OPTIMIZATION OF SUPERCRITICAL CARBONDIOXIDE EXTRACTION PARAMETERS OF ESSENTIAL OILS FROM VARIOUS PLANTS THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF DOCTOR OF PHILOSOPHY IN CHEMISTRY BY AARTI SINGH DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA) 2016 CANDIDATE’S DECLARATION I, Aarti Singh, Department of Chemistry, certify that the work embodied in this Ph.D thesis is my own bonafide work carried out by me under the supervision of Prof. Anees Ahmad at Aligarh Muslim University, Aligarh. The matter embodied in this Ph.D thesis has not been submitted for the award of any other degree. I declare that I have faithfully acknowledged, given credit to and referred to the research workers wherever their works have been cited in the text and the body of the thesis. I further certify that I have not willfully lifted up some other’s work, para, text, data, result, etc. reported in the journals, books, magazines, reports, dissertations, theses, etc., or available at web-sites and included them in this Ph.D. thesis and cited as my own work. Date: (Signature of the candidate) AARTI SINGH (Name of the candidate) Certificate from the Supervisor This is to certify that the above statement made by the candidate is correct to the best of my knowledge. Signature of the Supervisor: Name & Designation: Prof. Anees Ahmad Department: Chemistry (Signature of the Chairman of the Department with seal) COURSE/COMPREHENSIVE EXAMINATION/PRE-SUBMISSION SEMINAR COMPLETION CERTIFICATE This is to certify that Ms. Aarti Singh, Department of Chemistry has satisfactorily completed the course work/comprehensive examination and pre-submission seminar requirement which is part of her Ph.D. programme. Signature of the Supervisor Signature of the Chairman (Prof. Anees Ahmad) Department of Chemistry Date: COPYRIGHT TRANSFER CERTIFICATE Title of the Thesis: Optimization of Supercritical Carbondioxide extraction parameters of essential oils from various plants. Candidate’s Name: Aarti Singh Copyright Transfer The undersigned hereby assigns to the Aligarh Muslim University, Aligarh, copyright that may exist in and for the above thesis submitted for the award of Ph. D. degree. (Signature of the Candidate) AARTI SINGH Acknowledgement First and foremost, I express my deep gratitude to the Almighty God for giving me the courage and strength to complete this work. I express my sincere thanks to my supervisor Prof. Anees Ahmad for his guidance, support, advice and encouragements throughout my research work. I admire his invaluable comments and keen insight in research. I would like to express my deepest gratitude to Chairman, Department of Chemistry, A.M.U. Aligarh, for support, motivation and providing me research facilities. I am also thankful to UGC-BSR, New Delhi, India, for financial assistance. I also express my heartfelt gratitude to Dr. Abdul Latif for Identification and authentication of samples, Prof. Aquil Ahmed for providing me training on the MINITAB 14 statistical software package, Abbas F. M. Alkarkhi for clearing queries related to MINITAB 14 software, Dr. Sayeed Ahmad for my review article on carotenoids. I owe a debt of gratitude to you all for your kindness, active help and encouragements. I owe a great debt of gratitude to my parents Dr. B. Lal and Asha Singh, without your love, support and prayer it would not have come true! Special mention goes to my brother Mr. Surya Singh for his affectionate encouragement and help. I would also like to thank Dr. Deepak Singh for his care, patience, motivation and kind help throughout my research work. I do remember the prayers, support and wishes from my friends and lab mates Dr. Rani Bushra, Mr. Seraj Anwar Ansari, Ms. Fauzia Khan, Ms. Nishat Khan, Ms. Yashfeen Khan and Ms. Nehal Zehra. ‘Thank you’ is just not enough to say, I appreciate your kind help throughout the journey. Last but not least, I express my thanks to the staff from the Department of Chemistry, A.M.U., for their kind help and assistance during my research work. I am really grateful to you all. (Aarti Singh) Abstract The thesis comprises of seven chapters. First chapter deals with the general introduction of the subject matter of the thesis which describes about essential oils, sources of essential oil, chemical constituents of essential oils, Methods of extracting essential Oils (Conventional methods and non-conventional methods), uses of essential oils and aims and objectives of the work. Essential oils have a complex composition, containing from a few dozen to several hundred constituents, especially hydrocarbons and oxygenated compounds. The main group is composed of terpenes and terpenoids and the other aromatic and aliphatic constituents, all characterized by low molecular weight. Trace components are important, since they give the oil a characteristic and natural odour. They can be obtained from different parts of plants including flowers, buds, seeds, leaves, twigs, bark, herbs, fruits and roots. Oils are used in the embalming process, in medicine and in purification rituals. There are also over 200 references to aromatics, incense and ointments in the Old and New Testaments. Research has confirmed centuries of practical use of Essential Oils, and we now know that the 'fragrant pharmacy' contains compounds with an extremely broad range of biochemical effects. There are about three hundred essential oils in general use today by professional practitioners. Continual bombardment of viral, bacterial, parasitic and fungal contamination occurs in our body. Essential oils are of great benefit to help protect our bodies and homes from this onslaught of pathogens. Immune system needs support and these essential oils can give the required endorsement. The ability of plants to accumulate essential oils is quite high in both Gymnosperms and Angiosperms, although the most commercially important essential oil plant sources are related to the Angiosperms. Most of the aromatic plants and essential oil commodities in terms of world trade belong to the families of Lamiaceae, Umbelliferae and Compositae. An Essential Oil contains more than 200 chemical components, but some are many times more complex. Essential oils consist of chemical compounds which have hydrogen, carbon and oxygen as their building blocks. They can be essentially classified into two groups: 1 Volatile fraction: Essential oil constituting of 90-95% of the oil in weight, containing the monoterpene and sesquiterpene hydrocarbons, as well as their oxygenated derivatives along with aliphatic aldehydes, alcohols and esters. Non-volatile residue: This comprises 1-10% of the oil, containing hydrocarbons, fatty acids, sterols, carotenoids, waxes and flavonoids. The constituents can be again subdivided into 2 groups, such as the hydrocarbons which are made up of mostly terpenes and the oxygenated compounds which are mainly alcohols, aldehydes, esters, ketones, phenols and oxides. Some of the common components are listed below along with their properties. Methods of extracting essential oils are classified as: Conventional methods Maceration Cold Pressing Enfleurage Steam distillation and solvent extraction Non-conventional methods Microwave assisted extraction Ultrasonic assisted extraction Supercritical fluid extraction A supercritical fluid (SCF) is any compound at a temperature and pressure above the critical point. Above the critical temperature (Tc) of a compound, the pure, gaseous component cannot be liquefied regardless of the pressure applied. The critical pressure (Pc) is the vapor pressure of the gas at critical temperature. In the supercritical environment only one phase exists. The fluid, as it is termed, is neither a gas nor a liquid. This phase retains solvent power approximating liquids as well as the transport properties common to gases. Applications of essential oils Food industry Pharmaceutical industry Cosmetic industry Veterinary product industry 2 Industrial deodorants Tobacco industry Biocides and insecticides Antioxidants Chapter two describes the critical review of the existing literature on the extraction of essential oils from various parts of the plants using conventional and non-convention extraction techniques. Chapter three describes materials and methods which includes collection of plant material, identification and authentication of plant material, methods for extraction of essential oil (Hydro-distillation, Solvent Extraction, Ultrasonic Assisted Extraction and Supercritical Carbon-dioxide (SC-CO2)). It also includes optimization of SC-CO2 extraction parameters via Full Factorial Design (FFD), physiochemical characterization of essential oil, analytical Studies (SEM, HPTLC, GC-MS and FTIR) and antioxidant activity of essential oils. Use of natural antioxidants, as food additives for inactivating free radicals receiving a lot of attention nowadays, not only for their scavenging properties, but also because they are natural, non-synthetic products, and their appreciation by consumers is very favourable. Chapter four describes optimization of total essential oil yield of Cinnamomum zeylanicum N. by using SC-CO2 extraction. Essential oil was extracted via Hydro-distillation (HD), Solvent extraction (SE), Ultrasonic assisted extraction (UAE) and Supercritical Carbon-dioxide (SC-CO2) extraction. Among all these methods SC-CO2 gave the highest percentage yield (3.8 %) at optimum conditions of o pressure (200 bar), temperature (40 C) and flow rate of CO2 (4 g/min) by FFD. The analysis shows that pressure and flow rate have shown to