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 ABSTRACT 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 CO 2 ) 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 of SCFE system. The lemongrass was loaded into the 500ml extractor with an up flow rate of 0.5 m 3 per hour of CO 2 . The extraction temperature was varied from 35 0 C to 50 0 C 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 CO 2 and Steam Distillation. The extracts from both methods were analyzed by GC-MS and the variations of the compositions 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 were established with the extraction yield and citral content of the essential oil at the established responses. The optimum yields of essential oil from leaf and stem at the given extraction conditions were also determined. ii ACKNOWLEDGMENTS Firstly, I would like to express my grateful appreciation to the following institutions and laboratories that made this research work possible: Asian University Network / Southeast Asia Engineering Education and Development Network (AUN/SEED-Network) and Japanese International Cooperation Agency (JICA). De La Salle University, Host Institution of Chemical Engineering in AUN/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 Kawasaki Laboratory-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. iii 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 experience gathered 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 in Japan but also in the Philippines. Thank you for kindly sharing your knowledge as well 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 works accomplished. 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 iv 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. v 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. vi 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 CO 2 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 vii 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 CO 2 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 viii 4.3 The Steam distillation process 74 4.4 The Super critical CO 2 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 ix 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 CO 2 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 CO 2 Extraction of Lemongrass leaves 120 5.3.2 Analysis of SC CO 2 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 CO 2 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 35 0 C to 50 0 C) ……………………………………………………………………165 Appendix G: Percentage of Myrcene and Limonene (at 35 0 C 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 x [...]... 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. .. 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,... 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 xv Figure 44: GC results of essential oil from Lemongrass, stems at various extraction conditions at 350C, 110atm 112 Figure 45: GC results of essential oil. .. 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 350C, 90... atm 98 Figure 33: The curve of extract time of lemongrass versus cumulative essential oil (g/ 100g of material) at 500C, 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)... oil from Lemongrass, stems at various extraction conditions at 500C, 90atm 113 Figure 46: GC results of essential oil from Lemongrass, stems at various extraction conditions at 500C, 110atm 113 Figure 47: Effect of temperature to content of myrcene and limonene in lemongrass essential oil 116 Figure 48: Effect of Pressure to content of myrcene and limone in essential. .. component, which contained more than 90% in essential oil - as responses (leaf and stem separately) and the effect of three parameters: temperature, pressure of extraction process and length of material was observed from the result of the optimization process The models of the extraction process of lemongrass oil from leaf and stem were obtained from the results of the software for the experimental data as... 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. .. the essential oil then 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. .. mentioned about the compositions of essential oil, nothing is said about 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 The drying scheme . Physicochemical Properties of Lemongrass Oil 32 Table 5: Physicochemical Properties of main components of essential oil 34 Table 6: Investigation of SC CO 2 extraction of essential oil from lemongrass 38 Table. results of essential oil from Lemongrass, Leaves (RSM) 169 Appendix I: Analyses results of essential oil from Lemongrass, stems (RSM) 174 x Appendix J: Effects of extraction conditions on 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