes de qualité de la saveur de
Lavandula, les meilleurs extraits étaient ceux de lavande obtenu par SFE-CO2.
SFE effectuée à une basse pression de 2000 psi a été recommandé comme
conditions opérationnelles optimales. SFE-CO2 est une alternative durable
prometteuse pour la production d'extraits de lavande de haute qualité pour
application dans l'industrie des arômes comme un ingrédient naturel.
5
Table of Contents
LIST OF TABLES .......................................................................................................................................... 7
LIST OF FIGURES ........................................................................................................................................ 8
LIST OF ABBREVIATIONS ........................................................................................................................... 9
Statement from the Thesis Office ................................................................................................................ 10
Contribution of Authors ................................................................................................................................ 13
DEDICATION ............................................................................................................................................... 14
Chapter 1 Introduction ................................................................................................................................. 15
1.1 General Introduction ............................................................................................................................. 15
1.1.1 Commonly studied Lavandula species in research .................................................................... 15
1.2 Secondary metabolites in plants ........................................................................................................... 17
1.2.1 Chemical constituents found in Lavandula spp. ........................................................................ 19
1.3. Biological activities of the genus Lavandula ......................................................................................... 20
1.3.1. Flavour and Aroma impact ....................................................................................................... 21
1.3. 2 Toxicity studies .......................................................................................................................... 23
1.4. Lavandula and its applications ............................................................................................................. 23
1.4.1 Lavandula and food ................................................................................................................... 24
1.5 Quality characteristics of Lavandula spp. extractives .......................................................................... 25
1.6. Methods of isolation of natural flavour constituents from plants ....................................................... 28
1.6. 1 Critical fluid technology ............................................................................................................ 29
1.6.1. 1 Supercritical Fluid Extraction (SFE) ........................................................................................ 29
1.6. 1. 2 Subcritical water extraction (SWE) ....................................................................................... 32
1.7. Instrumental Analysis of Flavours ........................................................................................................ 33
Rationale and Objectives of the Proposed Research .................................................................................. 35
Chapter 2 Literature Review ........................................................................................................................ 37
2.1. Literature review on solvent extraction of Lavandula species using Soxhlet method......................... 37
2.2 Sub- and supercritical fluid extraction for the isolation of Lavandula spp. natural products............... 37
2.2.1 Literature review on SFE of Lavandula species ......................................................................... 38
2.2.2 Literature review on SWE of Lavandula species ........................................................................ 40
Chapter 3 Method Development of the Proposed Research ...................................................................... 42
3.1 Materials ................................................................................................................................................ 42
3.1.1 Plant material ............................................................................................................................. 42
6
3.1.2 Chemicals and reagents ............................................................................................................. 43
3.2 Extraction Methods ............................................................................................................................... 44
3.2.1 Soxhlet ....................................................................................................................................... 44
3.2.2 Supercritical fluid extraction using carbon dioxide (SFE-CO2) ................................................... 46
3.2.3 Subcritical water Extraction (SWE) ............................................................................................ 53
3.3 Analytical Procedure ............................................................................................................................. 59
3.3.1 Gas Chromatography Mass spectrometry (GC-MS)................................................................... 60
3.3.2 Fourier transform infrared spectroscopy .................................................................................. 62
3.4 Statistical Analysis ................................................................................................................................. 63
Chapter 4 Chemical composition and characterization of mobilized Lavandula spp. constituents
via selected conventional method and critical fluid technology ................................................................... 64
4.1 Qualitative studies of mobilized Lavandula spp. analytes via GC-MS ................................................... 64
4.1.1 Linear retention index (LRI) determination ............................................................................... 64
4.1.2 Electron ionization (EI) mass spectra of volatile target constituents ........................................ 67
4.2 Chemical composition of Lavandula spp. extract ................................................................................. 75
4.2.1 Soxhlet Method ......................................................................................................................... 75
4.2.2 SFE-CO2 ...................................................................................................................................... 77
4.2.3 SWE ............................................................................................................................................ 83
Chapter 5 Evaluation of extraction yield of Lavandula spp. isolates obtained by conventional
method and critical fluid technology ............................................................................................................ 86
5.1 Yield studies of Lavandula spp. extracts ............................................................................................... 86
5.1.1 Soxhlet Method ......................................................................................................................... 86
5.1.2 SFE-CO2 ...................................................................................................................................... 87
5.1.3 SWE ............................................................................................................................................ 91
Chapter 6 Study of selected quality parameters of the Lavandula spp. extracts obtained by
conventional method and critical fluid technology ....................................................................................... 93
Chapter 7 General Conclusions .................................................................................................................. 95
REFERENCES ............................................................................................................................................ 98
Appendix A ................................................................................................................................................ 106
7
LIST OF TABLES
Percentage of major constituents of lavender, lavandin, and spike lavender oils ......................... 20
Odour impact of selected Lavandula spp. terpenes ...................................................................... 22
Physical properties of fluid states .................................................................................................. 30
Classification of the Lavandula spp. samples ............................................................................... 42
Properties of solvents permitted for use in flavour extraction ....................................................... 46
Retention time of alkanes and bromo-alkanes for LRI determination ........................................... 65
Physicochemical properties of selected Lavandula spp. constituents .......................................... 66
Percentage yields obtained via Soxhlet using ethanol or hexane as solvent ............................... 88
Percentage yield of Lavandula spp. obtained via SFE-CO2 at various experimental conditions .. 88
Percentage yield of Lavandula spp. obtained via SWE at various extraction temperatures ......... 88
Summary of the classification of tentatively identified constituents ............................................. 106
Chemical Composition of Lavandula spp. extract obtained via Soxhlet...................................... 107
Chemical Composition of Lavender extract obtained via SFE-CO2 ........................................... 111
Chemical Composition of Lavandin extract obtained via SFE-CO2 ............................................ 115
Chemical Composition of Lavender extract obtained via SWE ................................................... 119
Chemical Composition of Lavandin extract obtained via SWE ................................................... 123
8
LIST OF FIGURES
Structural representation of important quality marker monoterpenes of Lavandula spp............... 27
Laboratory built supercritical fluid extraction system ..................................................................... 50
Laboratory built subcritical water extraction (SWE) system .......................................................... 56
Mass spectrum of linalyl acetate from NIST library (above) and component determined by GC-MS
(below) ........................................................................................................................................... 70
Mass spectrum of linalool from NIST library (above) and component determined by GC-MS
(below) ........................................................................................................................................... 71
Mass spectrum of camphor from NIST library (above) and component determined by GC-MS
(below) ........................................................................................................................................... 72
Mass spectrum of eucalyptol from NIST library (above) and component determined by GC-MS
(below) ........................................................................................................................................... 73
Mass spectrum of borneol from NIST library (above) and component determined by GC-MS
(below) ........................................................................................................................................... 74
Total ion count chromatogram of Lavender extract obtained via SFE-CO2 at 2000 psi and 80°C 80
FTIR spectra of co-extracted wax-like material (SFE-CO2; 3000psi and 60°C) ........................... 82
ATR-IR spectra of co-extracted wax-like material (SFE-CO2; 3000psi and 60C) ........................ 82
A 3D response surface plot of yields obtained via SFE-CO2 of lavender with respect to pressure
and temperature ............................................................................................................................ 90
A 3D response surface plot of yields obtained via SFE-CO2 of lavandin with respect to pressure
and temperature ............................................................................................................................ 90
9
LIST OF ABBREVIATIONS
CO2 Carbon dioxide
DDW Distilled de-ionised water
EI Electronic ionization
FTIR Fourier transform infrared spectroscopy
GC Gas Chromatography
LRI Linear retention index
MS Mass spectrometer
Rt Retention time
SFE Supercritical Fluid Extraction
SFE- CO2 Supercritical Carbon Dioxide Extraction
SWE Subcritical Water Extraction
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Statement from the Thesis Office
In accordance with the regulations of the Faculty of Graduate Studies and
Research of McGill University, the following statement from the Guidelines for
Thesis Preparation is included:
Candidates have the option of including, as part of the thesis, the text of one or
more papers submitted, or to be submitted, for publication, or the clearly-
duplicated text of one or more published papers. These texts must conform to the
“Guidelines for Thesis Preparation” and must be bound together as an integral
part of the thesis.
The thesis must be more than a collection of manuscripts. All components must
be integrated into a cohesive unit with a logical progression from one chapter to
the next. In order to ensure that the thesis has continuity, connecting texts that
provide logical bridges between the different papers are mandatory.
The thesis must conform to all other requirements of the “Guidelines for Thesis
Preparation” in addition to the manuscripts.
As manuscripts for publication are frequently very concise documents, where
appropriate, additional material must be provided in sufficient detail to allow a
clear and precise judgement to be made of the importance and originality of the
research reported in the thesis.
In general when co-authored papers are included in a thesis, the candidate must
have made a substantial contribution to all papers included in the thesis. In
addition, the candidate is required to make an explicit statement in the thesis as
to who contributed to such work and to what extent. This statement should
appear in a single section entitled “Contribution of Authors” as a preface of the
thesis.
11
When previously published copyright material is presented in a thesis, the
candidate must obtain, if necessary, signed waivers from the co-authors and
publishers and submit these to the Thesis Office with the final deposition.
12
Acknowledgements
I would like to take this opportunity to express my most sincere gratitude to the
many who have supported me throughout this journey of graduate studies. First
and foremost, I would like to recognize my supervisor Dr. W. D. Marshall for his
endless patience, positivity and support which have been sincerely appreciated.
The guidance provided in the final stage of my graduate studies by Dr. Yaylayan
has been important and for that, I am truly grateful. I am blessed with wonderful
family and friends who have always supported my endeavours, notably academic
ones. The love and encouragement demonstrated especially by my mom and my
fiancé, Matthew, have been invaluable to me. You two are my rock in life. I am
also grateful to all my fellow colleagues and departmental professors and staff
members at McGill for their support, collaboration and camaraderie. A special
thank you is merited to A. Rahn for helping me see the light at the end of the
tunnel with this thesis. I would like to thank D. Valtierra, P. Guerra, P. Owen, A.
Constantineau, M. Rivero-Huguet and T. Yuan for providing insight and sharing
their experiences with me. I would also like to thank Dr. S. Kermasha for his
encouragement throughout the years. Last but not least, I would like to express
my gratitude to the management team of the Montreal regional office of the
Canadian Food Inspection Agency for their support throughout my graduate
studies. It has been a memorable and valuable journey.
13
Contribution of Authors
This thesis is presented in the traditional, monograph format, and consists of
seven chapters.
The present author was responsible for the concepts, design of experiments,
experimental work and manuscript preparation. Thesis supervisor, Dr. William D.
Marshall had advisory input into the work in the earlier stage. Co-supervisor, Dr.
Varoujan A. Yaylayan guided manuscript completion while critically editing the
dissertation prior to submission. Thanks must be given to Dr. S. Prasher of
McGill University Department of Bioresource Engineering, Dr. T. A. Johns of
McGill University School of Dietetics and Human Nutrition, Dr. S. Karboune and
Dr. A. Ismail of McGill University Department of Food Science and Agricultural
Chemistry, for allowing me to use their laboratory equipment.
14
DEDICATION
This document is dedicated to the great mentors of my life, my parents, and to a
gracious supervisor, W. D. Marshall.
15
Chapter 1 Introduction
1.1 General Introduction
Belonging to the Labiatae (Lamiaceae) family, Lavandula spp. plant falls in the
same lineage as several aromatically renowned herbs such as rosemary
(Rosmarinus spp.), thyme (Thymus spp.), mint (Mentha spp.), and sage (Salvia
spp.). Essential oils of species of great economical significance originate from
this plant family (Kara and Baydar, 2013).
The Genus Lavandula is recognized as an aromatic shrub of characteristically
fragrant conical-shaped whorls of light purple to blue hued flowers held around a
central stem. The flowers, consisting of a five-lobed corolla (petals) tube
emerging through the calyx (sepals), are commonly used for various applications.
The characteristic qualities of the plant are attributed to the secondary
metabolites of the multicellular-headed glandular trichomes (Iriti et al., 2006).
These glandular trichomes are located on the superficial parts of the leaves and
the calyx of the Lavandula plant (Iriti et al., 2006). This blossom is recognized for
the very intense, tangy floral notes yet calming nuance lavender brings to
products.
1.1.1 Commonly studied Lavandula species in research
Reaching over thirty different species, the genus Lavandula is divided into six
sections: Lavandula, Stoechas, Dentata, Pterostoechas, Chaetostachys, and
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Subnuda (McNaughton, 2000). The Section Lavandula is frequently discussed in
literature as it represents the most cultivated lavenders in the world as well as
holding the most odoriferous examples of the Lavandula genus (McNaughton,
2000). Species within the Lavandula genus which are commonly studied and of
commercial and economic importance include Lavandula latifolia (Spike
lavender), Lavandula angustifolia (True lavender or English lavender), Lavandula
stoechas (Spanish lavender), and Lavandula x intermedia (lavandin) (Gonςalves
and Romano, 2013; McNaughton, 2000; Iriti et al., 2006; Topal et al., 2008). The
Section Lavandula cultivars which have been established for this research are
English or true lavender and lavandin.
1.1.1.1 True Lavender (Lavandula angustifolia)
True lavender, Lavandula angustifolia, gets its name from angustifolia meaning
“narrow-leaved”. This specie is popular for oil production as it is known to yield
high quality oils (Lis-Balchin, 2002). This high quality is based on the oil
constituents’ sweet contribution in fragrance. Moreover, it has been suggested
that this specie is the best for culinary use as Lavandula angustifolia has a sweet
aroma and taste (Platt, 2009). The specie is also known for its tolerance to
extreme conditions such as cold temperatures, winds, rain, and snow, thus
making it among the hardiest of lavanders (McNaughton, 2000).
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1.1.1.2 Lavandin (Lavandula x intermedia)
Lavandin is a result of crossbreeding between Lavandula latifolia and Lavandula
angustifolia (McNaughton, 2000). They are very hardy plants characteristic of its
parent, Lavandula angustifolia. Their increased spike production and greater oil
content, being about four to eight times that of lavender, are favourable
(McNaughton, 2000). For this reason, global lavandin oil production supersedes
lavender oil production being the primary Lavandula cultivar grown globally for its
oil (McNaughton, 2000). The oils from the most important lavandin varieties
include ‘Abrial’ and ‘Grosso’ (Surburg and Panten, 2006). Lavandin differs slightly
in aromatic notes from lavender. The former holds a characteristically strong but
reduced sweet lavender fragrance, due to the greater amount of camphor relative
to the Lavandula angustifolia cultivars. Section 1.2.1 further discusses the
contribution to the aromatic differences among the Lavandula spp..
1.2 Secondary metabolites in plants
Phytochemicals are secondary metabolites of plants which are produced for roles
other than primary functions such as growth, photosynthesis, reproduction. They
provide a protective value to the plants. For instance, they may have negative
impacts on other organisms such as herbivores and pathogens and inhibit the
18
growth of competitor plants (i.e. allelopathy) (Dudareva, 2006). Furthermore, they
are responsible for the colour contributions to flowers (i.e. pigments such as
delphinidin and malvidin flavonoids in lavender) and together with terpene and
phenolic odours, attract pollinators (Dudareva, 2006; Lis-Balchin, 2002). The
individual phytochemicals or mixture thereof within the Lavandula genus is of
significance to the biological activities of the plant material.
The terpenes are of the greatest importance among the bioactive compounds
found within the genus. The biosynthesis of terpenes or terpenoids in higher
plants is believed to be a function of two independent pathways; the cytoplasmic
pathway, mevalonate (MVA), and the plastidal pathway, methylerythritol (MEP)
(Biswas et al., 2009). These constituents are a group of compounds chemically
based on the number of isoprene (2-methyl-1,3 butadiene) units linked together
and comprise of hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15),
diterpenes (C20), sesterpenes (C25), triterpenes (C30), polyterpenes (>C30)
(Chizzola, 2013). Essential oils are a complex mixture of volatile secondary
metabolites which consist of aromatic and aliphatic monoterpenes and
sesquiterpenes (Biswas et al., 2009; Chizzola, 2013). Triterpenoids, diterpenes,
and polyterpenes are non-volatile secondary metabolites. Sesquiterpenes may be
volatile or non-volatile.
19
1.2.1 Chemical constituents found in Lavandula spp.
The chemical composition of Lavandula spp. can include over three hundred
chemical constituents (Lis-Balchin, 2002). They generally comprise of
monoterpenes (i.e. α- and β-pinene, camphene, limonene, p-cymene, sabinene,
terpinene, β-ocimene) and its alcohols (i.e. linalool, borneol, α-terpineol,
lavandulol, p-cymen-8-ol), its aldehydes (i.e. cumin aldehydes), its ether (i.e.
eucalyptol), its esters (i.e. linalyl acetate, terpenyl acetate), its ketones (i.e.
camphor, fenchone, carvone, coumarin, n-octanone) (Lis-Balchin, 2002; Torras-
Claveria et al., 2007). Sesquiterpenes (i.e. caryophyllene and its oxide, α-
santalal), phenols (i.e. eugenol, thymol, coumarin, carvacrol, hydroxycinnamic
acids, chlorogenic acid, rosmarinic acid), and other trace components (i.e. other
flavonoids) are also among the bioactive compounds (Lis-Balchin, 2002; Torras-
Claveria et al., 2007).
The chemical variation found within the Lavandula genus, for lavender, lavandin,
and spike lavender, as can be shown in Table 1, contributes to the difference in
its bioactive properties. The major constituents of True lavender include linalool
and linalyl acetate. The latter two constituents along with eucalyptol, and
camphor make up largely lavandin oil while spike lavender oil predominantly
holds high amounts of linalool, eucalyptol, and camphor.
20
Table 1 Percentage of major constituents of lavender, lavandin, and spike lavender oils
Percentage of essential oil in a
Constituents Lavender oil
(Lavandula angustifolia)
Lavandin oil (Lavandula x intermedia )
Spike lavender oil (Lavandula latifolia )
Linalyl acetate 12-54% 19-26% 0-1.5%
Linalool 10-50% 20-23% 26-44% cis-and/or trans-Ocimene 1.0-17% 1.0-3.0% 0-0.3% Eucalyptol (1,8-Cineole) 2.1-3.0% 10.00% 25-36%
Camphor 0-0.2% 12.00% 5.3-14.3% Lavandulol and acetate 0.1-1.4% 0.5-0.8% 0.2-1.5%
α- and β-Pinene 0.02-0.3% 0.6-0.9% 1.6-3.6% Borneol 1.0-4.0% 2.9-3.7% 0.8-4.9%
Caryophyllene and/or its oxide
3.0-8.0% 2.7-6.0% 0.1-0.3%
Myrcene 0.4-1.3% 1.2-1.5% 0.2-0.4% Farnesene Trace 1.1% 0.2-0.3%
Germacrene D 0.2-0.9% 1.0-1.2% - Camphene 0.1-0.2% 0.3-0.6% 0.2-1.8% Limonene 0.2-0.4% 0.9-1.5% 1.0-2.2%
Reference from aLis-Balchin, 2002
1.3. Biological activities of the genus Lavandula
According to the literature, lavender holds various potential bioactivities, including
clinical and therapeutic benefits. They include neurological or psychiatric (i.e.
sedative or hypnotic, anxiolytic, mood modulator, effect on cognitive function,
analgesic), cardiovascular, pulmonary, gastrointestinal or hepatic, endocrine,
rheumatological, reproductive, immune modulation, antimicrobial, pesticidal,
antineoplastic, antioxidant, and others (Lahlou, M., 2004; Cavanagh and
Wilkinson, 2002; Torras-Claveria et al., 2007; Ghoreishi et al., 2012). The two
21
characteristic chemical constituents of the Lavandula genus, linalyl acetate and
linalool, have been attributed to sedative action and local anesthetic effects
(Cavanagh and Wilkinson, 2002). The physiological properties of Lavandula
extractives are dependent upon stereochemistry (i.e. chirality and isomerism) as
well as chemotypes (Lahlou, M., 2004).
1.3.1. Flavour and Aroma impact
Flavour is a sensory experience which arises from a combination of taste and
smell. The aromatic properties of Lavandula extractives are owed to the
individual terpenes and mixture thereof which hold characteristic odours. These
can be seen in Table 2. Their odour thresholds are variable and dependant on
the matrix (Chizzola, 2013). True lavender oil is generally noted as a fresh,
sweet, floral, herbaceous odour of a woody balsamic base (Surburg and Panten,
2006). Lavandin and spike lavender oil are perceived as lavender-like with a
slightly camphoraceous note and a characteristic rough odour slightly like
eucalyptol and camphor respectively (Surburg and Panten, 2006).
22
Table 2 Odour impact of selected Lavandula spp. terpenes
Compound Aroma descriptors
Borneol Woody-camphoraceous, dry-minty a
Isoborneol Camphoraceous, weak peppery and woody a
Camphene Camphoraceous, mild-oily a
Camphor Camphoraceous, fresh, warm-minty, ethereal a
β-Caryophyllene Woody, spicy, terpene notes a
Caryophyllene oxide Weak woody, warm, mild, weak spicy a
Eucalyptol (1,8 cineole) Fresh notes, reminiscent of camphorb
trans-β-Farnesene Warm, mild, sweet a
trans-α-Farnesene Mild, warm, sweet a
Germacrene D Weak spicy, weak fruity, apple-like, weak dry-woody a
Limonene Fresh, citrus-like, mild lemon and orange notes a
Linalool Fresh, floral, clean, sweet, lemon notes a
Linalyl acetate Bergamot-lavenderb
Myrcene Mild, sweet, balsamic, plastic note a
cis-β-Ocimene Herbal, warm-herbaceous, sweet-floral, neroli-oil-like a
trans-β-Ocimene Herbal, weak floral a
α-Pinene Pine-like, sharp, woody, turpentine-like a
β-Pinene Dry-woody, pine-like, resinous-terpene-like, spicy a
a Reference from Chizzola, 2013 ;
b Reference from Surburg and Panten, 2006
23
1.3. 2 Toxicity studies
Given its historic use, Lavandula toxicity is deemed rare. The lethal Dose (LD50)
of lavender and lavandin oil in rats (oral dose) and lavender oil in rabbits (dermal
application) has been reported as higher than 5 g/kg body weight (Schulz et al.,
2004; Lis-Balchin, 2002). An account of lavandin poisoning has been reported in
an eighteen month old boy after the ingestion of home-produced lavandin extract
(Landelle et al., 2008). Sensitization has also been reported (Lis-Balchin, 2002).
1.4. Lavandula and its applications
The Genus Lavandula is distinguished for its versatility as it has seen various
uses over time. Its botanical designation or genus name, Lavandula, is inherited
from the Latin word lavare, meaning “to wash”, as the plant was commonly used
to perfume baths in Roman times (McNaughton, 2000). Lavandula spp. continue
to be popular in various applications in addition to pharmaceutical and medicinal
ones (Gonςalves and Romano, 2013). Lavender’s essential oil is commonly
employed in aromatherapy and massage where its major clinical benefits are on
the central nervous system (Cavanagh and Wilkinson, 2002). Lavender has
antimicrobial activity against bacteria, fungi, and some insects lending itself to
agricultural uses as a natural pesticide besides ornamental planting
(McNaughton, 2000). The fragrance industry also makes use of its fragrance
24
and/or oil for the production of perfumes, cosmetics, and household products
(Platt, 2009). Moreover, culinary uses are seen as it is found in haute cuisine and
food manufacturing. Flavour molecules within lavender impart a sweet floral note
to food and beverages which stimulate the gustatory and olfactory senses
(Surburg and Panten, 2006).
1.4.1 Lavandula and food
Lavender has continued to garner great interest for its use as a natural flavouring
substance. In food manufacturing, Lavandula species natural extractives are
generally recognized as safe (GRAS) (Burdock and Fenaroli, 2005). Culinary
uses are seen in dairy (ice cream and cheese), confectionary, baked goods,
jams, gelatins and puddings, chewing gum, and beverages (Burdock and
Fenaroli, 2005; McNaughton, 2000). Lavender has been said to bring a soothing,
aromatic quality to foods such as chocolate.
Natural flavouring substances contribute to the marketing of natural foods and
actually comprise the major portion of the food flavour market (Reineccius,
2006). Moreover, flowers represent beauty, naturalness and health and these
properties may be used for emotional product positioning in the food industry
(DöhlerGroup, 2014). Producer, marketer and provider of natural ingredients,
ingredient systems and integrated solutions for the food and beverage industry,
25
DöhlerGroup, have indicated that hints of florals such as lavender hit high notes
today among top trendsetters (2014).
1.5 Quality characteristics of Lavandula spp. extractives
Food and beverage formulators are considering specific value-added ingredients.
The quality and marketable value of a Lavandula natural extract is measured by
the chemical composition of the essential oil. Preparations rich in oxygenated
compounds often represent an important index for flavour quality. In Lavandula
spp. extracts, flavour quality is predominantly determined by the presence of
oxygenated compounds such as the ‘character impact’ compounds which
contribute to the characteristic sweet-floral aroma of lavender. They include (R)-
(-) linalool and linalyl acetate. The former is an acyclic unsaturated tertiary
alcohol which occurs in many essential oils and known for its fresh, clean,
flowery aroma quality. Due to its relatively high volatility, it is known to impart
naturalness to top notes. Together with its esters, linalool is one of the most
frequently used fragrance substances (Surburg and Panten, 2006). Linalyl
acetate is an acyclic terpene ester which imparts a bergamot-lavender odour
(Surburg and Panten, 2006). This acetate is recognized as the most important
fragrance and flavour substance among the linalyl esters (Surburg and Panten,
2006). High percentage of linalool and linalyl acetate, with minimal camphor
proportions are representative of a high quality Lavandula extract (Biswas et al.,
26
2009; Kara and Baydar, 2013). Camphor, a cyclic terpene ketone with slightly
minty odour (Surburg and Panten, 2006). Consequently, lavender oil is
considered to be of higher quality relative to lavandin oil. Moreover, linalyl
acetate holds a higher value to linalool as a food additive in the flavour market
(Martin et al., 2007; Lee and Shibamoto, 2002).). Wesolowska et al. (2010)
indicated that the ratio of linalyl acetate to linalool should be greater than one for
high quality Lavandula spp. extractives. These quality parameters are consistent
with ISO standards. ISO 3515:2002 standard states the percent composition of
distilled essential oil of true lavender to be 25.0-45.0%, 25.0-38.0%, 0-0.5% and
0-1.5% for linalyl acetate, linalool, and camphor respectively (Kara and Baydar,
2013; Lis-Balchin; 2002). The standard ISO 8902:1986 also sets compositional
ranges of 28.0-38.0%, 25.0-35.0%, and 6.0-8.0% for linalyl acetate, linalool, and
camphor respectively for lavandin ‘Grosso’ oil (Lis-Balchin, 2002).
27
Acyclic Bicyclic
(R)-linalool
Camphor
(R)-linalyl acetate
Figure 1 Structural representation of important quality marker monoterpenes of Lavandula spp.
28
1.6. Methods of isolation of natural flavour constituents from plants
As defined by the Codex Alimentarius Commission (2005), natural flavouring
substances are “flavouring substances obtained by physical processes that may
result in unavoidable but unintentional changes in the chemical structure of the
components of the flavouring (e.g. distillation and solvent extraction), or by
enzymatic or microbiological processes, from material of plant or animal origin.”
Natural flavouring substances derived from plant material commonly use
conventional methods such as distillation and solvent extraction. Distillation
processes and expression (for citrus fruits) give rise to essential oils (Chizzola,
2013). Despite their extensive use in flavour extraction from plant material, the
harsh heat treatment of the distillation techniques has been found to degrade
volatile compounds in the plant material yielding extracts with incomplete sets of
flavour profiles (Da Porto et al., 2009). Many disadvantages are associated with
existing conventional methods with the principle concern being that of organic
solvent use and contamination. A movement in minimizing the use of toxic
solvents in processing has been demonstrated, leading to the development of
alternative sustainable and greener processes such as critical fluid technology
including subcritical and supercritical fluid extraction.
29
1.6. 1 Critical fluid technology
Critical fluid technology has demonstrated its potential as an alternative
separation technique due to its extraction and fractionation ability using solvents
around their critical points. Potential applications in the isolation of bioactive
molecules in plants, including flavour and aroma compounds, have been
demonstrated (Herrero et al., 2006; Pourmortazavi and Hajimirsadeghi, 2007; Xia
et al. 2008).
1.6.1. 1 Supercritical Fluid Extraction (SFE)
Supercritical fluid extraction (SFE) exploits the liquid phase and gas-like
physicochemical properties of a solvent, maintained at temperatures and
pressures above their critical point, to mobilize a solute from a given matrix. The
extraction process occurs in four stages; the diffusion of the supercritical fluid
into the porous sample matrix, the separation of the solute-solute interaction
within the matrix, the diffusion of the solutes out of the matrix, and the recovery of
the analytes from the sample during decompression (Richter, 1992). Among the
parameters of SFE, pressure holds a fundamental role in the process due to its
solubility effect. An important relationship of supercritical fluids is that of solvent
strength to density. The solvent strength of pure supercritical fluids shows a
direct correlation to the density of the fluid (Richter, 1992). As a function of
30
pressure and temperature, changes in density can permit variable solvating
power, allowing for selective extractions. As can be observed in Table 3, the
density of supercritical fluids is comparable to that of a liquid (Reineccius, 2006).
A supercritical fluid possesses a higher molecular diffusion (diffusivity) and lower
viscosity relative to a liquid, which in turn closely resemble the properties of a
gas.
Table 3 Physical properties of fluid states
Densitya (kg/m3)
Diffusivity a (m3/s)
Viscositya (MPa s)
Gas (P= 101.3 kPa, T= 288-303K)
0.6-2 (0.1-0.4) x 10-4 (1-3) x 10-4
Supercritical (T= TcP =Pc) 200-500 (0.7) x 10-7 (1-3) x 10-4
(T= TcP =4Pc) 400-900 (0.2) x 10-7 (3-9) x 10-4
Liquid (T= 288-303K) 600-1600 (0.22) x 10-9 (0.2-3) x 10-2 Reference from
aReineccius, 2006
Moreover, no surface tension occurs due to the absence of a liquid and gas
interphase (Attokaran, 2011). Low kinematic viscosities (viscosity divided by
density) are said to promote free-convection mass transfer (i.e. buoyancy effects,
gravity effects, concentration gradients) as well as force-convective mass
transfer due to enhanced turbulence in a system (Richter, 1992). Rapid mass
transfer and solute mobilization from the matrix is achievable as a result. The
solvent as well as nature of the desired analyte(s) influence the operating
temperature of the extraction system.
31
SFE boasts of an environmentally-friendly, rapid technique, being accomplished
in minutes rather than hours. A significant advantage of this technology is its lack
of residual solvent in extracts. Solvents that are gaseous at atmospheric
conditions can be depressurized, allowing its escape into air or be re-
compressed and recycled back in the system, leaving a clean extract with no
need for an additional concentration step prior to analysis. SFE technology is
presently known for its industrial application to the large scale production of food
processes such as the decaffeination of coffee and tea and the extraction of
aroma contributing α- or β- acids from hops used in beer brewing, functional
ingredients such as naturally derived antioxidants and preservative agent
NatureGuardTM rosemary extract, and various pharmaceutical extractions
(Attokaran, 2011; Newly Weds Foods, 2014).
1.6. 1. 1.1 Supercritical fluid extraction using carbon dioxide (SFE-CO2)
Extraction using carbon dioxide in its supercritical state has become an attractive
method for isolation processes in food and biological applications. It is the
solvent of choice for food applications as it meets the requirements food safety
standards (Reineccius, 2006). Moreover, it is attractive due to its abundant,
inexpensive, inert towards oxidation, noncorrosive in dry environments, nontoxic,
odourless, tasteless, colorless, non-flammable and nonexplosive nature
32
(Beckman, 2004; Attokaran, 2011; Pallado et al., 1997). It permits extractions at
low temperatures and pressures with its critical temperature (Tc) of 31°C and
critical pressure (Pc) of 73atm (1073psi); hence a valuable tool for the extraction
of heat labile materials. SFE-CO2 is well recognized for its numerous advantages
in flavour extraction applications. It is also credited for its enhanced product
quality as it provides a more complete flavour profile with added “top-notes” (first
flavours or odours perceived) and “back-notes” (residue flavours or odours
perceived) in all producing a different profile than obtained with traditional
extraction methods (Reineccius, 2006).
1.6. 1. 2 Subcritical water extraction (SWE)
Also referred to as superheated or pressurized hot water extraction, SWE
employs pressurized water, maintained in its liquid state, between its boiling (Tb)
and critical temperature (Tc) of 100°C and 374°C respectively (Herrero et al.,
2006). This environmentally clean alternative extraction technique has
demonstrated its ability to selectively extract different classes of compounds from
natural sources by the thermal fine-tuning of water for nutraceutical and/or
pharmaceutical applications (Ozel and Gogus, 2014; Waseem and Kah, 2015).
The principles of SWE involve the behaviour of liquid water as a collection of
individual molecules with the dielectric constant closely resembling that of a gas
at the critical temperature. Subcritical water may mimic polar organic solvents
33
such as acetonitrile, ethanol, methanol and acetone with polarity changes
brought about by temperature. An increase in the latter imparts intrinsic
thermodynamic transformations to the solvent causing a fall in the high degree of
association in the liquid caused by van der Waals forces, hydrogen bonding and
dipole-dipole interactions (Herrero et al., 2006; Ozel and Gogus, 2014). A
decrease in the viscosity and surface tension of water renders the subcritical
solvent suitable for the mobilization of polar to non polar organic compounds
(Ozel and Gogus, 2014). The attractiveness of SWE is also a result of its rapid,
efficient, selective, inexpensive, environmentally friendly nature (Herrero et al.,
2006). The solvent is naturally occurring, non-toxic, and non-flammable. Higher
quality extractives may be produced due to the enhanced recovery of
oxygenated compounds which are valuable components as they contribute
significantly to the flavour and fragrance of essential oils. Minimized energy
requirements have also been demonstrated in comparison to conventional
methods such as steam distillation due to the absence of energy intensive latent
heat of evaporation (Herrero et al., 2006; Ozel and Gogus, 2014).
1.7. Instrumental Analysis of Flavours
Used as a standard method in determining many volatile constituents, gas
chromatography (GC) has been widely accepted in various industries including
34
the food industry, notably with groups working with flavours and aromas. The use
of GC for the identification of aromatic compounds has been well studied,
particularly with GC coupled with Mass Spectrometry (GC-MS) (Surburg and
Panten, 2006; Reverchon et al., 1995; Pallado et al., 1997; Iriti et al., 2006;
Matos et al., 2009; Lee and Shibamoto, 2002; Da Porto et al., 2009; Waseem
and Kah, 2015).
35
Rationale and Objectives of the Proposed Research
The concepts of health and wellness, natural, and environmentally clean
practices are leading themes today.
Credited for its positive contribution to human health and well-being, the
Lavandula genus shows potential for use as a natural ingredient in high value
(value added) food and beverage systems. Currently, the scientific research
which employs Lavandula plant species of diverse geographical range remains
relatively scarce as the majority of literature covers Lavandula spp. from the
European and particularly Mediterranean region. In this research, North
American Lavandula spp. cultivars were considered. Moreover, critical fluid
technology has demonstrated great promise as a sustainable processing
technique of biological materials. Little research has been performed on sub- and
supercritical fluid extraction of Lavandula spp. cultivars.
The overall objective of this research was to investigate critical fluid technology
for its use as a sustainable alternative for the extraction of high quality Lavandula
flavour for application as a natural ingredient in food industry.
36
The specific objectives of this study were:
I. To extract and characterize the mobilized flavour volatiles from North
American Lavandula Angustifolia ‘Royal Velvet’ and Lavandula x
Intermedia ‘Grosso’ by Gas Chromatography Mass Spectrometry
II. To investigate the chemical composition of the volatile extractives and the
mobilization of valuable oxygenated compounds of the Lavandula spp.
plant material extracted by Soxhlet, supercritical carbon dioxide, and
subcritical water
III. To study the extraction yield of Lavandula spp. isolates obtained by the
three proposed extraction methods
IV. To investigate the quality of the Lavandula spp. extracts obtained by the
three proposed extraction methods
V. To formulate conclusions regarding the relative merits of the three
proposed extraction methods
37
Chapter 2 Literature Review
2.1. Literature review on solvent extraction of Lavandula species using Soxhlet
method
Ghoreishi and associates carried out the traditional Soxhlet extraction of dried
Iranian Lavandula angustifolia flowers by the standard method for 8 h with 50 mL
of n-hexane. The total mass of four components (i.e. camphor, fenchone, linalyl
acetate, and linalool) in the plant material extracted using Soxhlet accounted for
80 % of the total mass of the lavender sample (Ghoreishi et al., 2012). In another
study, Soxhlet was performed for the isolation of volatiles in a comparative study
using spike lavender (Lavandula latifolia Medik) (Eikani et al., 2008). Essential oil
components were extracted from Iranian lavandin (Lavandula hybrida) dried
flowers using Soxhlet for 8h with 50 mL of ethanol solution (Kamali et al., 2012).
The oil content in the lavandin sample was determined to be 1.52% by mass
(Kamali et al., 2012).
2.2 Sub- and supercritical fluid extraction for the isolation of Lavandula spp.
natural products
Little research has been found in literature on the isolation of Lavandula spp.
natural products using sub- or supercritical fluid technology.
38
2.2.1 Literature review on SFE of Lavandula species
An earlier study of supercritical fluid extraction of Turkish Lavandula Stoechas
subspecies Cariensis Boiss flowers for the isolation of essential oils was
performed by Adasoglu, Dincer, and Bolat (1994). A comparative study was done
on the effect of pressure (70-110 bar (1015-1595 psi/ 69-109 atm)), temperature
(30-50°C), carbon dioxide flow rate (7.2-36 L/h) and particle size on the relative
yields with respect to steam distillation. The extraction time considered was 20,
40, and 60 minutes. It was demonstrated that the temperature and particle size
are major variables in the processing, with decreasing oil yields (loss of volatiles)
as a result of particle size reduction. It was suggested that the pressure and
solvent flow rate had little effect on the oil yields as extraction around the critical
point is favourable. The optimum conditions were found to be 85.77 bar (1244
psi/85 atm), 36.58°C, 10.11L/h, and -2143 μm (Adasoglu et al., 1994).
A primary study of dried lavandin, Lavandula x intermedia Emeric ex Loisel,
using supercritical carbon dioxide extraction was investigated by Oszagyan et al.
(1996). Stage wise separation (fractionation) of essential oils by varying
pressures (295-300 bar (4277-4351 psi/291-296 atm)) at a constant temperature
of 40°C was demonstrated. The effect of particle size of the plant material on
extraction rates was also considered. This study showed that yields of
39
supercritical fluid extracts were significantly greater than those of steam
distillation.
Akgün, Akgün, and Dincer (2000) also investigated the modeling and extraction
of Turkish Lavandula Stoechas, subspecies Cariensis Boiss, dried flowers for the
essential oil using supercritical carbon dioxide. The extraction process was
modeled using a quasi-steady-state model in order to demonstrate the effects of
solvent flow rate (1.092-2.184x10-3 kg/min), pressure (8-14 MPa (1161-2028 psi/
79 – 138 atm)), and temperature (308-323K (35-50°C)) on the extraction rate. An
extraction time maximum of 180 minutes was used, with 30 minutes intervals.
Under these conditions, this research demonstrated that extraction rate was not
significantly affected by the carbon dioxide flow rate. Moreover, the authors also
demonstrated that extraction rate increased with increasing temperature and
pressure (Akgün et al., 2000). From this research, extraction parameters to
consider included a pressure range of 10-14 MPa (1454-2028 psi/99-138 atm),
temperature of 323K (50°C), flow rate of 1.456x10-3 kg/min for an extraction
duration of 90-120 minutes (Akgun et al., 2000).
Da Porto, Decorti, and Kikic (2009) considered the SFE of Italian Lavandula
angustifolia L. flowers for use in food manufacturing. A comparative study was
done on the isolation of flavour compounds of lavender relative to
hydrodistillation and ultrasound-assisted solvent extraction. The supercritical
40
carbon dioxide conditions that were studied included a pressure range of 80-
120 bar (1160-1740 psi/ 79-118 atm), temperature between 35 and 60 °C. The
best overall conditions were a pressure of 120 bar (1740psi/118atm) and
temperature of 40°C. This was determined on the basis of the extract
composition analysed by GC-MS. The results of this study demonstrated that
supercritical fluid extracts were quantitatively the most rich with a shorter
extraction time and offer greatest food flavouring stability and quality (Da Porto et
al., 2009).
2.2.2 Literature review on SWE of Lavandula species
Eikani et al. (2008) performed a comparative study on SWE with conventional
isolation methods such as Soxhlet extraction and hydrodistillation on Iranian
dried spike lavender (Lavandula latifolia Medik). The authors demonstrated the
great potential of subcritical water as a solvent in the extraction process of
Lavandula spp. It was found to be comparable quantitatively to traditional
methods but more rapid and more selective towards valuable oxygenated
compounds. The processing variables used in this research were a temperature
range of 100-175°C, pressure range of 20-40 bar (290-580 psi/20-39 atm) with
solvent flow rate of 1,2,3,4 ml/min, and extraction time maximum of 120 minutes,
using 20 minutes intervals. SWE used ground lavender with particle size varying
41
from 250-500 μm. The kinetic data showed the working optimal conditions to be
at temperature, pressure and flow rate of 150°C, 20 bar (290 psi/20 atm), and 3
ml/min respectively (Eikani et al., 2008).
Giray et al. (2008) investigated the effects of SWE on the chemical composition
of dried Lavandula Stoechas flowers from Turkey. Kinetic studies demonstrated
the quick efficiency of subcritical water in isolating volatile constituents relative to
conventional methods including distillation and solvent extraction. SWE was
found to be selective towards small molecular weight oxygenated compounds
contribute significantly to the fragrance of the Lavandula oil (Giray et al., 2008).
Processing parameters that were considered in this study were temperatures of
100, 125, and 150°C, and pressure range of 40-90 atm (588-1323 psi). The flow
rate used was that of 1ml/min and a varying static extraction time 0-30 minutes,
followed by a 20 minute dynamic extraction. The optimum extraction temperature
was 100°C.
42
Chapter 3 Method Development of the Proposed Research
3.1 Materials
3.1.1 Plant material
Commercially available North American dried florets of Lavandula Angustifolia
‘Royal Velvet’ (Lavender) and Lavandula X Intermedia ‘Grosso’ (Lavandin) grown
in Sequim, Washington, USA were used for this research. Table 2 offers the
scientific and botanical classification of the Lavandula spp. samples used in this
study.
Table 4 Classification of the Lavandula spp. samples
Common Botanical Name Lavender Lavandin
Genus Lavandula Lavandula
Subgenus Lavandula Lavandula
Section Lavandula Lavandula
Species L. Angustifolia L. X Intermedia
Cultivar ‘Royal Velvet’ ‘Grosso’
3.1.1.1 Physical properties of plant material sample
3.1.1.1.1 Moisture content
The moisture content of the dried florets of Lavandula Angustifolia ‘Royal Velvet’
and Lavandula X Intermedia ‘Grosso’ were 9.274% ± 0.995 dry weight basis and
43
14.381% ±2.070 dry weight basis respectively. A sample size of 5.000g of each
Lavandula spp. was placed separately in a dessicator in order to remove the
available moisture from the dried florets. The moisture content was determined
once no significant change in moisture loss was observed over time.
3.1.1.1.2 Particle size
The plant material was ground using a Thomas Scientific Thomas Wiley mini mill,
with a mesh size 20, in order to facilitate solvent penetration through cell
rupturing and increased interfacial area. The particle size of the starting material
was determined to be 841 μm. This parameter was established while taking into
account the limitations of the instrumental grinding tool.
3.1.2 Chemicals and reagents
Hexane and dichloromethane were purchased from Caledon Laboratories Ltd.,
Georgetown, ON, Canada. Ethanol was obtained from Fisher Scientific, Ottawa,
ON, Canada. Nitrogen gas was purchased from MEGS, Saint-Laurent, QC,
Canada. All reagents were ACS reagent grade or better. All reference standards,
linalool (≥99.0%, GC, Fluka), linalyl acetate (≥95%, GC, Fluka), camphor
(≥99.0%, GC, Fluka) , eucalyptol (≥99.0%, GC, Fluka), (-)borneol (≥99.0%, GC,
Fluka), were purchased from Sigma-Aldrich Canada Ltd., Oakville, ON, Canada.
The analytical homologous series of n-alkane (C6-C16) standards were obtained
44
from PolyScience corp., Illinois, USA. The carbon dioxide (SFE grade) was
obtained by MEGS (Ville St-Laurent, QC, Canada). The solvent used in the
extraction process was distilled de-ionised water (DDW) obtained from a
Millipore-Q purification system (Millipore, MA, USA) with a resistivity of 18.0 MΩ
cm. Drierite® (8 mesh CaO4S) (Fisher Scientific) was used as the desiccant’s
drying agent.
3.2 Extraction Methods
3.2.1 Soxhlet
3.2.1.1 Experimental Procedure
Soxhlet extraction was carried out in a standard apparatus for 12h on 2.000g of
the dried Lavandula spp. plant material with 200 mL of solvent, depending on the
selected mobile phase system (see section 3.2.1.2). The rate of extraction was
an average of 15 and 12 minutes per cycle for lavender and lavandin
respectively. The addition of cotton to the top of the thimble was done in order to
prevent the residue of the sample from entering the receiving flask. The extract
was concentrated using a Büchi Rotavopor R-114 at 40°C to remove the bulk of
solvent, subsequently being placed under a stream of nitrogen where the
residual solvent was evaporated within 30 minutes. These conditions were
45
selected so as to minimize the loss of volatiles. Extract yield was determined
using the following equation
Equation 1
%100*(g) weight sample
(g) residue ofweight (%)
Yield
3.2.1.2 Mobile phase selection
The mobile phase for the reference method was carefully selected. Hexane and
ethanol were found to be suitable solvents in order to provide appropriate
comparables with the selected critical fluid extraction methods. The polarity of the
extractants as well as the nature of the solvent were considered. Important
chemical properties of various solvents permitted for use in the flavour extraction
processes which have been considered can be found in Table 3. Ideally, the
solvent should hold a relatively low boiling point so that it can easily be removed
by evaporation with minimal loss of volatiles. Furthermore, the solvent properties
need to be compatible with GC-MS analysis.
46
Table 5 Properties of solvents permitted for use in flavour extraction
Solvent Boiling Point (°C)
at 760mm Log P (octanol-
water) Permitted
Residue (ppm) a
Hexane 68.7 3.80 25
Methylene chloride
40.0 1.25 30
Acetone 56.5 -0.24 30
Ethanol 78.4 -0.32
Methanol 64.7 -0.82 50
Supercritical CO2 -56.6 a
Reineccius, 2006
Hexane was selected given its traditional use in solvent extraction due to its low
polarity (log P value of 3.80) and thus high selectivity toward lipophilic
components. It will also serve as a good reference for the SFE-CO2 analysis.
Ethanol, being a more polar organic solvent (log P value of -0.32), is useful for
the extraction of lipophilic materials with polar constituents thus providing a good
reference for the comparative analysis to the SWE process. Moreover, it is
considered a safe chemical solvent for use in food systems.
3.2.2 Supercritical fluid extraction using carbon dioxide (SFE-CO2)
3.2.2.1 Experimental Procedure
In the present work, the two Lavandula samples were subjected to a dynamic
batch extraction by an in house SFE system using carbon dioxide under various
process conditions.
47
The SFE-CO2 extraction system was a laboratory built assembly as shown in
Figure 2. The unit consisted of a high pressure capacity pump, a heated region
for temperature control of the extraction cell, and a region for the decompression
and collection of the mobilized analytes. The supercritical fluid was pumped via a
“superpressure” diaphragm compressor (Newport Scientific, Jessup, MD, U.S.A.)
through an empty stainless steel column assembly (10 mm id x 25 cm), which
acted as a temperature and pressure equilibration vessel (TPEV). The TPEV,
immersed in a heated serological water bath (Fisher Scientific, USA), was
connected in series to a preheating coil (1/16 in. stainless steel tubing) located
upstream from the stainless steel high pressure cylindrical extraction vessel (5
mL, Waters Corp, USA). The extraction cell contained stainless steel caps at
both ends with a frit assembly to provide even distribution of fluid. The
preheating coil and extraction cell, holding the 2.0 g of dried ground Lavandula
spp. material, were situated within a GC oven (Hewlett-Packard, 5890A).
Pressure within the assembly was maintained with a terminal restrictor made of
silica un-deactivated fused capillary (0.75 mm x 1 in.id length) tubing
(Chromatographic Specialties, Brockville, ON, Canada). A digital pressure meter
(Omega engineering Inc., CT, USA) was online to provide the pressure of the
incoming supercritical fluid stream located immediately out of the oven (pre-
preheating coil). The exit tip of the capillary tubing was immersed in 3.0 mL of
48
ethanol to trap analytes from the reactor effluent. High pressure needle valves
(SSI model 02-0120, Alltech, U.S.A.) were mounded throughout the assembly.
The second incoming solvent stream was designated as a pressure release
stream and was configured to permit venting of the system if the pressure within
the reactor column became excessive. A Varian 9010 solvent delivery system
(Varian Associates Inc., Walnut Creek, U.S.A.) was online in order to deliver
selected solvents of varying polarities for cleaning of the system in between runs.
A final purging of the system was completed with CO2 to dissolve any
contamination remaining.
The extract, collected in a dry and tarred vial, was concentrated under a N2
stream and weighed once all solvent was evaporated. These conditions were
selected so as to minimize the loss of volatiles. The percentage of extract
obtained was calculated using the following equation
Equation 2
%100*(g) weight sample
(g) residue ofweight (%)
Yield
3.2.2.2 Process parameters considerations
The extraction parameters that were considered include time, pressure,
temperature, solvent volumetric flow rate, and particle size.
49
3.2.2.2.1 Time
A preliminary study investigating the extraction time parameter demonstrated that
a 20 minutes extraction was feasible for an efficient extraction process. This first
evaluation consisted of the fractionation of the extracts over a one hour time
period at time intervals of 20 minutes at processing conditions of 2000 psi and
40°C. It was noted that the bulk of the characteristic compounds (linalyl acetate
and linalool) were extracted within the first 20 minutes. The quantitative
difference of these characteristic analytes collected between 20 to 60 minutes
was considered negligible based on GC-MS analysis results and therefore a
quick dynamic extraction process was considered.
50
Figure 2 Laboratory built supercritical fluid extraction system
51
3.2.2.2.2 Pressure
The pressure parameter investigated were ca 2000 psi, ca 2500 psi, ca 3000 psi.
In literature, pressures used in SFE-CO2 are relatively lower than those in the
proposed research (Adasoglu et al., 1994; Da Porto et al., 2009). The range
studied permitted for the attainment of an extract rather than exclusively the oil
as with lower operating pressures (Oszagyan et al., 1996). Higher pressures
were difficult to control manually given the instrumental (ie, powerful pump)
limitations. The experimental conditions reflected the range of ca 2000 psi (2000-
2100 psi), ca 2500 psi (2500-2600 psi) and ca 3000 psi (3000-3100 psi).
3.2.2.2.3 Temperature
The temperature conditions investigated included 40°C, 60°C, and 80°C.
Minimization of the loss of volatile flavour compounds was considered for the
selection of the temperature range. A preliminary study was performed with a
temperature range between 60°C to 100°C. At the latter temperature, thermal
degradation was apparent and a strong burn-like odour was perceived. Martin et
al. (2007) also suggests that 80°C is the highest feasible operating temperature
for SFE of plant material.
52
3.2.2.2.4 Solvent volumetric flow rate
The volumetric flow rate of the solvent was determined using water displacement
approach as the flow rates exceeded the working limits of the available digital
flow meter. Using the Boyle’s and Charles’ combined gas law, the compressed
volumetric flow rate was calculated from the experimentally measured
decompressed volumetric flow rate using the following equations
Equation 3
Equation 4
where P1 is the internal pressure of the system, P2 the atmospheric pressure
(14.7 psi), ύ1 the volumetric flow rate of compressed CO2, ύ2 the volumetric flow
rate of decompressed CO2, T1 temperature within the system, T2 atmospheric
temperature (25°C). The volumetric CO2 flow rates at the various process
conditions are shown in Table 4.
53
Table 4 Decompressed CO2 flow rate (mL/s) at experimental conditions
Flow rate at ATPa (mL/s)
(compressed CO2 flow rate, ml/minb) ±Std.Dev.c
Extraction Cell Temperature (°C)
Pressure (PSI) Lavender Lavandin
40
2000
36 (20) ± 8.68 37 (21) ± 14.80 2500
3000
60
2000
53 (45) ± 5.70 70 (59) ± 17.00 2500
3000
80
2000
51 (58) ± 4.76 57 (64) ± 3.46 2500
3000 a
ATP-ambient temperature and pressure ;b
calculated compressed CO2 flow rate (mL/min);c Std.Dev.- mean standard deviation
3.2.2.2.5 Particle size
The particle size of the sample was 841 μm as previously discussed in section
3.1.1.1.2.
3.2.3 Subcritical water Extraction (SWE)
3.2.3.1 Experimental Procedure
In the present work, the two Lavandula samples were subjected to a dynamic
fractionation process using an in house SWE system under various process
conditions.
SWE was performed in a laboratory built apparatus as shown in Figure 3. The
unit consisted of a heated water supply in its liquid state, a pump, a heated
54
region for temperature control of the extraction cell, and a region for the
collection of the mobilized analytes. Pre-heated at 100°C, distilled de-ionised
water (DDW) was delivered for an extraction process via a Varian 9010 solvent
delivery system (Varian Associates Inc., Walnut Creek, U.S.A.) at a constant flow
rate to the stainless steel high pressure cylindrical extraction vessel (5 mL,
Waters Corp, USA), holding the 1.0 g of ground Lavandula material. The
extraction cell contained a stainless steel cap, on each end, with a frit assembly
to provide even distribution of fluid. The extraction cell connected to a preheating
coil (1/16 in. stainless steel tubing) was mounted vertically within a gas
chromatographic oven (Hewlett-Packard, 5890 II) with solvent flowing from top to
bottom. The eluent was collected from the stainless steel tubing (1/16 in.) exiting
the heated chamber, in vials held in an ice bath in order to minimize thermal
degradation of the volatile constituents. Pressure within the assembly was
maintained with a terminal restrictor made of silica un-deactivated fused capillary
(0.75 mm x 1 in.id length) tubing (Chromatographic Specialties, Brockville, ON,
Canada) ensuring that the water remained in the liquid state. The pressure within
the system was monitored digitally throughout the extraction process via the
Varian 9010 solvent delivery system. High pressure needle valves (SSI model
02-0120, Alltech, U.S.A.) were mounded throughout the assembly. The SWE
55
system was purged and selected solvents of ranging polarities were run through
for the cleaning of the system in between runs.
The yield was determined by the solvent evaporation of 1 mL portion of aqueous
extract, collected in a dry and tarred vial, at atmospheric conditions and weighed
once all solvent was evaporated. These conditions were selected based on the
availability of laboratory equipment, all the while seeking to minimize the loss of
volatiles. The yield was calculated using the following equation
Equation 5
%100*
(g) weight sample
1
10ml (g) residue fractional ofweight
(%)Yield
ml
56
Figure 3 Laboratory built subcritical water extraction (SWE) system
57
3.2.3.2 Process parameters considerations
The extraction parameters that were considered include time, pressure, temperature,
solvent volumetric flow rate, and particle size.
3.2.3.2.1 Time
A fractionation of the extracts over 2 min time intervals for a maximum of 12 minute
period was considered. The objective of this study was to investigate an extraction
method suitable for a quick isolation process. A preliminary study investigating the
extraction time parameter demonstrated that the bulk of the coloured emitting and
odoriferous compounds were extracted within the first 6 to 8 minutes. SWE operating
conditions in literature consider short extraction times of under 60 minutes (Ayala and
Luque de Castro, 2000; Giray et al, 2008). Ayala and Luque de Castro (2000) suggest
that SWE is almost complete within 15 minutes.
3.2.3.2.2 Pressure
The pressure parameter studied ranged between 56 ± 6 to 66 ± 4 atm for the SWE
process of Lavandula spp. The latter range permits for an extraction of Lavandula
extracts with the solvent in its liquid state. Table 5 demonstrates the experimental
conditions for each temperature and time parameter respectively.
58
Table 5 Pressure (atm) conditions of the SWE of Lavandula spp.
Pressure (atm) ±Std.Dev.a
Temperature (°C) Lavender Lavandin
100 63 ± 5 66 ± 4
120 56 ± 6 63 ± 4
140 59 ± 6 58 ± 5 aStd.Dev.- mean standard deviation
3.2.3.2.3 Temperature
The temperature conditions investigated included 100°C, 120°C and140°C.
Minimization of the thermal degradation of flavour compounds was considered for the
selection of the temperature range below the critical temperature of the solvent of
374°C. A preliminary experimental study of the temperature condition of SWE
demonstrated that thermal degradation was apparent at 160°C with a strong burn-like
odour from the sample and darker coloured extract perceived. Giray et al. (2008)
discusses a similar observation at 150°C.
3.2.3.2.4 Solvent volumetric flow rate
The volumetric flow rate of the solvent was regulated digitally at a constant flow of 5
mL/min.
3.2.3.2.5 Particle size
The particle size of the sample was 841 μm as previously discussed in section
3.1.1.1.2.
59
3.3 Analytical Procedure
The extracts isolated by the three proposed methods were investigated using gas
chromatography mass spectrometry (GC-MS) with a primary focus on three key
Lavandula quality and flavour profile markers; linalool, linalyl acetate and camphor. The
SFE-CO2 isolated a two phase extract with a quasi-solid material in liquid. The co-
extracted wax-like material was subjected to further analysis with FTIR.
For qualitative analysis, the chromatographic peaks were tentatively identified through
National Institute of Standards and Technology (NIST) and Saturn library search
routines and structures were selected based on high matching factor. Five key
compounds, linalyl acetate, linalool, camphor, eucalyptol, and borneol, were identified
by comparing the retention time and mass spectra of the relevant chromatographic
peaks with that of authentic reference standards. The linear retention index (LRI) was
determined and used in the tentative identification of the eluting compounds.
Characterization of chromatographic peaks was further based on the calculated LRI in
comparison to LRI found in literature when reference standards were not available. An
analytical homologous series of n-alkanes (C6-C16) were used for the determination of
the LRI. The following equation was used
Equation 6
n+
tn - 1+tn
tn -t 100LRI
60
where t is retention time of target compound, tn is retention time of n-alkane eluting
before target compound, tn+1 is retention time of n-alkane eluting after target compound,
and n is the n-alkane carbon number. Linear retention index equation used is for a non-
isothermal temperature programming for GC analysis.
The normalization method was employed for the semi-quantitative analysis. The relative
percentage of each analyte was estimated by dividing the integrated peak area of the
individual compound by the sum of all integrated peaks within an extract. The equation
used is as follows
Equation 7
3.3.1 Gas Chromatography Mass spectrometry (GC-MS)
3.3.1.1 GC-MS analysis of extracts obtained via Soxhlet Method
The concentrated extract was reconstituted with 10 mL of selected solvent (see section
3.2.1.2). The reconstituted extract was then centrifuged in order to remove the solid
particles (waxes) in solution. An azeotropic mixture of 15:84:1 (v/v/v) was prepared with
the ethanolic extract, dichloromethane, and n-nonane respectively in order to take
advantage of the lower boiling properties of the azeotrope for GC-MS analysis. A ratio
of 15:84:1 (v/v/v) was prepared with the hexane derived extract, hexane, and n-nonane
61
respectively A final 15% (v/v) extract solution for all Soxhlet extracts was used for GC-
MS analysis.
GS-MS analysis was performed on a Varian GC instrument (model 3900) fitted with an
autosampler (8400 model) and coupled to a MS with ion trap detector (2100T model).
Chromatographic separations were performed on a Varian factorfour VF-5ms capillary
column (30m X 0.25mm ID X 0.25μm film thickness). The oven temperature
programming consisted of an initial temperature of 50°C, held for 5 min and raised to
130°C at a rate of 2°C min-1, subsequently the temperature was further raised to 200°C
at a rate of 3°C min-1, then finally to 250°C at a rate of 5°C min-1. Injector and transfer
line temperatures were 250°C and 250°C respectively. The carrier gas (He) flow rate
was 1.0 mL/min. The sample volume injected was 1.0 μL. A split injection with a ratio of
1:10 was used. The electron impact ionization mass spectrometer had an ionization
voltage of 70eV and ion source temperature of 150°C. The scan mode was in the mass
range of m/z 41 to 400 amu.
3.3.1.2 GC-MS analysis of extracts obtained via SFE-CO2
An azeotropic mixture of 50:49:1 (v/v/v) was prepared with the ethanolic extract,
dichloromethane, and n-nonane respectively for GC-MS analysis. The chemical boiling
properties of an azeotropic system between ethanol and dichloromethane were taken
62
advantage of for GC-MS analysis. Chromatographic analysis was performed under the
same GC-MS conditions as reported in section 3.3.1.1.
3.3.1.3 GC-MS analysis of extracts obtained via SWE
The extracts were centrifuged in order to separate the quasi-solid material which was
co-extracted by SWE. The supernatant (5 mL) of each extract was partitioned
successively with three alternate 5 mL aliquots of dichloromethane (DCM), thus
obtaining a DCM fraction. The latter non-polar fraction was collected and dried over with
NaSO4 (0.3g/mL sample) prior to analysis.
An azeotropic mixture of 50:49:1 (v/v/v) was prepared with the DCM fraction extract,
dichloromethane, and n-nonane respectively for GC-MS analysis. Chromatographic
analysis was performed under the same GC-MS conditions as reported in section
3.3.1.1.
3.3.2 Fourier transform infrared spectroscopy
An aliquot of SFE sample was placed in a centrifuge tube. The two phases consisting of
a liquid upper layer, and semi-solid bottom layer (wax-like material) were separated by
centrifugation. The semi-solid wax-like material was isolated, air dried at atmospheric
conditions and set aside for further evaluation.
63
The air dried semi-solid layer was evaluated using Fourier transform infrared (FTIR)
spectroscopy based on both transmission and attenuated total reflectance (ATR)
approaches. A Nicolet 8210 Fourier transform infrared spectrometer (Nicolet Instrument
Inc, Madison, WI) run under a DX operating system was used. A 100 μm (spacer) with
transmission CaF2 flow cell was used for FTIR analysis while the ATR used a Zn-Se
ATR crystal. The flow cell and ATR crystal were operated at ambient temperature.
Prior to analysis, the flow cell and the crystal surface in the ATR system were cleaned
thoroughly and rinsed with propanol. FTIR analysis was carried out by applying the
sample either onto the flow cell or the ATR crystal, recording the FTIR spectrum and
then wiping the cell with propanol. Spectra were collected by co-adding 128 scans at a
spectral resolution of 4 cm-1 in the range of 100 to 4000 cm-1 and a gain of 2.0. Ethanol
was used as to collect the spectrum of the solvent. The absorbance spectrum of the
solvent was subtracted from the absorbance spectrum of each sample to give its
differential spectrum. A baseline correction and a first derivative of the spectrum were
performed.
3.4 Statistical Analysis
The results reported in this study are means ± standard deviation (SD) of at least
duplicate measurements.
64
Chapter 4 Chemical composition and characterization of mobilized
Lavandula spp. constituents via selected conventional method and critical
fluid technology
4.1 Qualitative studies of mobilized Lavandula spp. analytes via GC-MS
4.1.1 Linear retention index (LRI) determination
Analysis of flavours may be complex due to numerous constituents or peaks and
interfering matrix signals. The characterization and tentative identification of compounds
in these complex chromatograms may be facilitated with the use of the LRI for GC-MS
analysis while overcoming the shortcomings of retention time (Rt) analysis due to the
numerous factors which can affect Rt.
A series of n-alkanes was used for the calculation of the LRI. The retention time of the
alkanes are shown in Table 6. Moreover, a comparative study using a series of bromo-
alkanes was considered in order to establish a model for potential use of bromo-alkanes
in LRI determination. The carbon number (n) of the selected alkanes was plotted
against the retention time. A linear correlation exists between the retention times of the
bromo-alkanes and the n-alkanes. A linear equation of y = 6.1863x - 26.025 and y =
6.1767x - 46.39 was obtained with a correlation coefficient of 0.9959 and 0.998 for the
series of bromo-alkanes and n-alkanes respectively. Differences between the retention
profile of the bromo-alkanes and the n-alkanes are evident. The bromine atom allows
65
for greater retention of the molecule to the column stationary phase relative to that of
the n-alkanes under the same gas chromatographic conditions. Thus, the bromo-
alkanes of a lower carbon number may be useful in complex analysis as they are less
volatile and less polar in comparison to the n-alkane counterpart.
Table 6 Retention time of alkanes and bromo-alkanes for LRI determination
n-alkanes Bromo-alkanes
carbon number Rt (min) carbon number Rt (min)
6 ND 5 5.224
7 ND 6 9.78
8 4.134 7 17.279 a
9 7.869 8 23.245
10 15.377 a 9 29.652 a
11 21.554 a 10 37.307
12 27.306 11 42.024 a
13 34.112 12 49.752
14 40.608 13 54.397 a
15 46.745 14 58.787
16 51.873 a
values are predicted values based on retention time modeling; ND = not detected
Results of LRI of each constituent mobilized within the extracts are shown in Tables 8-
12. The calculated LRI corroborate well with that found in published literature (Lee and
Shibamoto, 2002; Iriti et al., 2006; Chemat et al., 2006; Eikani et al, 2008; Matos et al.,
2009; Da Porto et al., 2009). The separation of the complex mixture of compounds of
volatile nature using a high resolution capillary column was achieved. The volatiles
eluted in the general order of their boiling point, as shown in Table 7, with increasing
GC oven temperature.
66
Table 7 Physicochemical properties of selected Lavandula spp. constituents
Constituent Chemical Name b Chemical
Formula a
Molecular
Weight
(g/mol) a
Boiling Point
(°C) a
at P =101.3kPa
Linalyl acetate 3,7-Dimethyl-1,6-
octadien-3-ol acetate C12H20O2 196.29 220
Linalool 1,6-Octadien-3-ol, 3,7-
dimethyl C10H18O 154.25 198
Camphor Bicyclo[2.2.1]heptan-2-
one, 1,7,7-trimethyl C10H16O 152.24 204
Eucalyptol
or 1, 8 Cineol
12-
Oxabicyclo[2.2.2]octane,
1,3,-trimethyl
C10H18O 154.25 176-177
Borneol Bicyclo[2.2.1]heptan-2-
ol,1,7,7-trimethyl C10H18O 154.25
Reference from a Surburg and Panten, 2006;
b NIST library
67
4.1.2 Electron ionization (EI) mass spectra of volatile target constituents
In GC-MS analysis, the characteristic mass spectral ions of each peak within the
chromatograms were studied. Characterization and tentative identification of extract
constituents was accomplished by studying the mass spectrum of each compound
considering the characteristic mass spectral ions such as the molecular ion, [M]+, the
highest peak at the highest m/z in the MS spectrum, the base peak representing the
largest and most intense peak in MS spectrum, and the fragment ions which are the
peaks formed from fragmentation of molecular ion with m/z values less than the
molecular ion with greater intensity than molecular ion. The elution pattern and mass to
charge (m/z) values of the mass spectrum of the authentic reference standards were
compared with that of the sample constituent’s mass spectrum for identification.
Similarities between the constituent’s mass spectrum and the library mass spectrum as
well as the calculated LRI were evaluated for the characterization of the remaining
compounds. It is important to note that the chromatograms were scanned for coeluting
peaks and background ions.
The identification of five key flavour compounds of Lavandula spp. including the
oxygenated hydrocarbon-derived compounds linalyl acetate, linalool, camphor,
eucalyptol, and borneol are discussed further. These compounds were identified using
authentic reference standards. A comparative analysis of the mass spectrum of each
key component in the extract and that given by the NIST library is considered in Figures
68
4-8. The mass spectral data of linalyl acetate from the extract supported that of the
NIST library. As can be seen, the base peak at m/z 93 is present however the molecular
ion at m/z 196 is absent. The characteristic mass spectral fragment ions determined by
GC-MS included the ions at m/z 43, 91, 41, 80, 79, 67, 121, 69 and 92. Considering the
spectral data of linalool found within the extract, the latter agreed well with that of the
NIST library. The base peak at m/z 71 is present however, the molecular ion of 154 is
not apparent. The characteristic mass spectral fragment ions determined by GC-MS
included ions at m/z 43, 93, 81, 41, 55, 80, 67, 69 and 121. In the evaluation of
camphor, the mass spectrum of this molecule corroborated well with the spectral data of
the library. In the mass spectrum of the eluting constituent, the base peak at m/z 95 and
the molecular ion at m/z 152 can be found. The characteristic mass spectral fragment
ions determined by GC-MS included ions at m/z 81, 108, 109, 67, 41, 55, 93 and153.
The evaluation of the mass spectra of eucalyptol demonstrates that the base peak at
m/z 43 and the molecular ion at m/z154 are both apparent. The characteristic mass
spectral fragment ions determined by GC-MS included ions at m/z 81, 93, 139, 108,
111, 67, 69 and 41. For the identification of borneol, the base peak at m/z 95 and the
molecular ion at m/z 154 are visible in the mass spectrum of the eluting constituent as
seen in Figure 8. The characteristic mass spectral fragment ions determined by GC-MS
included ions at m/z 67, 93, 41, 96, 43, 121, 136, 110 and 81.
69
Column bleed of the silicone phase accounts for the presence of high m/z values
around 281 and 355 in the experimental mass spectrum data.
70
Figure 4 Mass spectrum of linalyl acetate from NIST library (above) and component determined by GC-MS (below)
(mainlib) 1,6-Octadien-3-ol, 3,7-dimethyl-, acetate
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 2100
50
100
27
43
55
60
69
80
93
107
121
125
136
154
O
O
30.897 min, Scan: 1987 1,6-Octadien-3-ol, 3,7-dimethyl-, acetate
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
43
43
55
55
67
69
79
80
93
93
121
121
136
136
151
154
171 191 210 226 249 265 281 325 341 355 383 399
71
Figure 5 Mass spectrum of linalool from NIST library (above) and component determined by GC-MS (below)
(mainlib) 1,6-Octadien-3-ol, 3,7-dimethyl-
20 30 40 50 60 70 80 90 100 110 120 130 140 150 1600
50
100
27
29
31
39
41
45 51
53
55
59 65
67
69
71
80
83
93
96 107
117
121
127
136
153
OH
20.327 min, Scan: 1304 1,6-Octadien-3-ol, 3,7-dimethyl-
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290
41
4355
55
71
7180
81 93
93103
105 121
121
136
136
147 157 191 208 265 281
72
Figure 6 Mass spectrum of camphor from NIST library (above) and component determined by GC-MS (below)
(mainlib) Bicyclo[2.2.1]heptan-2-one, 1,7,7-trimethyl-, (1R)-
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 1600
50
100
15
27
36
39
41
43
45
53
55
61 65
67
69
81
83
91
93
95
108
123137
152
O
23.414 min, Scan: 1503 Bicyclo[2.2.1]heptan-2-one, 1,7,7-trimethyl-, (1R)-
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280
41
41
55
55
67
69
81
81
95
95
108
108
118
119
130
134
140 152
152
173 191 267
73
Figure 7 Mass spectrum of eucalyptol from NIST library (above) and component determined by GC-MS (below)
(mainlib) Eucalyptol
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 1700
50
100
15
27
31
39
41
43
45
53
55
59
65
69
71
79
81
84
93
96
108
111
115
121 136
139154
O
15.550 min, Scan: 995 Eucalyptol
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290
43
43
55
55
67
71
81
81
93
93
108
108121
125139
139
154
154
177 191 207 253 281
74
Figure 8 Mass spectrum of borneol from NIST library (above) and component determined by GC-MS (below)
MW: 154 (mainlib) Borneol
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
50
100
41
4355
5967
71 8286
93
95
110121 139
154
HO
25.225 min, Scan: 1620 Borneol
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190
41
41
49
50
55
55
61 67
67
72
79
81
84
86 93
93
95
95
98
98
103 110
110
121
121
136
136
146 153
154
176
75
4.2 Chemical composition of Lavandula spp. extract
The chemical composition of the Lavandula spp. extracts were found to be highly
complex with hundreds of components detected via AMDIS. The chemical constituents
mobilized by the various experimental extraction methods can be seen in Tables 12 to
16.
4.2.1 Soxhlet Method
Solvent extraction of Lavandula spp. flavour components from a solid matrix was
achieved using the Soxhlet extraction. The mature extraction process served as a
reference method and allowed for the estimation of the extract content of the plant
materials. It is also known to generate extractives with a complete flavour profile as well
as other intrinsic characteristics such as colour of the natural starting material
(Reineccius, 2006).
GC-MS analysis demonstrated the presence of 200 to 600 components in the
Lavandula spp. extracts obtained via Soxhlet using the two solvent systems (ethane,
hexane). A lower amount of components were detected by AMDIS from the lavender
samples. The Soxhlet extracts were found to be a complex composition of volatile
terpenes where monoterpenes such as linalool and linalyl acetate constituted the bulk of
the content. The percentage of linalool and linalyl acetate ranged from 22-33% and 33-
37% respectively. These relative percentages for the lavender and lavandin samples
76
agree well with literature (Da Porto et al., 2009; Lis-Balchin, 2002; Bisset, 1994). Linalyl
acetate and linalool accounted for up to 58-70% of the Lavandula spp extract.
GC-MS analysis indicated that the chemical composition of the extracts differed
qualitatively in the detection of minor constituents. Recurring constituents for the
Lavandula spp. samples included monoterpene, α-pinene, low molecular weight
oxygenated compounds such as eucalyptol, ocimenes, linalyl acetate, linalool and its
oxide, camphor, borneol, terpinen-4-ol, geraniol, and high molecular weight oxygenated
compounds including geranyl acetate, caryophyllene and its oxide, santalene,
farnescene, germacrene D. The chemical composition of the Soxhlet extracts can be
seen in Table 12.
The characteristic chromatographic differences in chemical composition between the
two cultivars due to the presence of camphor corroborated well with literature (Lis-
Balchin, 2002; Bisset, 1994). It was observed that the percentages of camphor were
lower than the expected percentage reported in Table 1 for the lavandin cultivar. This
finding suggests that there may be variability when working with biological system such
as plant materials. The lavandin extracts held prominent minor peaks of eucalyptol,
camphor, and borneol relative to lavender samples as expected given the characteristic
chemical composition of lavandin species.
It was also noted that the reference method gave rise to Lavandula spp. extracts
composed of predominantly oxygenated terpene compounds. The extracts held a
77
greater presence of oxygenated constituents in the ethanol solvent system extracts
relative to the hexane. The semi-polar nature of ethanol successfully extracted chemical
species that may be considered more polar than the hexane extract. This was apparent
as the Soxhlet extracts produced a greenish-yellow liquid, representative of the
presence of chlorophyll, with ethanol as solvent in comparison to a yellow tinged liquid
obtained with hexane. Moreover, the extraction of non-polar constituents such as β-
caryophyllene by the polar mediums has been previously reported and postulated to be
due to the molecule being loosely bound to the Lavandula plant material (Eikani et al.,
2008). The hexane solvent mobilized the most amounts of hydrocarbon molecules.
4.2.2 SFE-CO2
A light coloured extract with a true representation of the aroma of the starting Lavandula
plant material was obtained via SFE-CO2. The chemical compounds of Lavandula spp.
samples obtained by SFE and detected by GC-MS can be seen in Tables 13 and 14.
Linalool and linalyl acetate remained the predominant peaks within the chromatograms,
as illustrated in Figure 9. Linalool and linalyl acetate held percentages ranging from 8-
25% and 16-31% respectively. This corroborated well with those seen in literature (Lis-
Balchin, 2002). The lavandin cultivar demonstrated a different trend with respect to the
relative percentages of linalool and linalyl acetate ranging from 2-20% and 12-32%
respectively. The minimum range fell slightly lower than that reported in literature for this
78
cultivar (Lis-Balchin, 2002). Camphor was not apparent in the lavender samples as it is
characteristic of this cultivar’s composition. Higher molecular weight compounds such
as α-bisabolol were quite prominent within the lavandin samples.
Recurring constituents for the Lavandula spp. samples included monoterpene, α-
pinene, low molecular weight oxygenated compounds such as linalyl acetate, linalool,
borneol, terpinen-4-ol, geraniol and high molecular weight compounds like geranyl
acetate, caryophyllene and its oxide, santalene, farnescene, germacrene D, bisabolene,
and α-bisabolol.
An increase in the mobilization of oxygenated terpenes was noted with an increase in
temperature with respect to the lavender cultivar. This was a result of an increase in the
solute vapor pressure with temperature (Ghoreishi et al., 2012). A general decrease in
the percentage of oxygenated compounds was also observed with increase in pressure.
Ghoreishi et al. (2012) explains that this may be attributed to the simultaneous counter
effect of higher temperature which causes lower solvent density with respect to density
enhancement via pressure increase. The pressure of 2000 psi is thus suggested as
optimal pressure due to the highest percentage of oxygenated compounds extracted
with lowest operational input. This agrees well with findings by Da Porto et al. (2009)
who suggested that the best overall performance of SFE to be at a pressure of 120 bar
(1740psi), which was the highest pressure investigated in their study, and a temperature
of 40°C. Additionally, a consistent increase in oxygenated compounds at 60°C was
79
observed in the results within the lavandin cultivar. The effect of solvent flow rate on the
mobilization of volatiles may be attributed to this occurrence.
The supercritical solvent may not have mobilized a greater percentage of oxygenated
compounds relative to the reference method; however, it is suggested that this
percentage may be valuable taking into account the shorter extraction time required for
SFE (20 min vs. 12 hrs). Moreover, supercritical carbon dioxide may provide extracts
which are moderately free from polar, charged, and large molecules such as sugars,
polysaccharides, glycosides, tannins, chlorophyll, cellulose, organic and inorganic salts,
saponins, tannins, phospholipids, amino acids, and proteins with selective fine tuning of
operating parameters (Oszagyan et al, 1996; Pallado et al., 1997; Attokaran, 2011).
SFE with carbon dioxide showed an increased ability towards sesquiterpenes and high
molecular weight compounds with a carbon number of fifteen or greater relative to the
other proposed methods. The selective non-polar nature of SFE-CO2 was apparent as
carbon dioxide in its supercritical state is soluble to lipophilic organic compounds such
as cuticular waxes and resins and esters, ethers, and lactones (Oszagyan et al, 1996).
A quasi solid opaque wax-like material was co-extracted with the Lavandula spp. oil by
SFE-CO2.
80
Figure 9 Total ion count chromatogram of Lavender extract obtained via SFE-CO2 at 2000 psi and 80°C
81
4.2.2.1 Fourier transform infrared (FTIR) spectroscopy
Fourier transform infrared (FTIR) spectroscopy based on both transmission and
attenuated total reflectance (ATR) approaches was used to evaluate the chemical
composition of the quasi solid material that was precipitated within the supercritical
carbon dioxide extraction process. Both the FTIR and ATR-IR data support the notion
that the supercritical carbon dioxide extraction process gives rise to natural extractives
which may include long chain hydrocarbons such as waxes.
The data collected via ATR-IR demonstrated an overall low absorption. Nonetheless,
the spectra obtained from the solid wax-like sample via ATR-IR shows strong
absorption bands, characteristic of simple alkanes due to C-H stretching and bending.
Strong absorptions bands (2971 cm-1, 2921 cm-1, 2855 cm-1) are observed falling within
the 3000-2850 cm-1 C-H stretch region. C-H bend or scissoring from 1470-1450 cm-1, C-
H methyl rocking from 1370-1350 cm-1. No indication of bands above 3000 cm-1 can be
found, suggesting the presence of C-H stretches only, and none from alkenes and
aromatics. Moreover, the lack of other significant characteristic band further
corroborates with the notion of the presence of hydrocarbons such as terpenes and
waxes.
82
Figure 10 FTIR spectra of co-extracted wax-like material (SFE-CO2; 3000psi and 60°C)
Figure 11 ATR-IR spectra of co-extracted wax-like material (SFE-CO2; 3000psi and 60C)
83
4.2.3 SWE
Selective fractionation was achieved with SWE. This was deemed valuable for
generating extracts with selective polar compounds of interest. SWE seemed to
produce the degradation of thermolabile compounds and hydrolysis of water sensitive
compounds.
The chemical composition of the Lavandula spp. samples obtained via SWE can be
seen in Table 6 and 7. It was observed that linalool was the predominant peak in the
chromatograms of both Lavandula spp. samples obtained via SWE. The percentage of
linalool ranged between 48-88% and 42-67% for the lavender and lavandin sample
respectively. This was higher than those reported in literature for lavender essential oils
(Lis-Balchin, 2002). The character impact compound, linalyl acetate, is a minor
constituents within the extracts, when detected. The percentage of linalyl acetate
ranged between 0-8% for the Lavandula spp. extracts. The absence of linalyl acetate is
noted throughout the entire extraction time at a temperature of 100°C for both
Lavandula spp. Hydrolysis of this compound may have resulted during SWE. Esters can
be hydrolyzed by the release of H+ ions from water to the corresponding acids and
alcohols at high temperature (near its boiling point). This chemical reaction is a common
occurrence in hydro-distillation of essential oil from lavender (Reverchon et al., 1995). It
is also suggested that linalyl acetate was broken down into its biosynthesized precursor
84
molecule, linalool and accordingly, may account for the higher amount of the latter
constituent in the extract.
This finding may also suggest that SWE conditions did not give rise to a significant
change in polarity of water at 100°C with the experimental pressure of 56- 66 atm. The
solvent may not have been able to solubilize the least hydrophilic compounds or permit
for high diffusivity of linalyl acetate out from the plant matrix. Eikani et al. (2008)
suggested that some constituents such as linalyl acetate, borneol, α-terpineol were
extracted at slower rate within 50 minutes of the SWE as a result of their lower polarities
and solubilities in the solvent.
Furthermore, SWE greatly modified the extract with respect to the starting composition
of the plant material. The fractions were quite rich in percentage of camphor in the
lavandin sample relative to lavender, as expected. These ketone rich fractions held
percentages as high as 24% of the total extract. GC-MS analysis demonstrated that the
chemical composition of the extracts obtained was selective towards polar oxygenated
terpenes without plant waxes, as little to no hydrocarbons present. Eucalyptol and
borneol held a steady presence in the lavandin samples. Recurring constituents for the
Lavandula spp. samples included monoterpene, α-pinene (within the lavandin sample
only) and low molecular weight oxygenated compounds such as alcohol constituents
eucalyptol, linalool, borneol, terpinen-4-ol, and terpineol.
85
Selected organic compounds, such as aromatic compounds from Lavandula spp. plant
material, may be soluble in the solvent at subcritical conditions, particularly if they hold a
polar moiety. In comparison to the other proposed methods, the subcritical solvent was
able to produce highly odiferous fractions while mobilizing flavonoid pigments of
Lavandula spp. within 12 minutes of extraction. SWE gave rise to an array of colorful
extract fractions; a dark violet coloured extract was apparent after a 2 minute
fractionation and this subsequently faded following a 4 minute fractionation and then
reached nearly colourless after 12 minutes.
86
Chapter 5 Evaluation of extraction yield of Lavandula spp. isolates obtained
by conventional method and critical fluid technology
5.1 Yield studies of Lavandula spp. extracts
5.1.1 Soxhlet Method
In the present study, the percentage yield ranged between 11.75 to 16.08% (w/w) for
the Lavandula spp. as figured in Table 8. The experimental yields of the Lavandula spp.
samples obtained by the conventional method appeared to be relatively high, at first
glance, as compared to literature where lavender flowers are known to contain up to 3%
of essential oil (Bisset, 1994). Lis-Balchin (2002) has also reported values of 2.8-4.96%
and 6.25-10% (dry floral weight) of essential oil isolated by distillation processes for
lavender and lavandin respectively. The higher yield obtained experimentally was
attributed to the presence of constituents of high molecular masses such as waxes
similar to those found in an oleoresin content. Solvent extraction is a method employed
for the isolation of aromatic extractives, including essential oil, non-volatile compounds,
and/or waxes from materials through the use of solvents. Apolar solvents (hexane) and
ethanol are known to extract a certain amount of non-volatile compounds and natural
waxes from the plant (Reineccius, 2007; Chizzola, 2013).
Furthermore, the Soxhlet extraction using ethanol gave rise to a yield with
approximately a 4 percent difference between the Lavandula samples. The overall
87
lavandin yield was greater than that of lavender as expected. The presence of a
valuable difference in yields between the Lavandula species has been reported as
lavandin may hold about four to eight times a greater amount of essential oil relative to
lavender (McNaughton, 2000). No difference in the yield was observed between
Lavandula spp. with the Soxhlet extraction using hexane.
5.1.2 SFE-CO2
SFE using carbon dioxide was carried out on the two Lavandula spp. at different
experimental conditions. The yield obtained via SFE-CO2 of the Lavandula samples are
demonstrated in Table 9. The percentage yield ranged between 1.00 to 3.12% (w/w) for
the Lavandula spp. One may suggest that SFE-CO2 is able to successfully extract all
the essential oil from the plant as literature indicates up to 3% oil content in the
Lavandula spp. (Bisset, 1994). Nonetheless, little amounts of quasi solid material such
as waxes were co-extracted under some processing conditions. Similarly to Soxhlet
method with hexane as solvent, the characteristic differences between the two
Lavandula spp. are not observed with SFE under the selected processing conditions.
However, the highest yields obtained were 2.52% ± 0.530 and 3.12% ± 0.601 for
lavender and lavandin respectively, with the latter cultivar producing a greater maximum
yield relative to lavender.
88
Table 8 Percentage yields obtained via Soxhlet using ethanol or hexane as solvent
Solvent Percent Yield (w/w) ± Std.Dev.a
Lavender Lavandin
Ethanol 11.75±1.114 16.08±1.260
Hexane 12.60±1.341 12.28±1.166 a
Std.Dev.-mean standard deviation
Table 9 Percentage yield of Lavandula spp. obtained via SFE-CO2 at various experimental conditions
Cell Temperature
(°C)
Percent Yieldb (w/w) ± Std.Dev.a
Lavender Lavandin
Pressure (psi) 2000 2500 3000 2000 2500 3000
40 2.02±0
.531 1.97± 0.848 2.35±0.707 1.00± 0.212 2.75± 0.424
2.87± 0.176
60 1.42±0
.247 2.30± 0.217 2.52±0.530 2.17± 0.318 2.35± 0.212
3.12± 0.601
80 1.57±0
.459 1.97± 0.035 2.43±0.206 1.17± 0.318 1.30± 0.100
1.40± 0.281
aStd.Dev.-mean standard deviation;
bextraction time t= 20 min.
Table 10 Percentage yield of Lavandula spp. obtained via SWE at various extraction temperatures
Percent Yieldb (w/w) ± Std.Dev.a
Lavender Lavandin
Cell Temperature
(°C) 100 120 140 100 120 140
2.24±0.23 3.75±0.05 4.00±0.16 2.98±0.11 3.06±0.32 3.37± 0.01
aStd.Dev.-mean standard deviation;
bextraction time t= 12 mins
89
Changes in the pressure and temperature parameters demonstrated an influence on the
SFE-CO2 yield. A 3D response surface plot design of the SFE-CO2 of the Lavandula
samples yield using a two factor, temperature and pressure, response are illustrated in
Figures 12 and 13. The effect of supercritical fluid temperature and pressure on the
overall extraction yield was a linear relationship for the lavender sample. Density
enhancement via pressure increase was observed. An increase in yield was attributed
to the overlap of solute vapor pressure enhancement to supercritical fluid density
decrease with increasing temperature (Ghoreishi et al., 2012).
The response surface plot of the lavandin sample did not follow the same trend as
lavender. The yield increased with pressure as expected with density enhancement via
pressure, however two distinguished trends of the overall yield were observed with
increasing temperature. The yield increased until it reached a maximum value at 60°C,
followed by a decreasing region. Ghoreishi et al. (2012) suggests that the first region of
increasing temperature is a result of solute vapor pressure enhancement followed by
retrograde solubility as solute vapor pressure becomes equivalent to the effect of
carbon dioxide density and then subsequently the effect of density decrease prevails
the vapor pressure enhancement. Furthermore, the variability in solvent volumetric flow
rate within the given process conditions was noted and may explain the inconsistencies
observed during the lavandin extraction. Pare and Belanger (1997) have suggested that
the solvent flow rate does not influence the solvating power, given that density is a
90
Figure 12. A 3D response surface plot of yields obtained via SFE-CO2 of lavender with respect to pressure and temperature
Figure 13. A 3D response surface plot of yields obtained via SFE-CO2 of lavandin with respect to pressure and temperature
1.565 1.659 1.752 1.846 1.94 2.034 2.127 2.221 2.315 2.409 above
SFE-CO2 OF LAVENDER
1.328 1.519 1.71 1.901 2.092 2.282 2.473 2.664 2.855 3.046 above
SFE-CO2 OF LAVANDIN
91
function of pressure and temperature, but rather holds a secondary effect on mass
transfer properties. The study by Akgun, Akgun and Dincer (2000) also supports this
theory stating that the extraction process is controlled by intraparticle mass-transfer by
diffusion, which can be controlled by flow rate, in addition to solute solubility in the
supercritical carbon dioxide.
Additionally, some inconsistencies in the results may be explained by the sensitive
function of the pressure and temperature parameters. Pare and Belanger (1997) point
out that good thermal and pressure control are indispensable in SFE-CO2. The
temperature control was in check throughout the extraction process as it was digitally
set. In contrast, the pressure control was experimentally challenging and fluctuated,
falling within a given range as noted in section 3.2.2.2.2. The gas loading factor
responds analogously with pressure which in turn affects the extraction power of carbon
dioxide due to the solvent density and solute vapor pressure changes (Varona et al.,
2008; Akgun, Akgun and Dincer, 2000).
5.1.3 SWE
In this study, the yield was calculated based on a total extraction time of 12 minutes.
The yield obtained via SWE of the Lavandula samples are demonstrated in Table 10.
The percentage yield ranged between 2.24 and 4.00% (w/w). The overall yield trend
increased with increasing temperature for both Lavandula spp. This is in agreement with
reported literature (Giray et.al, 2008). The highest percentage was obtained by means
92
of SWE at cell temperature of 140°C with yield of 4.00% and 3.37% for lavender and
lavandin respectively. The characteristic differences between the two Lavandula
cultivars are not observed with SWE under the selected processing conditions. In this
research, the pressure was generally consistent and the pressure response was not
studied for the SWE. The experimental conditions fell within the pressure range studied
by Giray et al. (2008) who demonstrated that pressure ranging from 40-90 atm did not
have a significant effect on the yield in SWE. It is suggested that SWE is successful in
extracting all of the essential oil from the plant without the precipitation of cuticular
waxes.
93
Chapter 6 Study of selected quality parameters of the Lavandula spp.
extracts obtained by conventional method and critical fluid technology
In the present study, it was found that all extracts obtained by traditional Soxhlet and
SFE-CO2 gave rise to high quality Lavandula spp. extracts as the ratio of linalyl acetate
to linalool was greater than 1. This can be determined via Tables 12-16. The ester
content of the Lavandula spp. is of greatest significance in the flavour quality as linalyl
acetate, the ‘character impact’ compound, determines the fine quality and market value
of Lavandula spp. oils. The extracts obtained by SWE produced a hydrolysis reaction of
linalyl acetate to linalool and therefore the quality marker ratio was inferior to 1 or
unresolved.
The chromatographic results are reflective of the composition of each extract obtained
and thus could suggest similar oil composition in accordance to the ISO International
Standard for lavender and lavandin. The semi-quantitative data determined by GC-MS
area normalization allowed for an evaluation of the chemical composition of the
Lavandula spp. extracts with particular attention brought to the three key Lavandula
spp. quality markers (linalyl acetate, linalool, and camphor). In this study, not all of the
extracts were comparable to ISO 3515:2002 lavender oil and ISO 8902:1986 lavandin
oil standards. When comparing the three proposed methods, the lavender (Lavandula
Angustifolia ‘Royal Velvet’) gave rise to the highest Lavandula spp. Quality extracts
94
obtained by mean of Soxhlet technique with hexane as solvent and SFE-CO2 (at 2000
psi and 80°C) as it reflected all the ISO standards criteria with respect to the linalyl
acetate, linalool, and camphor.
Considering the individual constituents, the linalyl acetate content in lavender extracts
obtained via Soxhlet (using hexane and ethanol as solvents) and SFE-CO2 (2000 psi
and 40-80°C, 2500 psi and 80°C, 300 psi and 80°C) reflected the acceptable range set
by ISO 3515:2002 for lavender oil. The ISO 8902:1986 lavandin oil standard was
comparable with respect to linalyl acetate for all Soxhlet extracts and SFE-CO2 (2000
psi and 60°C, 2500 psi and 40-60°C).
The linalool percentage in the lavender extracts obtained via Soxhlet (hexane and
ethanol) and SFE-CO2 (2000 psi and 80°C) reflected the ISO 3515:2002 lavender oil
compositional standard. Soxhlet (ethanol) and SFE-CO2 extracts did not reflect the ISO
8902:1986 lavandin oil standard. The linalool content of the SWE extracts were
significantly above the two ISO Lavandula oil compositional standards for linalool due to
hydrolysis of linalyl acetate.
With respect to the camphor content, the lavender extracts obtained by Soxhlet
(hexane), SFE-CO2 and SWE (6-12 min fractions) reflected the ISO 3515:2002 lavender
oil standard. The ISO 8902:1986 lavandin oil standard was not met in relation to the
camphor.
95
Chapter 7 General Conclusions
Great interest in the application of critical fluid technology for the isolation of food and
biological products has been demonstrated by the chemical, pharmaceutical,
nutraceutical and food industries. This research was aimed to investigate the flavour
extractives obtained by natural processes such as traditional Soxhlet solvent extraction
and critical fluid technology, SFE-CO2 and SWE, using North American Lavandula
Angustifolia ‘Royal Velvet’ and Lavandula x Intermedia ‘Grosso’ cultivars. The chemical
composition of the volatile extractives of Lavandula species was determined by GC-MS.
The mobilization of critical quality components (linalyl acetate, linalool, camphor) of the
Lavandula spp. plant material were studied.
In summary,
1) Supercritical fluid technology using carbon dioxide can be suggested as a good
extractant for the extraction of natural Lavandula flavours. It is a rapid (20 min
vs. 12 hrs) and a sustainable extraction method relative to conventional
alternatives. Other advantages include improved product quality and safety, and
minimized energy requirements through the use of non-toxic solvent under mild
operating conditions.
96
2) High quality, natural Lavandula spp. extracts may be obtained via Soxhlet and
critical fluid technology by SFE-CO2 as the ratio of linalyl acetate to linalool was
greater than 1. Moreover, Lavandula Angustifolia extracts via Soxhlet using a
hexane as solvent and SFE-CO2 (2000 psi and 80°C) reflected the chemical
composition of ISO quality standards with respect to linalyl acetate, linalool, and
camphor content.
3) Despite its advocacy as a quick sustainable alternative in extraction techniques,
SWE did not give rise to high quality Lavandula spp. extracts with the
experimental conditions set in the present study.
4) Selective fractionation is easily achieved with SWE. Subcritical water is however
suggested as a good solvent for natural extractives which are selective towards
valuable oxygenated compounds.
The results fulfill the main objective of this project which was to evaluate sustainable
extraction techniques for the isolation of high quality Lavandula spp. extracts for
application in the flavour industry as a natural ingredient.
97
It is important to note that standardisation of natural flavour extracts is a great
challenge as their molecular composition can vary widely. Differences in the results
may be attributable to various sources such as environmental conditions (i.e. light
intensity, temperature, altitude, time of year, geographic distribution), horticultural
practices (i.e. watering practices, fertilizers), genotypes of the plant, drying conditions,
and extraction and separation technology.
In future work, further investigation is needed to understand the effect of solvent flow
rate in SFE-CO2. Quantitation studies should also be carried out. Further optimization
studies would be required on the extraction method efficiencies of SFE-CO2 for
industrial applications. Moreover, additional studies are required to understand the
physico-chemical properties of the natural isolated extracts is necessary for their direct
use in different food and beverage matrices.
98
REFERENCES
Adasoglu, N., S. Dincer, and E. Bolat. (1994). “Supercritical-Fluid Extraction of Essential
Oil from Turkish Lavender Flowers”. The Journal of Supercritical Fluids. 7 (2): 93.
Akgun, Mesut, Nalan A Akgun, and Salih Dincer. (2000). “Separations - Extraction and
Modeling of Lavender Flower Essential Oil Using Supercritical Carbon
Dioxide”. Industrial & Engineering Chemistry Research. 39(2): 473.
Attokaran, M.. Natural food flavors and colorants. Hoboken: Wiley. 2011.
Ayala, R.S. and Luque de Castro M.D. (2000) “Continuous Subcritical water extraction
as a useful tool for isolation of edible essential oils”. Food Chemistry. 75(1):109-113.
Beckman, Eric J. (2004). “Supercritical and near-critical CO2 in green chemical
synthesis and processing”. Journal of Supercritical Fluids. 28(2): 121–191
Bisset, N.L. Lavandulae floes herbal drugs and phytopharmaceuticals. Stuttgart: CRC
Press, 1994.
Biswas, K. K., Foster, A. J., Aung, T. and Mahmoud, S. S. (2009). “Essential oil
production: relationship with abundance of glandular trichomes in aerial surface of
plants”. Acta Physiologiae Plantarum. 31(1): 13-19.
Burdock, G. A. and Fenaroli, G. Fenaroli's handbook of flavor ingredients. Boca Raton,
Fla: CRC Press, 2005.
99
Cavanagh, H. M. A. and Wilkinson, J. N. (2002). “Biological activities of lavender
essential oil”. Phytotherapy Research. 16(4): 301-308.
Chemat, F., M.E. Lucchesi, J. Smadja, L. Favretto, G. Colnaghi, and F. Visinoni. (2006).
“Microwave accelerated steam distillation of essential oil from lavender: A rapid, clean
and environmentally friendly approach”. Analytica Chimica Acta. 555 (1): 157-160.
Chizzola, R. (2013). Regular Monoterpenes and Sesquiterpenes (Essential oils). In K.
G. Ramawat and J. M. Merillon (Eds.), Natural products: Phytochemistry, botany and
metabolism of alkaloids, phenolics and terpenes. (pp.2973-3008). Berlin: Springer.
Codex Alimentarius Commision, Joint FAO/WHO Food Standards Programme Codex
Committee on Food Additives and Contaminants.Discussion Paper on Flavouring
Agents. 37th Session, The Hague, the Netherlands, 25-29 April 2005.
Dapkevicius, A., Venskutonis, R., van Beek, T. A. and Linssen, J. P. H. (1998).
“Antioxidant activity of extracts obtained by different isolation procedures from some
aromatic herbs grown in Lithuania”. Journal of the Science of Food and Agriculture.
77(1): 140-146
Da Porto, C., Decorti, D. and Kikic, I. (2009). “Flavour compounds of Lavandula
angustifolia L. to use in food manufacturing: Comparison of three different extraction
methods”. Food Chemistry. 112(4): 1072-1078.
DöhlerGroup. Blossom Flavors. Retrieved October 27, 2014, from
www.doehler.com/en/landingpages/blossom-flavours.html
100
Dudareva, Natalia, Florence Negre, Dinesh A. Nagegowda, and Irina Orlova. (2006).
“Plant Volatiles: Recent Advances and Future Perspectives”. Critical Reviews in Plant
Sciences. 25(5): 417-440.
Eikani, Mohammad H., Fereshteh Golmohammad, Soheila Shokrollahzadeh, Mehdi
Mirza, and Soosan Rowshanzamir. (2008). “Superheated Water Extraction of Lavandula
Latifolia Medik Volatiles: Comparison with Conventional Techniques”. Journal of
Essential Oil Research. 20(6): 482-487.
Ghoreishi, Seyyed M., Kamali, Hossein, Ghaziaskar, Hasan S., and Dadkhah, Ali A.
(2012). “Optimization of Supercritical Extraction of Linalyl Acetate from Lavender via
Box-Behnken Design”. Chemical Engineering Technology. 35(9): 1641–1648.
Giray, E. Sultan, Saliha K r c , D. Alpaslan Kaya, Murat Türk, zgür Sönmez, and
Memet nan. (2008). “Comparing the effect of sub-critical water extraction with
conventional extraction methods on the chemical composition of Lavandula
stoechas". Talanta. 74(4): 930-935.
Gonςalves, S and Romano, A. (2013). Micropropagation of Lavandula spp.. In M.
Lambardi, E. A. Ozudogru and S. M. Jain (Eds.), Protocols for micropropagation of
selected economically-important horticultural plants (pp.189-198). New York: Humana
Press.
Hallahan, D. L. (2000). Monoterpenoid Biosynthesis in Gladular Trichomes of Labiate
Plants. In J. A. Callow (Ed.), Advances in Botanical Research incorporating advances in
plant pathology-Plant Trichomes (pp. 77-111). London: Academic press.
101
Herrero, Miguel, Alejandro Cifuentes, and Elena Iba ez. (2006). “Sub- and supercritical
fluid extraction of functional ingredients from different natural sources: Plants, food-by-
products, algae and microalgae:A review”. Food Chemistry. 98(1): 136-148.
Iriti, M., Colnaghi, G., Chemat, F., Smadja, J., Faoro, F. and Visinoni, F. A. (2006).
“Histo-cytochemistry and scanning electron microscopy of lavender glandular trichomes
following conventional and microwave-assisted hydrodistillation of essential oils: a
comparative study”. Flavour and Fragrance Journal. 21(4): 704-712.
Kamali H, Jalilvand, M.R. and Aminimoghadamfarouj, N. (2012). “Pressurized fluid
extraction of essential oil from Lavandula hybrida using a modified supercritical fluid
extractor and a central composite design for optimization”. Journal of Separation
Science. 35(12): 1479-85.
Kara, N. And Baydar, Hasan. (2013). “Determination of lavender and lavandin cultivars
(Lavandula sp.) containing high quality essential oil in Isparta, Turkey”. Turkish Journal
of Field Crops. 18(1): 58-65.
Lahlou, M. (2004). “Essential oils and fragrance compounds: bioactivity and
mechanisms of action”. Flavour and Fragrance Journal. 19(2): 159-165.
Landelle C, G Francony, NF Sam-La , Y Gaillard, F Vincent, I Wrobleski, and V Danel.
2008. “Poisoning by lavandin extract in a 18-month-old boy”. Clinical Toxicology
(Philadelphia, Pa.). 46(4): 279-81.
102
Lee, K. G. and Shibamoto, T. (2002). “Determination of antioxidant potential of volatile
extracts isolated from various herbs and spices”. Journal of Agricultural and Food
Chemistry. 50 (17): 4947-4952.
Lis-Balchin, Maria. Lavender, The Genus Lavandula. New York: Taylor & Francis, 2002.
Mart n, , Silva, V., Pérez, L., Garc a-Serna, J., and Cocero, M. J. (2007). “Direct
Synthesis of Linalyl Acetate from Linalool in Supercritical Carbon Dioxide: A
Thermodynamic Study”. Chemical Engineering & Technology. 30(6): 726-731.
Matos, F., Miguel, M. G., Duarte, J., Venancio, F., Moiteiro, C., Correia, A. I. D.,
Figueiredo, A. C., Barroso, J. G. And Pedro, L. G. (2009). “Antioxidant Capacity of the
Essential Oils From Lavandula luisieri, L. stoechas subsp lusitanica, L. stoechas subsp
lusitanica x L. luisieri and L. viridis Grown in Algarve (Portugal)”. Journal of Essential Oil
Research. 21 (4): 327-336.
McNaughton, Virginia. Lavender the Grower’s Guide. Portland: Timber Press Inc., 2000.
Newly Weds Foods. Food Safety & Functional Ingredients. Retrieved May 12, 2014
from http://www.newlywedsfoods.com/products/food-safety-functional-ingredients/
Oszagyan M, Simandi B, Sawinsky J, Kery A, Lemberkovics E. and Fekete J. (1996)
“Supercritical fluid extraction of volatile compounds from lavandin and thyme.” Flavour
and Fragrance Journal. 11(3):157-165.
103
Ozel, M. Z. and Gogus, F. (2014). Subcritical Water as a Green Solvent for Plant
Extraction. In F. Chémat and M. A. Vian (Eds.), Alternative solvents for natural products
extraction. (pp. 73-90). Heidelberg: Springer.
Pallado, Paolo, Graziano Tassinato, Martina D'Alpaos, and Pietro Traldi. (1997). “Gas
chromatography/mass spectrometry in aroma chemistry: a comparison of essential oils
and flavours extracted by classical and supercritical techniques”. Rapid
Communications in Mass Spectrometry. 11(12): 1335-1341.
Platt, Ellen Spector. Lavender, How to Grow and Use the Fragrant Herb.
Mechanicsburg: Stackpole Books, 2009.
Pourmortazavi, S.M., and S.S. Hajimirsadeghi. (2007). “Supercritical fluid extraction in
plant essential and volatile oil analysis”. Journal of Chromatography A. 1163(1-2): 2-24.
Reineccius. G. A. (2007). Flavour-Isolation Techniques. In R. G. Berger (Ed.), Flavours
and fragrances: Chemistry, bioprocessing and sustainability. (pp. 409-425). Berlin:
Springer.
Reverchon E, Della Porta G. and Senatore F. (1995) “Supercritical CO2 extraction and
fractionation of lavender essential oil and waxes”. Journal of Agricultural and Food
Chemistry. 43(6):1654-1658.
Richter, B. E. (1992). Supercritical Fluid Extraction Methods. In T. Cairns & J. Sherma
(Eds.), Emerging strategies for pesticide analysis (pp. 51-70). Boca Raton, Fla: CRC
Press.
104
Schulz, Volker, Hansel, Rudolf, and Blumenthal, Mark. Rational phytotherapy: reference
guide for physicians and pharmacists. Berlin: Springer. 2004.
Surburg, Horst and Panten, Johannes. Common Fragrance and Flavor Materials.
Weinheim: Wiley and Co., 2006.
Topal, U., Sasaki, M., Goto, M., and Otles, S. (2008). “Chemical compositions and
antioxidant properties of essential oils from nine species of Turkish plants obtained by
supercritical carbon dioxide extraction and steam distillation”. International Journal of
Food Sciences and Nutrition. 59(7-8): 619-634.
Torras-Claveria, L., Jauregui, O., Bastida, J., Codina, C. and Viladomat, F. (2007).
“Antioxidant activity and phenolic composition of lavandin (Lavandula x intermedia
emeric ex loiseleur) waste”. Journal of Agricultural and Food Chemistry. 55(21): 8436-
8443.
Varona, S., A. Martin, M.J. Cocero, and T. Gamse. (2008). “Supercritical carbon dioxide
fractionation of Lavandin essential oil: Experiments and modeling”. The Journal of
Supercritical Fluids. 45(2): 181-188.
Waseem, Rabia, and Kah Hin Low. (2015). “Advanced analytical techniques for the
extraction and characterization of plant-derived essential oils by gas chromatography
with mass spectrometry”. Journal of Separation Science. 38(3): 483-501.
Wesolowska, Aneta, Jadczak, Dorota, and Grzeszczuk, Monika. (2010) “Influence of
distillation time on the content and composition of essential oil isolated from lavender
(Lavandula angustifolia Mill.)”.Herba Polonica. 56: 3.
105
Xia, Yan, Mario E. Rivero-Huguet, Brianna H. Hughes, and William D. Marshall. (2008).
“Isolation of the sweet components from Siraitia grosvenorii”. Food Chemistry. 107(3):
1022-1028.
106
Appendix A
Table 11. Summary of the classification of tentatively identified constituents
Oxygenated Hemiterpenes 2-Pentanal
Monoterpene Hydrocarbons
Camphene α-Pinene Limonene
Z-Ocimene E-Ocimene
Oxygenated Monoterpenes
Eucalyptol cis-Linalool oxide
trans-Linalool oxide Linalool Camphor Borneol Terpinen-4-ol Terpineol Cis-Geraniol Carvacrol Eugenol 8-Hydroxylinalool
Sesquiterpene Hydrocarbons
β-Caryophyllene
α-Santalene α-Bergamotene Farnesene Germacrene D Bisabolene Cedrene
Oxygenated Sesquiterpenes
Santalol
α-Caryophyllene oxide α-Cadinol α-Bisabolol
Other Oxygenated Compounds
2-Hexenal Linalyl acetate Lavandulyl acetate Geranyl acetate
107
Table 12. Chemical Composition of Lavandula spp. extract obtained via Soxhlet
Peak number
Tentative Identification LRIb LRIrefc
Lavender Lavandin
Ethanol Hexane Ethanol Hexane
1 2-Hexenal d, 866
2 2-Pentanal d, 911
3 N.I.g 933
4 Camphene d 965
0.34
5 α-Pinened, e 968 968 0.67 2.56 1.36 2.87
6 N.I.g 981
7 N.I.g 990
8 Limonene d 1000
0.57
9 Eucalyptol d, f 1002 1005 0.45 0.48
2.06
10 Z-Ocimene d, f 1010 1017 0.53 2.43
1.69
11 E-Ocimene d,f 1021 1027 0.47 1.65 0.9 1.72
12 cis-Linalool oxide d,f 1046 1045 1.53 1.4
0.33
13 trans-Linalool oxide d,f 1064 1059 1.38 1.19
0.17
14 Linalool d,f 1082 1074 33.2 31.6 22.16 23.96
15 Octen-acetate d,e 1090 1107 0.62 0.68
0.44
16 Camphor d,e 1132 1137 0.6
3.54 5.94
17 N.I.g 1156
0.39
0.7 0.57
18 Borneol d,e 1163 1161 2.27 2.21 6.11 4.64
108
19 N.I.g 1170
20 Terpinen-4-ol d,j 1173 1186 2.41 2.15 1.72 1.67
21 N.I.g 1182
1.12 1.02
22 Terpineolj 1190
1.08 0.78
0.33
23 N.I.g 1224
24 N.I.g 1231
25 Linalyl acetate d,e 1252 1254 36.8 35.96 36.5 32.77
26 Geraniol d 1284
1.91 2.05 2.73 2.54
27 Carvacrold 1291
28 N.I.g 1323
0.47 0.78
29 Eugenol d, f 1331 1327
30 8-Hydroxylinaloold 1334
1.22 1.04 0.84 0.19
31 N.I.g 1345
0.88
32 Limonene oxide/dioxided 1348
0.57
33 Neryl Acetatej 1359
0.8 0.7 0.93
34 N.I.g 1373
35 Geranyl acetatee,d 1377 1377 0.56 1.72 1.3 1.79
36 β-Caryophyllenee,d 1411 1413 2.19 2.54 2.21 2.3
37 α-Santalenee,d 1413 1414 3.39 4.04
0.38
38 α-Bergamotenej 1427
0.55 0.86
0.27
39 N.I.g 1445
1.88
109
40 Farnesenee,d 1453 1453 1.08 0.88 2.25 0.23
41 Germacrene D d, f 1474 1474 0.5
2.55 2.07
42 N.I.g 1504
1.11 0.62
43 Bisabolene d,j 1507
1.03 0.66
44 N.I.g 1511
1.33 0.76
45 Santalold 1515
46 Cedrene d 1522
1.3 0.84
47 α-Caryophyllene oxidee,d 1575 1573 0.91 1.19
48 Ethyl phthalate d 1587
1.89
49 α-Cadinol d N.D.h
50 N.I.g N.D.h
3.77 1.79
51 α-Bisabolol d,e N.D.h 1677
5.89 3.1
Monoterpene Hydrocarbons 2.01 6.64 2.26 6.85
Oxygenated Monoterpenes 46.62 42.9 37.1 41.83
Sesquiterpene Hydrocarbons 7.71 8.32 9.34 6.75
Oxygenated Sesquiterpenes 0.91 1.19 5.89 3.1
110
Other Oxygenated Compounds 39.27 39.73 40.53 37.1
Total of characterized compounds (%) 96.52 98.78 95.12 95.63
Total of Oxygenated Compounds (%) 86.8 83.82 83.52 82.03 Total of Hydrocarbons (%) 9.72 14.96 11.6 13.6
% Figures are their relative proportions as percent of total area; The % RSD values ranged from 2-20%; b Linear Retention Index;
d NIST library;
e- Iriti et al., 2006;
f Matos et al.,
2009; gN.I.-not identified;
hN.D.-not determined;
iLee and Shibamoto, 2002;
j Da Porto et al., 2009
111
Table 13. Chemical Composition of Lavender extract obtained via SFE-CO2
Lavender
Peak number
Tentative Identification
LRIb LRIrefc
2000 psi 2500 psi 3000 psi
40°C 60°C 80°C 40°C 60°C 80°C 40°C 60°C 80°C
1 2-Hexenal d 866
2 2-Pentanal d 911
3 N.I.g 933
4 Camphened 965
5 α-Pinened, e 968 968 1.42 1.21 1.45
0.88
6 N.I.g 981
7 N.I.g 990
8 Limonene d, 1000
9 Eucalyptol d, f 1002 1005
10 Z-Ocimene d, f 1010 1017
11 E-Ocimene d,f 1021 1027
0.96
12 cis-Linalool
oxide d,f 1046 1045
0.71
13 trans-Linalool
oxide d,f 1064 1059
0.69
14 Linalool d,f 1082 1074 18.87 23.59 24.92 7.79 13.06 18.52 15.4 16.8 20.62
15 Octen-acetate
d,e 1090 1107
112
16 Camphor d,e 1132 1137
17 N.I.g 1156
0.51
18 Borneol d,e 1163 1161 3.24 3.68 2.09 2.7 3.02 3.42 3.42 3.64 4.07
19 N.I.g 1170
0.57
20 Terpinen-4-ol d,j 1173 1178 1.89 1.73 1.27 1.73 1.56 1.6 1.9
21 N.I.g 1182
1.45 1.32 6.12 1.54 1.43 1.71 1.87 2
22 Terpineolj 1190 1196 2.27 2.26 3.53 3.37 3.74 3.94 4.03
23 N.I.g 1224
24 N.I.g 1231
25 Linalyl acetate
d,e 1252 1254 30 30.6 29.88 15.96 19.68 27.09 24.31 22.7 27.53
26 Geraniol d 1284
2.38 2.02 1.7 2.13 2.29 2.01
27 Carvacrold 1291
0.95 2.64 1.58 1.51 1.69 1.83
28 N.I.g 1323
2.8 2.32 6.14 3.39 3.15 3.48 3.81
29 Eugenol d, f 1331 1327
30 8-
Hydroxylinaloold 1334
3.05 3.53 2.73 7.27 4.52 4.19 4.51 4.71 4.1
31 N.I.g 1345
2.52 2.82 1.95 4.75 2.79 2.55 2.85 2.95
32 Limonene
oxide/dioxided 1348
1.4 1.52 1.08 2.27 1.49 1.53 1.59 1.46
33 Neryl Acetatej 1359 1366
0.5
34 N.I.g 1373
35 Geranyl 1377 1377 1.51 1.36 1.37
1.31
113
acetatee,d
36 β-
Caryophyllenee,d 1411 1413 3.42 2.9 2.68 2.72 2.21 2.92 2.93 2.45
37 α-Santalenee,d 1413 1414 5.78 5.04 4.5 5.48 4.02 4.71 4.97 4.24 3.47
38 α-Bergamotenej 1427 1418 0.9 0.8 0.62 0.61 0.61 0.4 0.38
39 N.I.g 1445
1.54
1.63
1.75 1.95
40 Farnesenee,d 1453 1453 3.2 2.96 1.87 3.2 2.69 2.4 3 2.64 2.25
41 Germacrene D d,
f 1474 1474 1.37 1.11
1.1
1.34 0.93
42 N.I.g 1504
43 Bisabolene d,j 1507 1500 0.89 1.06
1.41 1.16 1.16 1.22 1.58
44 N.I.g 1511
0.89
45 Santalold 1515
1.2 0.7 2.12 1.52 1.08 1.27 1.31 1.33
46 Cedrene d 1522
47 α-Caryophyllene
oxidee,d 1575 1573 3.59 4.3 2.89 7.61 4.76 4.17 4.63 4.53 5.34
48 Ethyl phthalate 1587
49 α-Cadinol d N.D.h
50 N.I.g N.D.h
1.75 1.99 1.31 4.04 2.03 1.8 2.27 2.22 2.4
51 α-Bisabolol d,e N.D.h 1677 1.23 1.43 0.95 2.8 1.54 1.34 1.6 1.63 1.74
52-61 N.I.g (C>15)
5.05 7.36 5.55 16.4 16.95 8.31 6.82 9.16 17.66
114
Monoterpene Hydrocarbons 1.42 1.21 2.41 0 0 0.88 0 0 0
Oxygenated Monoterpenes 33.1 32.32 39.18 22.67 30.17 36.4 34.2 35.99 34.72
Sesquiterpene Hydrocarbons 15.56 13.87 9.67 11.4 12.04 11.8 13.8 11.86 7.3
Oxygenated Sesquiterpenes 4.82 6.93 4.54 12.53 7.82 6.59 7.5 7.47 8.41
Other Oxygenated Compounds 33.89 31.96 33.27 15.96 21.38 29.22 27.91 24.71 27.53
Total of characterized compounds (%) 88.79 86.29 89.07 62.56 71.41 84.89 83.41 80.03 77.96
Total of Oxygenated Compounds (%) 71.81 71.21 76.99 51.16 59.37 72.21 69.61 68.17 70.66 Total of Hydrocarbons (%) 16.98 15.08 12.08 11.4 12.04 12.68 13.8 11.86 7.3
% Figures are their relative proportions as percent of total area; The % RSD values ranged from 2-28%; b Linear Retention Index;
d NIST library;
e- Iriti et al., 2006;
f Matos et al.,
2009; gN.I.-not identified;
hN.D.-not determined;
iLee and Shibamoto, 2002;
j Da Porto et al., 2009
115
Table 14. Chemical Composition of Lavandin extract obtained via SFE-CO2
Lavandin
Peak number
Tentative Identification
LRIb LRIrefc
2000 psi 2500 psi 3000 psi
40°C 60°C 80°C 40°C 60°C 80°C 40°C 60°C 80°C
1 2-Hexenal d 866
2 2-Pentanal d 911
3 N.I.g 933
4 Camphene d 965
5 α-Pinened, e 968 968
1.77 0.92 1.65 1.38 0.99 0.61 0.82 0.7
6 N.I.g 981
7 N.I.g 990
8 Limonene d, 1000
9 Eucalyptol d, f 1002 1005
10 Z-Ocimene d, f 1010 1017
0.69
0.67 0.56
11 E-Ocimene d,f 1021 1027
1.28 0.65 1.21 1.02 0.73 0.58
12 cis-Linalool
oxide d,f 1046 1045
13 trans-Linalool
oxide d,f 1064 1059
14 Linalool d,f 1082 1074 1.73 20.29 12.57 12.92 17.14 14.62 3.63 10.3 6.74
15 Octen-acetate
d,e 1090 1107
116
16 Camphor d,e 1132 1137
1.97 0.89 0.37 1.29 0.85
17 N.I.g 1156
0.77
0.49
18 Borneol d,e 1163 1161 2.37 5.72 6.34 4.85 5.84 5.53 3.97 4.65 5.1
19 N.I.g 1170
20 Terpinen-4-ol d,j 1173 1178
1.62 1.32 1.27 1.53 1.34 0.51 1
21 N.I.g 1182
22 Terpineolj 1190 1196
23 N.I.g 1224
24 N.I.g 1231
25 Linalyl acetate
d,e 1252 1254 11.7 32.46 23.97 31.75 33.2 29.82 16.85 25.17 21.59
26 Geraniol d 1284
1.9 3.08 2.6 2.93 2.03 3.07 2.48 2.55 2.65
27 Carvacrold 1291
28 N.I.g 1323
29 Eugenol d, f 1331 1327
30 8-
Hydroxylinaloold 1334
1.62 0.46 1.16
1.07 1.35 1.08 1.5
31 N.I.g 1345
1.38
32 Limonene
oxide/dioxided 1348
33 Neryl Acetatej 1359 1366
0.63
34 N.I.g 1373
35 Geranyl 1377 1377
1.22
1.24 1.03 0.91
117
acetatee,d
36 β-
Caryophyllenee,d 1411 1413
3.57 2.34 4.11 4.14
3.04 2.12 2.39
37 α-Santalenee,d 1413 1414 1.77
0.57
2.21 0.91
38 α-Bergamotenej 1427 1418
39 N.I.g 1445
40 Farnesenee,d 1453 1453 5.23 3.03 4.4 4.64 3.55 4.03 6.29 4.08 5.18
41 Germacrene D d,
f 1474 1474 4.94 3.45 4.57 5.07 3.87 4.11 5.7 4.23 5.09
42 N.I.g 1504
3.49 1.32 2.35 2.3 1.79 2.08 3.18 2.39 3
43 Bisabolene d,j 1507 1500 2.54 1.26 2.01 1.94 1.49 1.79 2.54 1.82 2.55
44 N.I.g 1511
3.06 1.38 2.52 2.4 1.9 2.16 3.02 2.37 2.84
45 Santalold 1515
1.68
46 Cedrene d 1522
3.73 2.78 2.65 2.12 2.42 3.94 2.59 3.31
47 α-Caryophyllene
oxidee,d 1575 1573 1.36 0.99 0.67
1.2 0.98 1.09
48 Ethyl phthalate 1587
49 α-Cadinol d N.D.h
0.96
0.78 0.66
50 N.I.g N.D.h
13.94 3.94 8.9 6.93 5.34 7.08 11.23 9.78 11.32
51 α-Bisabolol d,e N.D.h 1677 11.24 6.47 13.83 10.53 8.51 11.31 18.24 15.55 18.46
52-61 N.I.g (C>15)
28.42 1.64 4.9 0 1.61 3.88 8.64 7.25 6.5
118
Monoterpene Hydrocarbons 0 3.74 1.57 3.53 2.96 1.72 0.61 1.4 0.7
Oxygenated Monoterpenes 7.62 33.14 24.88 22.34 27.83 26.48 11.94 19.58 15.99
Sesquiterpene Hydrocarbons 18.21 11.31 16.1 18.98 15.17 14.56 22.42 14.84 18.52
Oxygenated Sesquiterpenes 13.56 8.15 14.82 10.53 9.18 11.31 20.22 17.19 19.55
Other Oxygenated Compounds 13.6 36.76 26.57 35.92 36.26 33.8 19.33 27.72 24.24
Total of characterized compounds (%) 52.99 93.1 83.94 91.3 91.4 87.87 74.52 80.73 79
Total of Oxygenated Compounds (%) 34.78 78.05 66.27 68.79 73.27 71.59 51.49 64.49 59.78 Total of Hydrocarbons (%) 18.21 15.05 17.67 22.51 18.13 16.28 23.03 16.24 19.22
% Figures are their relative proportions as percent of total area; The % RSD values ranged from 4-25%;
b Linear Retention Index;
d NIST library;
e- Iriti et al., 2006;
f Matos et al.,
2009; gN.I.-not identified;
hN.D.-not determined;
iLee and Shibamoto, 2002;
j Da Porto et al., 2009
119
Table 15 Chemical Composition of Lavender extract obtained via SWE
Lavender
Peak number
Tentative Identificati
on LRI
b
LRI
refc
100°C 120°C 140°C
2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12
1 2-Hexenal
d
866
2 2-Pentanal
d
911
3 N.I.g 933
4 Camphene
d
965
0.92
5 α-Pinene
d,
e
968 968
6 N.I.g 981
7 N.I.g 990
8 Limonene
d,
1000
9 Eucalyptol
d, f
1002 100
5 1.06
1.57
1.35
1.15 1.37
1.18
1.14
10 Z-Ocimene
d, f
1010 101
7
0.75
11 E-Ocimene
d,f
1021 102
7
12 cis-Linalool
oxide d,f
1046
1045
8.03
8.29
6.91 0.92
13 trans-
Linalool oxide
d,f
1064 105
9 7.4
7.42
6.23
14 Linalool d,f
1082 107 49. 81. 85. 87. 80. 67. 48.2 81.2 86. 84. 68. 65. 55.6 80. 75. 75. 66. 73.
120
4 24 59 97 89 82 86 2 1 66 55 66 04 7 25 44 52 11 38
15 Octen-
acetate d,e
1090
1107
16 Camphor
d,e
1132 113
7 1.42
1.67
1.4 1.69
1.49 1.41
17 N.I.g 1156
5.42
18 Borneol d,e
1163 116
1 5.13
5.43
4.14
4.22
5.1 5.18 4.64
3.57
1.53 5.26
19 N.I.g 1170
1.73
1.67
4.45 5.89
20 Terpinen-
4-ol d,j
1173
1178
4.66
6.21
5.2 4.44 5.81 5.68
4.72
3.86 1.65
2.62
21 N.I.g 1182
3.89
1.89
3.89 1.7
6.84 0.69
22 Terpineolj 1190
1196
6.5 1.65
3.34
7.89
19.18
32.14
6.87 2.12 3.02
5.98
31.34
34.96
3.12 2.79
21.94
24.48
33.89
26.62
23 N.I.g 1224
24 N.I.g 1231
25 Linalyl
acetate d,e
1252
1254
1.04
26 Geraniol d
1284
27 Carvacrold 1291
28 N.I.g 1323
5.06
4.63
4.48
29 Eugenol d,
f 1331
1327
30 8-
Hydroxylinalool
d
1334
5.89
5.13
31 N.I.g 1345
121
32 Limonene
oxide/dioxide
d
1348
33 Neryl
Acetatej
1359 136
6
34 N.I.g 1373
35 Geranyl
acetatee,d
1377
1377
36 β-
Caryophyllene
e,d
1411 141
3
37 α-
Santalenee,
d
1413 141
4
38 α-
Bergamotene
j
1427 141
8
39 N.I.g 1445
40 Farnesene
e
,d
1453 145
3
41 Germacren
e D d,
f
1474 147
4
42 N.I.g 1504
43 Bisabolene
d,j
1507 150
0
44 N.I.g 1511
45 Santalold 1515
46 Cedrene d
1522
47 α-
Caryophyllene
1575 157
3
122
oxidee,d
48 Ethyl
phthalate 1587
49 α-Cadinol d
N.D.h
50 N.I.g N.D.
h
51 α-Bisabolol
d,e
N.D.h
1677
52-61
N.I.g (C>15)
Monoterpene Hydrocarbons 0 0 0 0 0 0 0.75 0.92 0 0 0 0 0 0 0 0 0 0
Oxygenated Monoterpenes 89.33
98.12
100 100 100 100 88.0
2 97.3
8 100 100 100 100
81.11
90.63
78.06
75.52
66.11
73.38
Sesquiterpene Hydrocarbons 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Oxygenated Sesquiterpenes 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Other Oxygenated Compounds 0 0 0 0 0 0 1.04 0 0 0 0 0 0 0 0 0 0 0
Total of characterized compounds (%)
89.33
98.12
100 100 100 100 89.8
1 98.3 100 100 100 100
81.11
90.63
78.06
75.52
66.11
73.38
Total of Oxygenated Compounds (%)
89.33
98.12
100 100 100 100 89.0
6 97.3
8 100 100 100 100
81.11
90.63
78.06
75.52
66.11
73.38
Total of Hydrocarbons (%) 0 0 0 0 0 0 0.75 0.92 0 0 0 0 0 0 0 0 0 0
% Figures are their relative proportions as percent of total area; The % RSD values ranged from 1-22%; b Linear Retention Index;
d NIST library;
e- Iriti et al., 2006;
f Matos et al.,
2009; gN.I.-not identified;
hN.D.-not determined;
iLee and Shibamoto, 2002;
j Da Porto et al., 2009
123
Table 16. Chemical Composition of Lavandin extract obtained via SWE
Lavandin
Peak numb
er
Tentative Identificat
ion LRI
b
LRI
refc
100°C 120°C 140°C
2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12
1 2-Hexenal
d
866
0.1
0.06
2 2-Pentanal
d
911
1.17
0.2 0.44
0.08
3 N.I.g 933
0.08
4 Camphene
d
965
0.47
5 α-Pinene
d,
e
968 968 1.31
1.69
1.27
1.99
4 0.94 1.37 1.88
2.97
3.53
3.24
1.24
1.82
2.49
3.77
2.79
6 N.I.g 981
0.1
7 N.I.g 990
0.17
8 Limonene
d,
1000
0.48
1.18
1.51
1.6 1.2 2.13
1.36
9 Eucalyptol
d, f
1002 100
5 5.21
4.69
4.51
4.87
2.29
4.07 4.39 5.13
4.5 5.31
3.98
4.44 3.72
3.48
3.77
3.5 2.78
10 Z-Ocimene
d, f
1010 101
7 0.13
0.65
0.6 0.51
0.54
11 E-Ocimene
d,f
1021 102
7 0.55
1.13
1.06
0.97
0.91
1.04
12 cis-
Linalool oxide
d,f
1046 104
5 2.36
2.69
3.04 0.09
13 trans-
Linalool oxide
d,f
1064 105
9 1.93
2.34
2.51 0.19
124
14 Linalool d,f
1082 107
4 42.29
46.62
53.87
55.47
57.83
60.85
42.12
49.16
52.53
58.17
66.73
63.41
46.43
50.31
57.49
59.59
63.98
56.77
15 Octen-
acetate d,e
1090
1107
0.32
0.66
0.2
16 Camphor
d,e
1132 113
7 19.38
11 20.45
18.77
15.23
9.75
19.58
24.4 19.5
9.93
3.12
0.81
23.15
22.83
14.44
3.84
0.53
17 N.I.g 1156
1.24
1.19 0.87 0.98
1.15
1 1.02
0.86
0.08
18 Borneol d,e
1163 116
1 11.85
26.65
12.28
12.04
11.64
12.12
14.33
13.77
13.42
11.24
6.17
2.28
13.98
14.21
12.32
6.56
1.93
0.72
19 N.I.g 1170
0.38
20 Terpinen-
4-ol d,j
1173
1178
4.37
8.05
4 3.88
3.7 4.13
4.35 4.1 3.95
3.79
2.84
1.8 4.19 3.99
3.92
3.26
1.78
1
21 N.I.g 1182
0.73
2.28
22 Terpineolj 1190
1196
1.88
6.5 3.03
4.06
4.74
6.85
2.55 1.93 2.61
5.08
9.72
16.93
2.26 2.7 5.5 12.96
19.97
18.83
23 N.I.g 1224
0.93
0.93
2.69
24 N.I.g 1231
0.11
25 Linalyl
acetate d,e
1252
1254
2.59
2.75
2.94
8.29
26 Geraniol d
1284
0.42
0.62
27 Carvacrold 1291
28 N.I.g 1323
29 Eugenol d,
f 1331
1327
30 8-
Hydroxylinalool
d
1334
0.6
125
31 N.I.g 1345
0.38
32 Limonene oxide/diox
ided
1348
33 Neryl
Acetatej
1359 136
6
0.19
34 N.I.g 1373
35 Geranyl
acetatee,d
1377
1377
0.32
36 β-
Caryophyllene
e,d
1411 141
3
37 α-
Santalenee
,d
1413 141
4
38 α-
Bergamotene
j
1427 141
8 1.16
39 N.I.g 1445
40 Farnesene
e,d
1453 145
3
41 Germacre
ne D d,
f
1474 147
4
42 N.I.g 1504
43 Bisabolen
e d,j
1507
1500
44 N.I.g 1511
45 Santalold 1515
46 Cedrene d
1522
126
47
α-Caryophyll
ene oxide
e,d
1575 157
3
48 Ethyl
phthalate 1587
49 α-Cadinol d
N.D.h
50 N.I.g N.D.
h
0.14
51 α-
Bisabolol
d,e
N.D.h
1677
52-61 N.I.g (C>15)
2.81
2.91
0.38
0.2
Monoterpene Hydrocarbons 2.94
0 1.69
1.27
1.99
4 0.94 1.37 1.88
5.93
6.1 6.41
0 1.24
1.82
5.11
5.9 5.73
Oxygenated Monoterpenes 89.33
93.49
95.29
94.67
93.27
89.14
90.67
96.69
95.51
88.98
84.17
73.14
97.74
96.06
92.67
77.88
71.19
62.92
Sesquiterpene Hydrocarbons 1.16
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Oxygenated Sesquiterpenes 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Other Oxygenated Compounds 0 0 0 0 0 0 0 0 0 0 0 3.01
0 0 0 2.75
2.94
9.23
Total of characterized compounds (%)
93.43
93.49
96.98
95.94
95.26
93.14
91.61
98.06
97.39
94.91
90.27
82.56
97.74
97.3
94.49
85.74
80.03
77.88
Total of Oxygenated Compounds (%)
89.33
93.49
95.29
94.67
93.27
89.14
90.67
96.69
95.51
88.98
84.17
76.15
97.74
96.06
92.67
80.63
74.13
72.15
127
Total of Hydrocarbons (%) 4.1 0 1.69
1.27
1.99
4 0.94 1.37 1.88
5.93
6.1 6.41
0 1.24
1.82
5.11
5.9 5.73
% Figures are their relative proportions as percent of total area; The % RSD values ranged from 1-15%; b Linear Retention Index;
d NIST library;
e- Iriti et al., 2006;
f Matos et al.,
2009; gN.I.-not identified;
hN.D.-not determined;
iLee and Shibamoto, 2002;
j Da Porto et al., 2009
