DEDICATION This work is dedicated to my son Teshan who is suffering from Duchene Muscular Dystrophy A Comparative Study of Solvent Extraction, Soxhlet Extraction, Steam Distillation, Headspace Analysis and Headspace Solid Phase Microextraction for the Extraction of Volatile Terpenoid Compounds in the Curry Leaf Plant (Murraya koenigii ) by Hogantharanni Govender Submitted in partial fulfilment of the academic requirements for the degree of Master of Science in the School of Chemistry, Faculty of Science and Agriculture, University of KwaZulu-Natal, Durban December 2010 As the candidate’s supervisors we have approved this thesis for submission. Signed: ---------------------------- Name:---------------------------- Date:------------ Signed: ---------------------------- Name:---------------------------- Date:------------ i ABSTRACT A comparative study was undertaken of different extraction methods for the isolation of volatile organic compounds from Murraya koenigii (curry leaf plant). The techniques studied included the traditional methods of extraction, namely, Soxhlet and solvent extraction as well as steam distillation. The solvent–free extraction techniques of headspace analysis and headspace solid phase micro-extraction (HS-SPME) were also investigated. In the evaluation of SPME, two different fibre coatings, poly(dimethylsiloxane) and poly(acrylate), were compared. Preliminary work to determine the effect of extraction parameters, such as extraction time, was carried out. The volatile oils in the fresh leaves of Murraya koenigii were isolated by the above- mentioned extraction methods and analysed by gas chromatography-mass spectrometry. The main aroma contributing compounds were identified by comparison of their retention times with those of standards and their mass spectra with those of known compounds contained in the National Institute of Science and Technology Standard Reference Database 1A (NIST 98). The essential oil contained mainly terpenes: monoterpene and sesquiterpene hydrocarbons. The constituents were identified and only the five selected analytes of interest, α-pinene, β-pinene, α-phellandrene, β-caryophyllene and α-caryophyllene were quantified in three of the methods, namely solvent extraction, soxhlet extraction and steam distillation. From the quantitative determination of the compounds of interest, steam distillation favoured the extraction of β-caryophyllene. The solvent and Soxhlet extractions showed no significant differences between the quantities obtained for α- and β- caryophyllene. The steam distillation and Soxhlet methods showed similar quantities of α-caryophyllene extracted. The extraction of the monoterpenes, α-pinene, β-pinene, and α-phellandrene, was favoured by the Soxhlet method of extraction. Quantification was difficult with HS-SPME and headspace analysis. ii Headspace analysis proved effective in the detection of the very volatile analytes. Headspace-SPME combined with GC-MS was found to be suitable for the identification of both monoterpenes and sesquiterpenes of M. koenigii . From this study, solvent extraction and Soxhlet extraction were found to be superior to the other methods studied for the characterisation and quantitation of the volatile organic compounds in essential oils of Murraya koenigii. iii PREFACE The experimental work described in this thesis was conducted in the School of Chemistry, University of KwaZulu-Natal, Durban, under the supervision of Professor B.S. Martincigh and Professor A. Kindness. These studies represent original work by the author and have not been submitted in any other form to another university. Where use was made of the work of others it has been duly acknowledged in the text. iv ACKNOWLEDGEMENTS I would like to express my sincerest gratitude to the following people for their contributory role in this study and to whom I am indebted to: Firstly, my supervisors, Prof. B. S. Martincigh and Prof. A. Kindness, for their invaluable expertise, meticulous guidance and advice throughout the duration of this project. Also, I thank Prof. S. Baijnath for the identification of the curry leaf trees and Dr D. H. Pienaar for his contributory role in this project. I would like to extend my gratitude to Mr S Naidoo for his encouragement and proof- reading of my thesis. Mr B Parel for the training on the GC/MS. Mr A Bissessur for his encouragement and support. Research Administration of the University of KwaZulu-Natal for financial assistance towards this project and Dr S. Singh. My colleagues in the School of Chemistry for their assistance, more especially Mrs T Naidoo, Mrs V Reddy and Mrs R Moodley. My late father for being an inspiration in my life. My mother, brothers and sister for their constant moral support and encouragement. Finally, my husband Thagaraj and my sons Teshan and Sherwyn Chester for their love and sacrifice. I thank both Thagaraj and Sherwyn for helping to take care of Teshan in my absence and most importantly, to Teshan, for his patience and understanding. v FACULTY OF SCIENCE AND AGRICULTURE DECLARATION 1 - PLAGIARISM I, Hogantharanni Govender, declare that 1. The research reported in this thesis, except where otherwise indicated, is my original research. 2. This thesis has not been submitted for any degree or examination at any other university. 3. This thesis does not contain other persons’ data, pictures, graphs or other information, unless specifically acknowledged as being sourced from other persons. 4. This thesis does not contain other persons' writing, unless specifically acknowledged as being sourced from other researchers. Where other written sources have been quoted, then: a. Their words have been re-written but the general information attributed to them has been referenced. b. Where their exact words have been used, then their writing has been placed in italics and inside quotation marks, and referenced. 5. This thesis does not contain text, graphics or tables copied and pasted from the Internet, unless specifically acknowledged, and the source being detailed in the thesis and in the References sections. Signed ……………………………………………………………………………… vi TABLE OF CONTENTS List of Figures xiii List of Tables xviii Abbreviations xxii CHAPTER 1 INTRODUCTION 1 1.1 Curry leaf, Murraya koenigii 2 1.1.1 Societal value 3 1.1.2 Phytochemistry 4 1.1.3 Compounds that give rise to aroma 6 1.1.4 The biogenesis of terpenes in plants 7 1.1.4.1 Biogenesis of monoterpenes 8 1.1.4.2 Biogenesis of sesquiterpenes 10 1.2 The determination of volatile organic compounds in M. koenigii 10 1.3 Sample preparation 12 1.4 Extraction techniques used in this study 13 1.4.1 Solvent extraction 13 1.4.2 Soxhlet extraction 14 1.4.3 Steam distillation 15 1.4.4 Gas phase extraction 15 1.4.4.1 Headspace analysis 15 1.4.4.2 Solid phase microextraction (SPME) 18 1.5 Other extraction methods 23 1.5.1 Supercritical fluid extraction (SFE) 23 1.5.2 Membrane extractions 24 1.5.3 Sorbent extraction 25 1.5.4 Solid–phase extraction (SPE) 25 1.5.5 Stir bar sorptive extraction 25 1.5.6 Microwave distillation-solid-phase microextraction 26 vii 1.5.7 Microwave-assisted solvent extraction 26 1.5.8 Microwave accelerated steam distillation (MASD) 26 1.6 Gas chromatography 26 1.6.1 The gas chromatographic system 27 1.6.1.1 Carrier gas 28 1.6.1.2 Sample injection 28 1.6.1.3 Types of columns 28 1.6.1.4 Oven 29 1.6.1.5 Detectors 30 1.7 Gas chromatography-mass spectrometry analysis 31 1.7.1 Brief overview of the gas chromatograph-mass spectrometer 31 1.7.2 Inlets 33 1.7.3 Ion sources 33 1.7.3.1 Electron impact ion source 33 1.7.3.2 Chemical ionization source 34 1.7.4 Mass analyser 35 1.7.5 Ion detectors 36 1.7.5.1 Electron multipliers 36 1.8 Mass spectra of the terpenes 37 1.8.1 Fragmentation 37 1.8.1.1 Factors influencing the fragmentation of an ion 38 1.8.2 The mass fragmentation patterns 39 1.8.2.1 Acyclic terpenes 39 1.8.2.2 Cyclic terpenes 41 1.8.2.3 Sesquiterpenes 45 1.8.3 Identification of compounds using the mass spectral library 45 1.9 The advantages of using GC-MS 46 1.10 Outline of this project 47 viii CHAPTER 2 EXPERIMENTAL 48 2.1 Materials and equipment 48 2.2 Sample collection 48 2.3 Preliminary work 48 2.3.1 Choice of fresh or frozen leaves 49 2.3.2 Solvent choice for solvent and Soxhlet extraction 50 2.3.3 Headspace analysis and headspace solid phase microextraction 50 2.3.3.1 Equilibration time 50 2.3.3.2 Desorption time 51 2.3.3.3 Fibre coating 51 2.4 Extraction and isolation of the volatile oils 52 2.4.1 Extraction time 52 2.4.2 Procedure for solvent extraction 53 2.4.3 Procedure for Soxhlet extraction 53 2.4.4 Steam distillation 55 2.4.5 Vapour enrichment procedure 55 2.4.5.1 Extraction temperature 56 2.4.5.2 Headspace analysis procedure 56 2.4.5.3 HS-SPME analytical procedure 56 2.5 GC-MS parameters 57 2.5.1 GC-MS sampling technique 58 2.5.1.1 Liquid phase 58 2.5.1.2 Vapour phase 58 2.5.2 Quantitative determinations 59 2.5.3 Data analysis 60 2.5.4 Component identification 61 2.6 Summary of conditions 63 ix CHAPTER 3 RESULTS AND DISCUSSION 64 3.1 Organisation of results 64 3.2 Preliminary study 64 3.2.1 Identification of the volatile components in M. koenigii 66 3.2.2 Choice of solvent for solvent and Soxhlet extractions 68 3.2.2.1 Solvent extraction 68 3.2.2.2 Soxhlet extraction 71 3.2.3 Headspace and HS-SPME extraction conditions 72 3.2.3.1 Equilibration time 73 3.2.3.2 Fibre desorption time 75 3.2.3.3 PDMS and PA fibre coating 76 3.3 Essential oil analysis by solvent and Soxhlet extraction 84 3.3.1 Extraction period 84 3.3.1.1 Extraction period for solvent extraction