extraction of essential oil from lemongrass using supercritical carbon dioxide

In this study, Supercritical fluid Carbon Dioxide SC CO2 was used in the extraction of essential oil from two different parts of the lemongrass plant, the leaves and the stems.. 106 Figu

EXTRACTION OF ESSENTIAL OIL FROM LEMONGRASS

USING SUPERCRITICAL CARBON DIOXIDE

In Partial Fulfillment of the Requirements for the Degree of Doctor

of Philosophy in Chemical Engineering

Submitted by

HUYNH KY PHUONG HA

Chemical Engineering Department

De La Salle University – Manila

March 2008

The Supercritical Fluid Extraction (SCFE) process is a powerful technique to develop for separating products with high added value In this study, Supercritical fluid Carbon Dioxide (SC CO2) was used in the extraction of essential oil from two different parts of the lemongrass plant, the leaves and the stems

The batch extraction process was carried out in a stand alone compact unit ofSCFE system The lemongrass was loaded into the 500ml extractor with an up flow rate of 0.5 m3 per hour of CO2 The extraction temperature was varied from 350C to

500C while the extraction pressure applied was from 90 to 110 atmospheres and the length of material was varied from 4 mm to 8 mm The extraction time was set at 3.0 hours

This study intends to compare the composition of essential oil extracted from lemongrass leaves and lemongrass stems using SC CO2 and Steam Distillation The extracts from both methods were analyzed by GC-MS and the variations of thecompositions were reported

Furthermore, the study attempts to formulate statistical experimental design using Design-Expert Software (version 7.0.1) The effects of temperature, pressure and length of material were analyzed employing the Response Surface Methodology (RSM) technique The models of extraction for both leaves and stems wereestablished with the extraction yield and citral content of the essential oil at theestablished responses The optimum yields of essential oil from leaf and stem at the given extraction conditions were also determined

De La Salle University, Host Institution of Chemical Engineering inAUN/SEED-Net program

Ho Chi Minh City University of Technology

Chemical Engineering Laboratory and Chemistry Department Laboratory of

De La Salle University (DLSU)– Manila

Chemical Technology Laboratory of Mindanao State University – Iligan Institute of Technology (MSU-IIT)

Laboratory of Tokyo Institute of Technology (TIT), and KawasakiLaboratory-TIT, Tokyo, Japan

I would like to sincerely thank my research advisor, Prof Dr Julius B Maridable, Ph.D, Vice-Chancellor for Academics, DLSU, who kindly guided me on

how to conduct my research in the best way, who always took care of me not only as

a student but also as a son, who taught me not only knowledge in research but also behavior in a new environment Thank you for your advice, guidance, direction and endless enthusiasm for the subject I will forever look at you with deep awe and respect and I am very fortunate and happy for having the chance to study as his advisee

Also, I would like to gratefully thank my advisor, Prof Pag-asa D Gaspillo, Ph.D, Dean of the College of Engineering, who essentially inspired me to

do my best in performing my experiment as well as in writing my thesis and papers

Thank you for your appreciated support, valuable encouragement and considerable recommendations for my thesis I deepest thank you not only for the perfect knowledge, but also for the nice working atmosphere, and the large experiencegathered there, which all serves as a stable basis for further scientific research Thank you for your kindly in reviewing my papers, presentations and manuscript Thanking you is not really enough to convey the gratitude I feel I always have the deepest respect for you

Prof Junjiro Kawasaki, Ph D, my Japanese thesis adviser, who has taken

care of me as an adviser, Sensei, not only during the time I stayed and researched inJapan but also in the Philippines Thank you for kindly sharing your knowledge aswell as experience in research, Thank you for cherishing and supervising my research, as well as my life throughout my stay at TIT Thank you for being happy to communicate your knowledge and experience to students, who always have the deepest respect for you I wish that you will always have good health

Dr Roberto M Malaluan, my thesis co-adviser from Mindanao State

University – Iligan Institute of Technology (MSU-IIT), thank you for being an anchor of strength during some of the most trying times of this research Thank you for giving me free reign in the supercritical laboratory Thank you for unselfishly sharing with me your veritable knowledge in the supercritical area, which proved to

be very essential for my research, and for always appreciating the worksaccomplished

From my heart, I deeply thank the members of my Thesis Examination Panel for their valuable time, comments and suggestions to improve my research: Prof Dr Leonila C Abella, Chair of the panel, Prof Dr Raymond G Tan, Prof.

Dr Bonifacio Doma, Prof Dr Jonathan Salvacion, Dr Carmela Centeno and

Prof Dr Servillano Olano Jr., for his comments and suggestions during the

proposal defense

Maraming salamat Po.

I would also like to extend my deepest gratitude to:

Ms Gladys Paz Cruz, coordinator of AUN/SEED-Net scholars, for being

very supportive and encouraging during my two years in the Philippines Thank you for helping me every time, especially when I had concerns Thank you for your warmth and kindness in helping me not only as a scholar but also as a brother Best Wishes to You and Your Family!

Ms Marie Ann Mercado, for your help during my studies at DLSU.

Prof Hitoshi Kosuge and Dr Hiroaki Habaki, TIT for your help during

the time I stayed in Tokyo and for your advising in my research as well as in writing

my paper

Thank you to all faculty members from the ChE Department of DLSU – Manila, Chet and Chemistry Department of Mindanao State University – Iligan Institute of Technology (MSU-IIT), Tokyo Institute of Technology (TIT)

Friends under AUN/SEED-Net program, and all the members of Kawasaki

Lab, TIT, for sharing with me research documents, for exchanging ideas and for your supportive encouragement

To my wonderful family, who never failed to support me in all of my

endeavors Thank you for the faith you have in me and the love you have provided

me You have always been my strength, physically and mentally Your love has always been my inspiration

I also would like to thank my Faculty (Faculty of Chemical Engineering) and all my Teachers, Friends, Colleagues, who sharing their relevant documents,knowledge and encouragement.

TABLE OF CONTENTS

Page

Abstract……… ……… ii

Acknowledgments… ……… iii

Table of Contents vii

List of Tables ……… ……… xii

List of Figures ………xiv

Chapter 1 Introduction 1

1.1 Overview 1

1.2 Statement of the problem 5

1.3 Significance of the study 7

1.4 Objectives of the study 9

1.5 Delimitations of the study 10

Chapter 2 Review of Related Literature 12

2.1 Development of supercritical and supercritical CO2 extraction 12

2.2 Application of Supercritical Extraction 15

2.2.1 Applications in food technology 15

2.2.2 Applications in medicine 18

2.2.3 Applications in cosmetic 19

2.2.4 Applications in Environmental Engineering 19

2.2.5 Applications in Organic Chemistry 20

2.2.6 Applications in Inorganic Chemistry 20

2.3 Lemongrass 25

2.3.1 Taxonomy 25

2.3.2 Morphological characteristics 26

2.3.3 Basic Agronomy Soils and climate 28

2.3.4 Lemongrass Oil 30

2.3.5 Steam distillation processing 34

2.4 Related studies 36

2.4.1 On extraction of Lemongrass Oil 36

2.4.2 On Modeling Optimization process in SCFE 39

Chapter 3 Theorical Consideration 41

3.1 Supercritical state of a fluid 41

3.2 Properties of supercritical fluid 44

3.2.1 Properties of Supercritical fluid affect Extraction process 44

3.2.2 Physical properties 45

3.2.3 Chemical properties 55

3.2.4 Biochemical properties 56

3.3 Supercritical fluid CO2 extraction 56

3.3.1 The Supercritical Fluid Extraction (SCFE) Process 56

3.3.2 Separator system 58

3.4 Factors affecting the Supercritical extraction process 59

3.4.1 Extraction time 61

3.4.2 Temperature 62

3.4.3 Pressure 62

3.4.4 Average length 62

3.5 Response Surface Methodology (RSM) of Design-Expert 7.0.1 software 63 3.5.1 Experiment Design process using RSM 64

3.5.2 Method of Analyses 66

3.5.3 Examine Model Graphs 67

3.5.4 Setting the Optimization Criteria 69

Chapter 4 Methodology 70

4.1 Phases of the study 70

4.2 Material 72

4.2.1 Raw material and sample preparations 72

4.2.2 Moisture content of lemongrass 73

4.3 The Steam distillation process 74

4.4 The Super critical CO2 extraction 75

4.5 The rotary vacuum evaporator 79

4.6 Characterization of essential oil properties 80

4.6.1 Physicochemical Properties 80

4.6.1.1 Determination of Refractive Index 80

4.6.1.2 Determination of Specific Gravity at 20qC/20qC 81

4.6.1.3 Determination of Acid Value 82

4.6.1.4 Determination of Ester Value 82

4.6.2 Chemical composition of essential oil 83

4.6.2.1 Determination of the composition of essential oil 83

4.6.2.2 Determination of the percentages of components (citral, myrcene and limonene) 84

4.6.2.3 Gas Chromatography and Mass Spectrometry equipment 84

4.7 Experiment design and Optimization process 85

4.7.1 Design for experiment using Design-Expert software 85

4.7.2 Optimization Process 88

4.8 Mass transfer and Dimensionless groups 89

4.8.1 Initial assumptions 89

4.8.2 Dimensionless groups 90

4.8.3 Diffusivity 90

4.8.4 Mass transfer coefficient 91

Chapter 5 Results and Discussions 93

5.1 Pre-Extraction stage 93

5.1.1 Comparative Study on the Process of Drying the Raw Materials 93

5.1.2 Extraction time 97

5.1.3 The GC-MS results of standards and reference 99

5.2 The Extraction operation 103

5.2.1 Steam Distillation Process 103

5.2.1.1 Physico-Chemical Properties 104

5.2.1.2 Composition of the Essential Oil by GC-MS Analysis 105

5.2.2 The Supercritical CO2 Extraction 107

5.2.2.1 GC-MS results and chemical compositions 109

5.2.2.2 Effect of extraction conditions on light components 114

5.2.2.2.1 Effect of Temperature 115

5.2.2.2.2 Effect of Pressure 116

5.2.2.3 Physicochemical properties of essential oil from SCFE 117

5.3 Optimization and Modeling 120

5.3.1 Analysis of SC CO2 Extraction of Lemongrass leaves 120

5.3.2 Analysis of SC CO2 Extraction of Lemongrass stems 127

5.3.3 Optimization Process 132

5.3.4 Dimensionless group and Mass transfer estimation 134

5.3.4.1 Dimensionless Group 134

5.3.4.2 Diffusivity and Mass transfer coefficient 135

Chapter 6 Conclusions and Recommendations 140

6.1 Conclusions 140

6.2 Recommendations 144

References ………146

Appendix A: List of Symbols 160

Appendix B: Setup the SC CO2 equipment system 161

Appendix C: Using the rotary evaporation system 162

Appendix E: Preliminary Experiment to determine extraction time 164

Appendix F: Percentage of Myrcene and Limonen (at 90 atm and from 350C to 500C) ………165

Appendix G: Percentage of Myrcene and Limonene (at 350C and from 90 atm to 110 atm) ………167

Appendix H: Analyses results of essential oil from Lemongrass, Leaves (RSM) 169

Appendix I: Analyses results of essential oil from Lemongrass, stems (RSM) 174

Appendix J: Effects of extraction conditions on extraction yield of essential oil from

Lemongrass, Leaves (RSM) 177

Appendix K: Effects of extraction conditions on extraction yield of essential oil from

Lemongrass, Stems (RSM) 184

Appendix L: Analysis results (RSM) of Lemongrass, Leaves 190

Appendix M: Analysis results (RSM) of Lemongrass, Stems 195

Appendix N: GC results of essential oil from Lemongrass, leaves (extra experiments)

………201

LIST OF TABLES

Table 1: Criteria determining implementation of SFC CO2 extraction 4

Table 2: Summary of Supercritical and Supercritical extraction development 14

Table 3: Summary of SC CO2 extraction process 22

Table 4: Physicochemical Properties of Lemongrass Oil 32

Table 5: Physicochemical Properties of main components of essential oil 34

Table 6: Investigation of SC CO2 extraction of essential oil from lemongrass 38

Table 7:Summary of previous researches in modeling and optimization in SCFE 40

Table 8: Characteristics of Supercritical fluid 43

Table 9 : The Critical temperature and pressure of some substances 43

Table 10: Experimental variables affecting Supercritical Fluid Extraction efficiency (Source: Luque de Castro et al., 1994) 60

Table 11: Experimental factors in code variables and original variables 65

Table 12: Matrix of experiment design in code variables 66

Table 13: Experimental conditions of lemongrass (leaves and stems) extraction by SC CO2 78

Table 14: The code and the original variables used in 23 factorials design 86

Table 15: The 23 factorials design table with two center points including the corresponding responses 87

Table 16: Set goal for each of the factors and responses for Optimization formulation 88

Table 17: Average extraction yield from air and heat dried lemongrass 95

Table 18: The information of lemongrass essential oil from NHR Organic Oils Company 100

Table 19: Properties of Lemongrass essential oil from steam distillation processing compare to values from internet 104

Table 20: The experiment results using 23 factorials design with two center points including the corresponding responses 108Table 21: Physicochemical properties of lemongrass SC CO2 extracted essential oil compare to the commercial standards 119Table 22: Sequential Model Sum of Square (Type I), selected the highest order polynomial where the addition terms are signifcant and the model is not aliased 121Table 23: Analysis of variance (ANOVA) table for Lemongrass, leaves 122Table 24: Analysis of variance (ANOVA) table for Lemongrass, stems 128Table 25: Set goal for each of factors and responses for the Optimization of

Formulation 133Table 26: The result of the optimization formulation from the Design-Expert

software with the selected optimum conditions 133Table 27: Results of dimensionless groups (Re, Sc, Sh) 135Table 28: Results of Re, Sc, Sh and values of DAB and k 137

LIST OF FIGURES

Figure 1: Number of SCFE in the world since 1980 to 2005 (Brunner, 2005) 5

Figure 2: Lemongrass (Cymbopogon citratus) after 6 months planted 26

Figure 3: Chemical structures of the major constituents of lemongrass oil 31

Figure 4: Solid-liquid-gas-supercritical fluid phase diagram of Carbon Dioxide (Source: Luque de Castro et al., 1994) 41

Figure 5: Relationship between basic properties and the features of Supercritical Extraction (Source: Luque de Castro et al., 1994) 44

Figure 6: Variation of carbon dioxide density with pressure 46

Figure 7: Viscosity isotherm for SCF CO2 (the dash line represents a fluid density of 770 kg.m-3) (Source: Johnston et al., 1987) 47

Figure 8: Binary diffusion coefficient of SCF CO2 at 40oC 49

Figure 9: Solubility of hydrocarbon in SCF CO2 versus number of carbon in structure (Source: Allada et al., 1986) 52

Figure 10: Variations in the solubility of a low-volatility substance 53

Figure 11: Effect of extraction time on extraction yield 61

Figure 12: Significance of average length at S1 and S2 (S is average length, mm) 63

Figure 13: Flowchart of experiment design, modeling and optimization 64

Figure 14: Central Composite Design for three factors 65

Figure 15: An example of Response surface contour plot 68

Figure 16: An example of 3D response surface plot 68

Figure 17: Flowchart of different phases of the research project 71

Figure 18: Lemongrass after drying (stems and leaves, respectively) 72

Figure 19: Air-dried lemongrass after cutting 73

Figure 20: Steam distillation set up in laboratory 74

Figure 21: The Super Critical Fluid Extraction (SCFE) System 76

Figure 22: Diagram of Experimental Set-up/Supercritical CO2 Extraction Unit 76

Figure 23: The 500 ml Reactor Vessel (Extractor) in a rectangular tank that served as

the water bath 77

Figure 24: The High Pressure Liquid Pump coupled to a Cooler System 77

Figure 25: The Rotary Vacuum Evaporator system 79

Figure 26: Abbé Refractometer used to determine Refractive Index 81

Figure 27: Gas Chromatography-Mass Spectrometry 84

Figure 28: GC result of heat dried lemongrass essential leaves 95

Figure 29: GC result of extract from heat dried lemongrass stems 96

Figure 30: GC result of extract from air dried lemongrass leaves 96

Figure 31: GC result of extract from air dried lemongrass stems 97

Figure 32: The curve of extract time of lemongrass versus cumulative essential oil (g/ 100g of material) at 35 0 C, 90 atm 98

Figure 33: The curve of extract time of lemongrass versus cumulative essential oil (g/ 100g of material) at 50 0 C, 110 atm 99

Figure 34: GC result of lemongrass essential oil from U.K 101

Figure 35: GC result of Citral component (neral and geranial) 102

Figure 36: Typical chromatogram of lemongrass essential oil obtained by dense CO2 extraction (L Paviani et al., 2006) 102

Figure 37: GC result of Lemongrass essential oil from steam distillation, stem 106

Figure 38: GC result of Lemongrass essential oil from steam distillation, leaf 106

Figure 39: GC results of essential oil from Lemongrass, leaves at various extraction conditions at 350C, 90atm 109

Figure 40: GC results of essential oil from Lemongrass, leaves at various extraction conditions at 350C, 110atm 110

Figure 42: GC results of essential oil from Lemongrass, leaves at various extraction conditions at 500C, 110atm 111

Figure 43: GC results of essential oil from Lemongrass, stems at various extraction conditions at 350C, 90atm 112

Figure 44: GC results of essential oil from Lemongrass, stems at various extraction conditions at 350C, 110atm 112Figure 45: GC results of essential oil from Lemongrass, stems at various extraction conditions at 500C, 90atm 113Figure 46: GC results of essential oil from Lemongrass, stems at various extraction conditions at 500C, 110atm 113Figure 47: Effect of temperature to content of myrcene and limonene in lemongrassessential oil 116Figure 48: Effect of Pressure to content of myrcene and limone in essential oil 117Figure 49: The 3D surface graph of Temperature vs Pressure for length 4mm of lemongrass from the optimization model with response as extraction yield 123Figure 50: The 3D surface graph of Temperature vs Pressure for length 5mm of lemongrass from the optimization model with response as Extraction yield 123Figure 51: The 3D surface graph of Temperature vs Pressure for length 6 mm of lemongrass from the optimization model with response as Extraction yield 124Figure 52: The 3D surface graph of Temperature vs Pressure for length 7 mm of lemongrass from the optimization model with response as Extraction yield 124Figure 53: The 3D surface graph of Temperature vs Pressure for length 8 mm of lemongrass from the optimization model with response as Extraction yield 125Figure 54: Extraction yield of lemongrass, leaves at 350C, 90 atm (1: 8mm, 2: 4mm

and 3: 0.5mm) 126

Figure 55: The 3D surface graph of Temperature vs Pressure at the length 6mm of lemongrass from the optimization model with response as Citral’s content,

lemongrass leaves 127Figure 56: The 3D surface graph of Temperature vs Pressure for length 4 mm of lemongrass from the optimization model with response as Extraction yield 129Figure 57: The 3D surface graph of Temperature vs Pressure for length 4 mm of lemongrass from the optimization model with response as Extraction yield 129

Figure 58: The 3D surface graph of Temperature vs Pressure for length 4 mm of lemongrass from the optimization model with response as Extraction yield 130Figure 59: The cell length of material (leaf on the left and stem on the right) 131Figure 60: The 3D surface graph of Temperature vs Pressure at the length 6mm of material particles from the optimization model with response as Citral’s content, lemongrass stems 132

CHAPTER 1

INTRODUCTION1.1 OVERVIEW

The Supercritical Fluid Extraction (SCFE) is a powerful technique developed

in the field of separation technology since the discovery of Supercritical state

(Hannay and Horgarth, 1879, Villard, 1896, Buchner, 1906…) Notably, there are

numerous studies and investigations conducted in recent years, especially on its probable industrial applications A hundred researches on SCFE were found SCFEhas become popular and is the preferred choice for separating products with high added value than the conventional methods of separation, like solvent extraction or

distillation The process is commonly applied in the areas of food (Froning et al.,

1990; H Sovova et al., 1994; J Statova et al., 1996; E Reverchon et al., 2001; S.G Ozkal et al., 2004), pharmaceuticals (Martin Danahera et al., 2001; N Vedaraman

essential oil industries (Verónica Arancibia et al., 2004; Paramita Bhattacharjee et

or volatile oils from herbs and spices operates at low operating temperature, is free from contamination of products by the solvents and does not require large plant areafor the equipment

The supercritical fluid CO2 is popularly used to extract flavor fromcompounds, essential oil or unnecessary fats from food Recent research and developments are continuously growing to find other applications, especially in the food industries It has several advantages relative to the existing conventional

processes such as it is non-toxic, so it is readily used in food industries (Gerd

Brunner et al., 2005), and most importantly, it is environmentally friendly However,

SCFE is an expensive process; but for products that require high purity likemedicines and food supplements, the operating costs are competitive relative to thetraditional operations like distillation and solvent extraction.

Supercritical fluid technology has also been widely used, as supercritical fluid chromatography In this process, SC CO has been used as a mobile phase Recently, there has been a significant increase in interest of the use of supercritical

2

carbon dioxide as a substitute for chlorofluorocarbons (CFCs) for a variety of

specific and specialized applications in which the choices of environmentallyacceptable alternatives are quite limited

Supercritical fluids were also found to be attractive cleaning solvents because

of their excellent mass transport characteristics and their unique physical properties

that allow extracts to be isolated with relative ease (A Marsal et al., 2000) Carbon

dioxide is especially attractive since it is inexpensive and environmentally benign in comparison to liquid solvents that it may replace It also has a relatively low critical temperature which makes it an ideal solvent for extracting thermally labile substances There is, however, a limitation of supercritical fluids having moderatecritical temperatures in that they are poor solvents for polar or ionic species that aretypically one of the target components in a cleaning operation

In the last decades, a rapid growth in research activity in the fields of SCF extraction has been observed Although many growing reviews and hundreds of publications have appeared covering a wide variety of applications, there are surprisingly few processes and plants operating on a commercial scale It is possiblethat many of the early publications overstressed the potential advantages of SCFs without addressing the limitations It is certainly the case that many reports of applications do not differentiate between the extraction/identification of trace amounts of components and realistic quantities on which a process could be based Without the necessary data it is often difficult or impossible to quantify theefficiency of a reported SCF extraction process in terms of mass transfer and

throughput of CO2 Against this background, it is not surprising that many popular misconceptions abound regarding SCFs

SCF extraction is not a cheap process (in terms of cost of system), but it hasmany advantages The one clear advantage that CO2 does offer for food applications

is its lack of toxicity Supercritical carbon dioxide (SC CO2) extraction offers theadvantages of using non-toxic, non-explosive and cost effective solvent It enables extraction at low temperatures and easy and complete removal of solvent from the final product In the current climate of growing consumer concern regarding food

safety, this feature will undoubtedly promote its use in the food industry Table 1

presents the novel features that serve to illustrate the potential of SCF extraction

The mass product nature of oil seed makes a continuous supercritical extraction essential if operation is to become economical relative to the conventional hexane extraction However, at present only batch processing is possible, although several attempts have been made to design a plant in which seeds are transported

continuously into, through and out of a pressure vessel (Eggers et al., 1985) On the

other hand, the advantages of supercritical fluid extraction prevail when smalleramounts of materials yielding high quality products are processed

SCF extraction (SCFE) processes can be carried out in different modes of operation In most cases, extraction from solids is concerned, which usually is carried out in batch and single-stage mode, since solids are difficult to handle continuously in pressurized vessels and separation factors are high Fluid mixturesoften have separation factors which make necessary the application of multi-stagecontacting, which is carried out most effectively in a counter-current mode Ifseparation factors are approaching, many theoretical stages are necessary for separating the components Chromatography is a magnificent tool for separating similar compounds Although chromatography with SCFs as solvents and mobilephases is presently mostly applied in analytical separations, the advantageous

possibilities of gaseous solvents should also be applied in chromatographic processes

on a preparative and process scale

Table 1: Criteria determining implementation of SFC CO 2 extraction

Economic Can save energy

Cheap Stable market

High capital cost

Non flammable

High pressure

Physical properties Enhances control through T

and P (fractionation) High vapor pressure enablesseparation at low temperature Low viscosity can provideenhanced mass transfer

Low solubility

Chemical properties Non oxidative

Environment

Water will effect on pH

Quite a large number of industrials plants (maybe around 100) of different lengths have been built during the last 20 years for the extraction of solid materialwith SCFs in a batch mode Since the early 80s, a total number of about 100 vessels

bigger than 100 liters in volume have been ordered for about 50 plants, Figure 1

shows for SCFE build up the statistical data They are mostly distributed in Europe,

the USA, Japan, and in the South East Asian Countries (Gehrig, 1998; Fukuzato,

2003; Brunner, 2005)

Figure 1: Number of SCFE in the world since 1980 to 2005 (Brunner, 2005)

Nowadays, many researches are using supercritical process, not only in the field of extraction but also in many other fields such as cosmetics, medicine,chemistry or environment

1.2 STATEMENT OF THE PROBLEM

The extraction process using supercritical (Supercritical Extraction – SCE)carbon dioxide has been studied as a potential alternative to the conventional extraction methods The interest in this application of supercritical extraction hasgrown due to the rapidly decreasing process costs and the increasing quantities of products, as well as the competitive advantage of this process in the commercial market

The conventional ways are generally acceptable, if done well by effecting extractors using premier ingredients There are some serious problems, however, with conventional, non-supercritical extraction The extracts from conventional

methods such as separation by extractive solvents (hexane, methanol), cold pressing

or steam distillation, require after extraction a stage of purification to remove theremaining solvent which is potentially hazardous Most importantly, the chemicalsolvents often used for such conventional extraction can be rather toxic Thepurification process is done through filtration, distillation, centrifugation and

rectification (Rodriques et al., 1997) This additional step in essential oil extraction

contributes to the cost of the final product since the operational cost is initially high Other disadvantages of the conventional methods are the remaining of solvent in extract, as well as the thermo-degeneration of extracts

The supercritical process does not present any of these problems Rather than using a chemical solvent as the dissolving fluid, the supercritical process uses compressed fluids and one of the popular solvent used in SCE is CO2 The harmless,natural gas, heats to temperature over 31.1 degrees C, and the gas is highly compressed at pressure higher than 73.8 atm The compressed gas has the density of

a liquid, but is able to penetrate deeply into the plant and dissolves the lipophilic constituents The pressure is then carefully released and the gas just harmlesslydissipates into the atmosphere and all that is left behind is the pure, concentrated extract The cycle in completed without pollution, no heat stress or damage, and no solvent residue

The material for extraction process, Lemongrass is used widely in food technology as well as in medicine (especially in traditional medicine) Lemongrassessential oil relieves some of the symptoms of jetlag, clears headaches and helps with nervous exhaustion and stress-related conditions It is useful with respiratory infections, such as sore throats, laryngitis and fever It helps prevent the spreading of

infectious diseases Recently, the research from Allison Kaplan Sommer (April 02,

2006) has mentioned about the lemon aroma in herbs like lemon grass (citral) can

kill cancer cells Lemongrass oil is also used in Food Technology and this is one of the delicious spices in the meal of Asian families.

But with Lemongrass, only a few research materials have been found; these

articles just mention the process of extraction from lemongrass leaves (Ariovaldo

Bolzan et al., 2001) There is a recent research from Brazil about the extraction of

Lemongrass Essential oil by using Supercritical CO2, entitled “Application of

molecular sieves in the fractionation of lemongrass oil from high-pressure carbon dioxide extraction” co-authored by are L Paviani, S B.C Pergher, and C Dariva

date June 2006 (although this topic has been started in September 2004) However, the authors just mentioned about the compositions of essential oil, nothing is saidabout the optimization process or what properties of the stems differ from the leaves

of lemongrass, as well as effects of extraction conditions on the extraction process.This present study focuses on the extraction of Lemongrass by SC CO2, the extraction process will be done separately on the stem and leaf of Lemongrass Thedrying scheme will be investigated to observe the difference between the air-driedand heat-dried lemongrass The extract from SCFE process will be compared withthe extract from Steam Distillation process in terms of extraction yield, physicochemical properties, as well as compositions The variation in content of light components with extraction temperature and pressure was investigated Theoptimization of the process (on the parameters temperature, pressure and the length

of material) was done by using Design-Expert software (version 7.0.1)

1.3 SIGNIFICANCE OF THE STUDY

Processing materials with Supercritical Fluid CO2 Extraction (SCFE) is aproven and industrially applied technology It is readily available for extraction from solids using batch reactor

The extraction of essential or volatile oils using supercritical CO2 from herbs and spices has been considered as a possible applied field of the SCFE because of

low operating temperature, avoiding the unfavorable effect of temperature on compositions of essential oil, especially light components There is no contamination

of product by the usual solvents because all the solvent (supercritical CO2) will beremoved very easily by expansion using the expansion valve or separation system.The Supercritical system is small so it does not need a large plant area Besides, its operation is not so complicated High-pressure equipment is more expensive than those for the conventional separation processes; however, the operating cost is usually lower than that of the process to collect pure essential oil by other methods.Hence, the total costs are comparable if the process is carried out at optimumconditions and sufficient extractor volume

Lemongrass has usually been extracted by steam distillation This method is considered cheap but can not supply the very pure essential oil to satisfy some high quality standard required in food and especially in medicine industry In addition,steam distillation gives very low extraction yield and it takes a long time (about 2 to

3 days after distillation) just to separate the essential oil from mixture with water In most countries, high purity essential oil commands a very high price: In the year

2005, 50 ml lemongrass essential oil from NHR Organic Oil Company (U.K.) cost

49 EURO Therefore, research on the Supercritical CO2 Extraction as a new and effective method to extract essential oil from Lemongrass is being encouraged and this study give the way for making comparison between essential oil from steamdistillation and SCFE

In previous research only the leaf of lemongrass was used, in this new research, both the leaf and stem of lemongrass were used for doing comparison studies on essential oil Furthermore, the heat-drying under vacuum condition and air-drying of Lemongrass were also considered and these processes were observed comparatively.This study used GC-MS results of extract to determine the compositions of essential oil as well as to determine the content of the main component (citral) which was used in optimization as one response These results were used to determine the

difference in characteristics of the essential oil from SCFE (leaf and stem), from steam distillation, and that from U.K.

The percentage change of light components, like myrcene and limonene with respect to extraction conditions were also determined using GC results

One of the extraction parameters, extraction time, was determined based on theexperimental data and it was made constant in the process

Optimization process was done by using Design-Expert software version 7.0.1 The process considered extraction yield of essential oil (mass of essential oil permass of raw material) and citral - the main component, which contained more than90% in essential oil - as responses (leaf and stem separately) and the effect of threeparameters: temperature, pressure of extraction process and length of material was observed from the result of the optimization process The models of the extractionprocess of lemongrass oil from leaf and stem were obtained from the results of thesoftware for the experimental data as well as the optimum operating conditions forthe process

This study provides a baseline data of the compositions (components and variation of light components), as well as mass transfer coefficients of SC CO2

extraction of essential oil from Lemongrass, which have not been mentioned in any literature before as far as this investigation is concerned

1.4 OBJECTIVES OF THE STUDY

This study investigated the compositions of essential oil separately from the leaf and stem of Lemongrass using SCFE process, as well as investigate the effect of the process parameters of extraction relative to yield and compositions of the extract This study includes optimization of the process conditions and establish appropriate model of extraction for lemongrass

In order to attain the general objective, the following specific objectives were done:

1) To carry out the experimental procedures for the extraction of Essential Oilfrom Lemongrass (separately from leaf and stem) using SC CO2

2) To determine the effect of drying schemes (heat-dried and air-dried) and then select the best drying method for the extraction process

3) To determine the physico-chemical properties of the product using various techniques (refractive index, acid value, ester value, specific gravity, GC-MS) 4) To compare the essential oil obtained from SFE and steam distillation in terms of extraction yield, Physicochemical Properties and Compositions

5) To investigate the effect of different parameters on the extraction process (temperature, pressure and length of material) and to optimize the processbased on the experimental data using the Design-Expert software with Response Surface Methodology

6) To determine the components of essential oil, as well as the content of Citral

as a main component, and investigate on how the percentage of light components (Myrcene, Limonene) varies with extraction conditions

1.5 DELIMITATIONS OF THE STUDY

This work uses the Supercritical process of CO2 extraction to get the essentialoil from plants The material is lemongrass (leaf and stem) processed on an experimental scale using a 500 ml extractor The source of lemongrass for all experiments was in Iligan City

This study investigates and optimizes the operating parameters (temperature,pressure, and the length of material) using Design-Expert software

After harvesting, the leaves of lemongrass were separated from the stems then dried (by air-dried and heat-dried) Then dried material were cut to small pieces at

the length of 4, 6 and 8 mm, their moisture will be determined Lemongrass was cut and weighed accurately each load of extraction is 100 grs

The steam distillation was set up on a laboratory scale to get the essential oilthen compared to the essential oil collected from Supercritical CO2 extraction in terms of extraction yield and physicochemical properties as well as composition.The components of the leaf of lemongrass were compared to those of its stem and the optimization calculations were applied to each material

This study focuses on the analysis of results of experiments on the essential oil properties, such as refractive index, acid value, ester value, specific gravity, GC-MS.The results of these experiments on essential oil were compared to theproperties of pure essential oil of lemongrass from NHR Organic Company (U.K)

The experiment used pure CO2 The other chemicals have high purity (analytical grade) The conditions for the experiments are high pressures (90 to 110 atm) and temperatures in the range of 35 – 500C, and length of material are from 4 to

8 mm

CHAPTER 2

REVIEW OF RELATED LITERATURE

2.1 DEVELOPMENT OF SUPERCRITICAL AND SUPERCRITICAL CO 2 EXTRACTION

Supercritical (SC) state has been known many years ago and gradually it has been applied in many science or industry fields It has experienced a long time of development process since its discovery until its use in Supercritical Fluid Extraction (SCFE) and in other fields The story of the SCFE development process can be summarized based on its historical data

As early as the 80 decades of the 19th century, many scientists started to research the behavior of highly compressed gases At that time, this field was new and very interesting to them, including even Mendeleev, who had also spent a lot of time on this problem After many researches and experiments, the first success result

in this field was found in 1869; this was the existence of an additional state of matterwhich became known when Andrews, the English Scientist, discovered the criticalphenomenon and supercritical state The values for the critical point of carbondioxide which he reported were in close agreement to the presently accepted values

After the preliminary results from the previous scientists, Hannay and

Horgarth studied the phenomenon of supercritical fluid (SCF) solubility in 1879

They found that gases could be good solvents under supercritical conditions and the dissolving power of a SCF was highly pressure-dependent Small changes in pressure continuously altered the density of these fluids from gas-like to liquid-like, thereby allowing their solubility power to be adjusted over wide ranges At that time

Hannay and Hogarth based their studies on the SC of ethanol They found that

increasing the pressure on the system caused the solutes to dissolve and that decreasing the pressure caused the dissolved materials to nucleate and precipitate “as

a snow.” They described their experiments, carried out in small-diameter glass tubes,

in which they observed that changes in pressure caused several inorganic salts (for example cobalt chloride, potassium iodide, and potassium bromide) to dissolve in orprecipitate from ethanol at a temperature above the critical temperature of ethanol

At the beginning of the 20th century, several years after Villard’s paper, E H.

Buchner (one of the famous German Scientists) reviewed the existing literature and

also made significant additions to the experimental data base of high-pressure

Supercritical Fluid-solute mixtures (Buchner, 1906) He carried out his studies over a

wide temperature range, and he used observations of cloud points, freezing points, and the number of phases present for his solubility determinations Based on theseobservations, he described very clearly the SC state In this research, he showed the phenomena that gas changed to supercritical state and he gave some supercritical conditions (temperature and pressure) of some substances, such as CO2, NO2 Todaymost chemists and scientists are familiar with the critical point of a substance as

defined by the critical temperature and pressure.

Table 2: Summary of Supercritical and Supercritical extraction development

1906 E H Buchner Reviewed the existing literature and also made

significant additions to the experimental data base of high-pressure Supercritical Fluid-solute mixtures

1920 U.S Companies The first application study of SCF has been

applied in the petrochemistry fields

1950 Max Planck Institute Used SCF in food, petroleum and chemical

1960 Kurt Zosel Applied SCFE of CO2 into natural products:

coffee decaffeination and tea decaffeinationprocesses in Europe, America and Australia

1970 U.S Companies Offering supercritical fluid development

Flavor Co and CEA

Applied SCFE into extraction of Tobacco, Aromas and Pharmaceuticals

Since the Supercritical Fluid technique (SCFT) has been applied into extractionprocess (SCFE), it has become useful in many fields of science as well as in industry

Table 2 above shows the summary of history and development of SCFE since

Andrew found the Supercritical state

Supercritical CO2 is also one of the SC substances The SC state of CO2 was found along with that of the other Supercritical Substances, and since the middle of the 20 century up to now, it was applied in many fields It can be applied in medicinefor processes such as extracting medicine from plants or enrichment of vitamin fromnatural sources, and encapsulation In the cosmetic field, it is used in products like soap, perfume and lipstick The SC CO2 process is also used for some otherindustrial and chemical processes, such as cleaning, aerogels, impregnation, particle generation and micro-encapsulation SC CO2 can be applied especially in Food Processing of decaffeinated coffee, decaffeinated tea, vitamin additives (E, A, n-3-fatty Acids), de-alcoholized wine, beer, defatted meat and spice extraction Since

1990 up to now, there have been many applications of the essential oil extraction of

SC including CO2

2.2 APPLICATION OF SUPERCRITICAL EXTRACTION

Since the end of 20 century, the SCFE has been developed very fast and thisprocess has been applied into many fields, in science, engineering, as well as in industry

2.2.1 Applications in food technology

The earliest and most important field that SCFE was applied in food technology and essential oil extraction In this field, many researches have beenpublished One example shown is the application of SCFE in coffee and tea becausedecaffeination of coffee and tea are the first process that SCFE has been used in food

technology Over the past decade there has been a growing consumer aversion to the levels of stimulants in beverages and there is now a large market in decaffeinated products Although there are some notable differences, the conventional solvent and SCF extraction processes for coffee and tea share many common features.

In the production of coffee, ‘green’ beans are roasted to generate the coffee oils that later impart flavor to the infusion To avoid co-extraction of flavor components

in the decaffeination process, green beans are therefore generally extracted Moist beans are used since it is found that dry beans do not allow effective extraction The crucial role of water in the process has not been unambiguously established but is thought to ‘free’ the caffeine from adsorption on the surface, reduce complexity with other molecules and reduce the complicacy factor by swelling the cellular structure

In the conventional decaffeination process, organic solvents such as methylenechloride or ethyl acetate are employed to reduce the level of caffeine from approximately 1% w/w to 0.06%

Heightened awareness of the potentially harmful effects of residual levels of these solvents has provided some impetus to examine alternative safer solvents such

as SCF CO2 Judging by the scale of its implementation, decaffeination of coffee isone of the most successful commercial applications of CO2 extraction in large plantsoperating in West Germany (Bremerhaven, 27.3 million kg per year) and in the USA (Dallas) At first sight CO2 extraction would not appear to be promising since thesolubility of caffeine in SCF CO2, is relatively low (< 0.2% w/w) according to Ebelling and Franck (1984), but this is offset by the high added value of the process

Moreover, CO2 provides a very selective solvent for decaffeination which does not remove as many of the desirable flavor-precursor components, as alternative organicsolvents (e.g ethyl acetate)

Selectivity for caffeine is probably greatest in SCF CO2, though solubility is low (approximately 0.05%) Increasing the pressure and temperature serves to increase the solubility of caffeine but reduces the separation factor In some

processes for production of decaffeinated instant coffee, flavor components that are

co-extracted are later separated and added back at the drying stage (Roselius et al.,

1974) It is often stated that the water added in decaffeination serves to increase the

solubility of caffeine in SCF CO2 This hypothesis is not borne out by experimentalevidence, since increasing the levels of water to saturation does not significantly affect the solubility of caffeine in SCF CO2 (Moulson, 1988).

Decaffeination illustrates well the adverse effects of solute adsorption, since it

is observed that considerably more CO2 is required to effect decaffeination than would be expected on solubility grounds alone This is because caffeine adsorbs onto the beans and prevents equilibrium solubility being reached in the extraction vessel

(McHugh and Krukonis, 1986).

Decaffeination of coffee represents one of the most widely patented applications of SCF extraction with innumerable variations and permutationsclaimed In contrast the SCF decaffeination of tea has been less well documented

(Vitzthum and Hubert, 1979) This may well be due to the more delicate flavor

profile of tea, which is more susceptible to damage during the extraction process The higher levels of caffeine in tea (3% compared to 1% in case of coffee) may also

be a contributing factor

The other important application is the reduction of the cholesterol content infood; it is very important to people who are on a diet The SCFE process also helpsthem in this field On solubility grounds there would appear to be a good chance of selectively extracting cholesterol from oils and fats using SCF CO2 Chrastll et al.,

(1982) has measured and collated the solubility of a variety of food components in

SCF CO2, and correlated his data using a simple ‘mass action’ model Even from the limited pressure range used in this study, it is clear that the solubility of cholesterol is

significantly greater than that of triglyceride oils Then in 1988, Krukonis, an other

scientist, tested the feasibility of removing cholesterol from butter, egg yolk and beeftallow by measuring the partition coefficients of the individual components in SCF

CO2 In the SCFE process, 90% removal of the cholesterol from butter was reported with an overall yield of 70% of cholesterol-reduced product Distribution coefficients

of cholesterol between milk fat and SCF CO2 reported by Bradley (1989) supported

the viability of selective separation Studies on the effect of extraction conditions upon the composition of SCF CO2 extracted egg yolk powder (Froning et al., 1990)

were in broad agreement with available solubility data

In addition, there were many applications of SCFE in food technology and especially in the field of essential oil extraction Some papers published were in the

field of food technology and herbs/ spices: H Sovova et al (1994); J Statova et al

(1996) and E Reverchon et al (2001) on vegetable oil extraction; Amra Uzunalic et al (2004) on extraction of chilli pepper; Edgar Uquiche et al (2004) on

Perva-extraction of red pepper; and A Guvenc et al (1998) applied SCFE to extract ethanol from fermentation broth Recently Gerd Brunner et al (2005) carried out the general

researches about Supercritical fluids- technology and application to food processing

In the field of essential oil extraction, many materials have been used to extract, such

as almond (C Marrone et al., 1998), palm kernel (M N Hassan et al., 2000), hiprose seed (E Reverchon et al., 2001), sunflower seeds (H.K Kiriamiti et al.,

2001), chamomile (Nanci P Povh et al., 2001), Philippine Cananga odorata Hook fil

et (Sheeva M Yahcob et al., 2003), hazelnut (S.G Ozkal et al., 2004) and so on

2.2.2 Applications in medicine

SCFE was used to extract the drug from plants or animals It was also usedfor the process of enrichment of vitamin from natural sources or in the encapsulationprocess There were many papers published such as: “Formulation of an effective

mosquito – repellent topical product from Lemongrass Oil” (A.O.Oyedele et al.,

2002), extraction and isolation of avermectins and milbemycins from liver samples (Martin Danahera et al., 2001), or extraction of cholesterol from cattle brain (N Vedaraman et al., 2004) S.M Ghoreishi et al (2001) applied SCFE in the extraction

of mannitol from plane tree leaf; and Rui L Mendes et al (2003) in the extraction of

compounds with pharmaceutical importance from microalgae On the other hand,

SCFE could also be applied to encapsulate medicine tablet (Yulu Wan et al., 2002).

2.2.3 Applications in cosmetic

SCFE process can be used to extract many materials to produce soap, perfume,lipstick One application is the extraction process of flavors and fragrances used in cosmetic and perfume Flavors and fragrances are conventionally isolated from botanical sources either as an absolute, using solvent extraction, or by steam distillation The main drawbacks to these methods are thermal degradation, loss of volatile ‘top notes’ and indiscriminate separation of high molecular weight components It was realized at an early stage in the development of SCF extraction that these problems could be largely overcome by using CO2

Most of the early work on the use of liquid CO2 for flavor extraction wascarried out in the Soviet Union in the 1960s and reported in Russia Since then the variety of flavors and fragrances examined has grown enormously and now represents probably the largest class of researched applications for CO2 extraction in the food industry Of all examples, that of hop oil extraction deserves special mention, since it is one of the few applications regularly operating on a largecommercial scale

There are many researches that have been carried out in this field such as

essential oil from orange peel (A.Berna et al., 2000)

2.2.4 Applications in Environmental Engineering

The environmental pollution has recently become one of the most important problems in the world Many organizations, governmental as well as non-governmental organizations, have been set up to protect our earth to avoid a global

environmental disaster The SC technique can also be applied to EnvironmentalEngineering Many researches in this problem have been listed as examples, such as

cleaning degreasing process (A Marsal et al., 2000), purification of used frying oil (Jungro Yoon et al., 2000), removal of organophosphate pesticides from wastewater (Jya-Jyun Yu et al., 2002), recovery of antioxidants ( J M del Valle et al., 2004).

SCF can also be used as cleaning solvent for some special cases

2.2.5 Applications in Organic Chemistry

SCFE is mainly used in organic chemistry to extract organic compounds from

natural materials, such as extraction of synthetic pyrethroids from wool (A.M.

Nguyen et al., 2000), extraction of lycopene and b-carotene from ripe tomatoes (Enzo Cadoni et al., 2000), separation of astaxanthin from red yeast Phaffia rhodozyma

(Gio-Bin Lim et al., 2002), extraction of 2-acetyl-1-pyrroline Pandanus amaryllifolius Roxb (Paramita Bhattacharjee et al., 2005), extraction of J-linolenic acid (GLA) from the cyanobacterium Arthrospira (Rui L Mendes et al., 2005), or in

the field of dyes, like investigation on the solubilities of dispersed dyes of blue 79,

red 153, and yellow 119 in supercritical carbon dioxide (Ho-mu Lin et al., 2001), this

research also can be applied to treat the waste water from dye factory

2.2.6 Applications in Inorganic Chemistry

Recently, SCF technique is also applied to Inorganic Chemistry Some researches have been promulgated, such as extraction of metals from aqueous

solutions (Can Erkey et al., 2000), extraction of uranium from solid matrices (Mojtaba Shamsipur et al., 2001), extraction of crystal water (Ken-Ichi Akao et al.,

2002), extraction equilibrium of gallium (III) (Sung-Yong Choi et al., 2002), or

extraction of cadmium as Cd–oxine complex (Verónica Arancibia et al., 2004).

Experience in high-pressure technology show that a lack of design data and high capital costs have all contributed to limit the application of SCF extraction in the food industry However, selected applications making use of the unique properties of SCFs have been, and are being, profitably exploited It is noteworthy that the products of such processes, although sometimes more expensive, have found

a place in the market by virtue of their improved quality This reflects the current trends of the consumer towards purer and more natural processed foods It seemslikely that these consumer demands, combined with increasing legislativerestrictions, will dictate greater implementation of SCF extraction technology in the future

In the field of SFFE, there are many plants produce different products which contribute actually to commercial products such as natural colorant for food products, food additive (odor or flavor), antioxidant, edible oil and markedly essential oil These products are widely extracted from different part of wide variety of crops and forest species which can be plant’s leaves, flowers, fruits, atmk, wood, and roots as

well as the seed Table 3 summarizes some major studies on the supercritical carbon

dioxide extraction from different materials

In this study, SC of CO2 was applied in the essential oil extraction process The material used was Lemongrass This study also considered the influence of someparameters such as temperature, pressure and length of material particles on the extraction process The Design-Expert software was used to analyze and model the extraction results

Table 3: Summary of SC CO 2 extraction process

References Materials Main work’s content Conditions

Sovová et

al., 1994 Grape seed

Oil extraction, effect of solvent flow rate and flow direction of extraction

T = 40qC

P = 280 atm

Reverchon

et al., 1995 Sage leave

Influence of CO2 density andextraction on extract composition

Two separators operated in series

to eliminate cuticular waxes

Oil extraction from seed and pulp, effect of extraction conditions on the solubility and mass transferrate

B Mira et

al., 1998 Orange peel

Investigation effect of CO2 feed rate, pressure, temperature andaverage length on composition and linalool

T=293-232KP=80-280 atm G=0.5-3.5kg/hS=0.1-10mmJ.M

A Marsal

et al., 2000 Sheepskin

The fractionation of the different components of the natural fat as a function of the CO2 pressure and temperature has been studied

T=450C

CO2 density = 0.70-0.75g/ml

4 and 5 extractors

40qC; 280 atm

110 – 220 atm 0.15 – 0.24 m3/h

T=45-700CP=335-450 atm

Uquiche et

al., 2004 Red pepper

Evaluate and model the effect of average length, solvent flow rate and pressure on extraction rate and yield of oleoresin

40qC

320 - 540 atm0.273- 3.90 mm 0.57-1.25 mm/s

Loui et al.,

2004 Parsley seed

Parsley seed oil extraction, effect

of pressure, temperature, average length and CO2 flow rate on oil extraction

308qK – 318qK

100 – 150 atm 0.7, 1.1, 2 kg/h

CO2 and hydrodistillation oil

35, 45 & 55qC

100, 200 & 300 atm

40qC; 350 atm

2 h Modifier Ethanol