CRITICAL FLUID TECHNOLOGY IN FOOD INDUSTRY FOR EXTRACTION AND PARTICLE GENERATION A Dissertation Submitted to the Faculty of Graduate School of Science and Technology of Kumamoto University

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(1)Kumamoto University. CRITICAL FLUID TECHNOLOGY IN FOOD INDUSTRY FOR EXTRACTION AND PARTICLE GENERATION A Dissertation Submitted to the Faculty of Graduate School of Science and Technology of Kumamoto University. By Ruhan ASKIN UZEL. In Partial Fulfillment of Doctor’s Degree of Philosophy in Chemical Engineering Field of New Frontier Sciences Department of Pulsed Power Sciences March, 2010.

(2) CRITICAL FLUID TECHNOLOGY IN FOOD INDUSTRY FOR EXTRACTION AND PARTICLE GENERATION. A Dissertation Submitted to the Faculty of Graduate School of Science and Technology of Kumamoto University. By Ruhan ASKIN UZEL. In Partial Fulfillment of Doctor’s Degree of Philosophy in Chemical Engineering Field of New Frontier Sciences Department of Pulsed Power Sciences March, 2010.

(3) ABSTRACT Extraction with supercritical fluids is a unit operation in chemical engineering that could be employed for a variety of applications. The goal of this project was to establish new approaches in supercritical technology in food industry. Therefore, supercritical fluids and their common applications for both carbon dioxide and water were briefly explained. Furthermore, supercritical fluid technology was applied on basis for natural products from mushroom called Ganoderma lucidum and a new hybrid vegetable called Petit vert. In the following sections, a general overview of supercritical fluids, the importance and recent industrial applications of supercritical fluid technology, as well as fundamental aspects which were studied in this work was described in more detail. Moreover, as carrying crucial importance for food packaging industry, removal of oligomers from PET (polyethylene terephthalate) by hydrothermal treatment has been studied in following chapters. Finally, a novel system design for particle generation has been investigated in a separate chapter. Being one of the objectives of this work, a method was established for extraction from grinded form of Ganoderma lucidum with water in sub-critical conditions and with SCCO2 at distinct temperatures to obtain extracts rich in watersoluble organic compounds (WSOC), including mostly beta glucans and triterpenoids including ganoderic acids and alcohols respectively. SCCO2 experiments were carried out at pressures of 10, 20 and 30 MPa and temperatures ranging from 40 to 60 oC with and without modifier. Ethanol was used as modifier at flow rates of 0.1, 0.2 and 0.4 mL/min. The CO2 flow rate was maintained at 4 mL/min for 2 hours extraction time. The extracts were analyzed by HPLC. The temperatures for batch-scale sub-critical water extraction kept constant at different time intervals starting from 373 up to 573 K. For semi-continuous scale experiments, samples were extracted once with water at 373, 423, 448 and 473 K, respectively, working at pressure around 10 MPa to keep the water in the liquid state. The flow rate used was 1 mL/min. The extracts were analyzed by TOC for water soluble organic compound recovery. The highest yield obtained at 473 K as 78.1 % and as 57.4 % for batch- and semi continuous scale experiments respectively. It was also. i.

(4) demonstrated that the modified SCCO2 extraction is suitable for the extraction of Ganoderma lucidum. Water has been shown to be capable of extracting different classes of compounds from Petit vert depending on the temperature used in hydrothermal treatment. Hot compressed water was continuously delivered into a reactor while gradually rising the temperature from 25 to 200 oC at 5 MPa and its flow rate was 1 ml/min for different experimental time combinations. The extract from the reactor was cooled and each fraction was collected at the volume having range from 10 ml up to 40 ml. Next, pectin was separated from Petit vert using semi continuous flow reactor. The quantitative analysis of the pectin was measured as the amount of a galacturonic acid according to the dimethylphenol method. It was found that the pectin was rapidly extracted when the temperature of the reactor reached 200 °C from the analysis of each fraction. As a result of the cation analysis, K+ was detected as an indicator element in a large amount having peak a little ahead from the extraction peak of pectin. A rapid, alternative technique for the extraction of low molecular weight oligomers from PET (polyethylene terephthalate) polymer film was reported. Traditionally, PET has been extracted using liquid–solid Soxhlet extraction but this has proved to be extremely time consuming. In this work, cyclic trimer and other low molecular weight oligomers have been extracted from PET film using water at subcritical conditions. The determination and identification of the extracts has been performed using HPLC and GPC analysis. Finally, in order to combine the extraction technique with particle formation process, a novel system was designed. The apparatus is divided into three sections: a supercritical fluid delivery unit (a water source, a circulating heater, an HPLC pump), a solute dissolving and extracting unit (a pre-heater, an extraction vessel and an oven), and a crystallizing-separating unit (nozzle and collector). The extraction vessel placed in the oven was heated to the desired temperature and accurately controlled to within range of 2 °C difference. The pressure in the stainless steel extraction vessel (10 ml) was monitored by a pressure gauge. The solution left the. ii.

(5) extraction vessel and was led to the crystallizer where it was throttled across an expansion nozzle. The pre-expansion temperature was monitored by a temperature controller (T1 -C). When the supercritical solution depressurized through the nozzle to ambient conditions, it experienced a rapid expansion and resulted in precipitation of the solute in the collection chamber as powder. Precipitated sample particles were analyzed with laser diffraction in order to have an idea about the size distribution of the product. SEM analysis was done respect to particle size and shape. Molecular weight distributions were determined via size-exclusion chromatography (SEC). The sample weight change with increasing temperature was evaluated by a thermogravimetric analyzer (TGA 2050, TA Instruments). The crystal structures and melting points of original and precipitated powder were examined by an X-raydiffractiometer.. iii.

(6) PREFACE This thesis is the final work of my Ph.D. study at the Field of New Frontier Sciences, Department of Pulsed Power Sciences, Faculty of Graduate School of Science and Technology, Kumamoto University. It serves as documentation of my work during the study, which has been made from autumn 2007 until spring 2010. The thesis consists of seven chapters. Five chapters contain papers that are accepted by or intended for an international journal or proceedings. These chapters cover information of critical fluids, application areas of supercritical fluids on laboratory and industrial basis, as well as demonstration of a novel system design on particle formation technology in food industry. Some of these papers are written jointly with other scientists, of which some are more food experts than chemists, and some are more chemists that analysts. In the first chapter, however, I have given a general introduction to critical fluid technology and a survey of the new scientific results, which are presented in following chapters in this thesis and in the final chapter; I did an overall summary and conclusion for all research.. iv.

(7) CERTIFICATION This is to certify that the thesis entitled “Critical Fluid Technology in Food Industry for Extraction and Particle Generation” submitted by Ruhan ASKIN UZEL to Kumamoto University, Graduate School of Science and Technology for the award of the degree of Doctor of Philosophy is a bona fide record of the research work carried out by her under our supervision and guidance. The content of the thesis, in full or parts have not been submitted to any other Institute or University for the award of any other degree or diploma.. Prof. Motonobu GOTO. Assoc. Prof. Mitsuru SASAKI. Place: Kumamoto University, Graduate School of Science and Technology, Department of Applied Chemistry and Biochemistry Date: 2010.03.25. v.

(8) DECLARATION I herewith declare that I autonomously carried out the PhD-thesis entitled “Critical Fluid Technology in Food Industry for Extraction and Particle Generation”. The following third party assistance has been enlisted:. (i) The thesis comprises only my original work towards the PhD except where indicated in the Preface, (ii) Due acknowledgement has been made in the text to all other material used, (iii)The thesis is less than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices.. I hereby affirm the above statements to be complete and true to the best of my knowledge and belief, it contains no material previously published or written by another person nor material which has been accepted for the award of any other degree or diploma of the university or other institute of higher learning, except where due acknowledgment has been made in the text.. 2010.03.25 Ruhan ASKIN UZEL. Signature. vi.

(9) ACKNOWLEDGEMENTS Doing a project is similar to entering a long-term relationship with educational side of daily life. Throughout it all, the project demanded constant attention. Therefore, I'd like to thank the people that made it possible to focus on the subject. I wish to acknowledge all for their role in my life; however, this page is specifically designed to note my appreciation of those people who stand out most notably in my mind as contributing to the content of what you will find on this graduation project. I deeply thank my supervisors Prof. Motonobu GOTO and Assoc. Prof. Mitsuru SASAKI, for giving me the opportunity to work and to learn from their expertise in the experimental field. Their advice, discussions and personal and professional support are greatly appreciated. I will keep you always as a good example of success. This project was supported in part by Kumamoto University Global COE “Center of Excellence” Program, Global Initiative Center for Pulsed Power Engineering. I am grateful for the financial support. I wish to express my deepest sense of gratitude and respect to Re. Farmer Co., Ltd. (Kumamoto, Japan) and Kagome Corporation (Tokyo, Japan) for providing working materials. I acknowledge my indebtedness and gratefulness to Monbukagakusho (MEXT, Ministry of Education, Science and Culture, Japan) for providing me a financial support to complete my master thesis in Faculty of Graduate School of Science and Technology, Department of Pulsed Power Sciences, Kumamoto University. This essay was improved by conversations with a large number of people who helped debug it. Particular thanks to Dr. Tao Fang, Dr. Masahiro Tanaka and Dr. Wahyudiono, for their support, advice and valuable discussions on both the experimental and computational fields. I would like to also thank all of my. vii.

(10) officemates for their help in kind suggestions, and all other chemical engineering laboratory members for their friendship and support. Finally I deeply thank my precious Caner UZEL and my family for the tremendous amount of patience they have had over the last 2 years and their continuous encouragement to succeed. I hope the relationships we have built will continue through our professional careers in the field of Supercritical Fluids.. viii.

(11) DEDICATION This thesis would be incomplete without a mention of the support given me by my family and my husband, Caner UZEL, to whom this thesis is dedicated. You have been with me every step of the way, through good times and bad. Thank you for all the unconditional love, guidance, and support that you have always given me, helping me to succeed and instilling in me the confidence that I am capable of doing anything I put my mind to. You were my own "soul out of my soul," who kept my spirits up when the muses failed me. Without your lifting me up when this thesis seemed interminable, I doubt it should ever have been completed.. ix.

(12) TABLE OF CONTENTS Pages Abstract. i. Preface. iv. Certification. v. Declaration. vi. Acknowledgements. vii. Dedication. ix. Table of Contents. x. List of Tables. xvii. List of Figures. xix. Chapter 1: Critical Fluid Technology 1. 1.. Introduction. 1. 1. 2.. Supercritical Fluids (SCFs). 3. 1. 2. 1.. Supercritical Carbon Dioxide (SCCO2). 4. 1. 2. 2.. Supercritical Water (SCW). 7. 1. 3.. Modification of Supercritical Fluids. 11. 1. 4.. Other States of Fluids. 13. 1. 5.. Basic Properties of SCFs. 14. 1. 6.. Extraction with Supercritical Fluids. 20. x.

(13) 1. 7.. 1. 6. 1.. Batch Extraction. 20. 1. 6. 2.. Continuous Extraction. 22. Applications and Commercial Processes of SCFs. 23. 1. 7. 1.. 24. Food Applications. 1. 8.. A New Application: Supercritical Particle Formation. 27. 1. 9.. References. 29. Chapter 2: Supercritical Carbon Dioxide Extraction of Ganoderma lucidum 2.1.. Introduction. 33. 2.2.. Experimental. 34. 2.2.1.. 2.3.. Materials and Chemicals 2.2.1.1.. Materials- Ganoderma lucidum. 34. 2.2.1.2.. Chemicals. 42. 2.2.2.. SCCO2 Extraction. 43. 2.2.3.. Analysis. 45. Results and Discussion. 46. 2.3.1.. Conventional Solvent Extraction and SCCO2 Extraction. 49. 2.3.2.. Non-modified and Modified SCCO2 Extraction of Ganoderma lucidum. 2.3.3.. 51. Temperature and Pressure Effects in Modified Supercritical Extraction. 2.4.. 34. Conclusion. 58 62. xi.

(14) 2.5.. Acknowledgements. 64. 2.5.. References. 64. Chapter 3: Recovery of water-soluble Compounds from Ganoderma lucidum by Hydrothermal Treatment 3.1.. Introduction. 68. 3.1.1. Advantage of Sub-critical Water (Hot Water) Extraction of Ganoderma lucidum 3.2.. 70. Experimental. 71. 3.2.1.. Materials and Chemicals. 71. 3.2.1.1.. Materials- Ganoderma lucidum. 71. 3.2.1.2.. Chemicals. 72. 3.2.2. Sub-critical Water Extraction (SWE) 3.2.2.1.. Batch type extraction. 73. 3.2.2.2.. Semi-continuous type extraction. 73. 3.2.3. Spray Drying of Ganoderma Extracts 3.2.3.1. 3.2.4.. 72. Drying conditions. Analysis. 77 78 79. 3.3.. Results and Discussion. 81. 3.4.. Conclusion. 102. 3.5.. Acknowledgements. 103. 3.6.. References. 103. xii.

(15) Chapter 4: Hydrothermal Treatment of Petit vert for the extraction of Pectin and Minerals 4.1.. Introduction. 106. 4.2.. Experimental. 107. 4.2.1. Materials and Chemicals. 108. 4.2.1.1.. Material: Petit vert. 108. 4.2.1.2.. Chemicals. 115. 4. 2. 2. Experimental Procedure. 115. 4.2.3. Analysis. 118. 4.3.. Results and discussion. 120. 4.4.. Conclusion. 135. 4.5.. Acknowledgements. 136. 4.6.. References. 136. Chapter 5: Removal of Oligomers from PET (Polyethyleneterephthalate) Materials in Food Packaging Industry 5.1.. 5.2.. Introduction. 138. 5.1.1. Raw materials. 139. 5.1.2. Polymerization methods. 140. 5.1.2.1.. First step: prepolymerization. 140. 5.1.2.2.. Second step: polycondensation. 141. 5.1.3. Polymerization kinetics under various reaction conditions. 142. Experimental. 143. xiii.

(16) 5.2.1. Materials and Chemicals 5.2.1.1.. 143. Material- PET pellets 5.2.1.1. a.. Food Contact Considerations for Virgin PET. 5.2.1.1. b.. 5.2.1.2.. 147. Migration Considerations in Food Contact Applications. 5.2.1.1. d.. 146. Sorption Considerations in Food Contact Applications. 5.2.1.1. c.. 143. 149. Reaction By-products Formed During PET Manufacture. 150. Acetaldehyde. 151. Oligomers in PET. 151. Chemicals. 152. 5.2.2. Experimental Procedure. 153. 5.2.3. Analysis. 157. 5.3.. Results and Discussion. 161. 5.4.. Conclusion. 183. 5.5.. Acknowledgements. 184. 5.6.. References. 184. Chapter 6: Particle Generation with Air-Assisted Sub-critical Water Extraction Technology 6.1.. Introduction. 188. xiv.

(17) 6.1.1. Summary of Supercritical Particle Formation Methodologies 6.1.1.1.. Rapid Expansion of Supercritical Solutions (RESS) 194 6.1.1.1. a.. 6.1.1.2.. Advantages and disadvantages. Particle from Gas Saturated Solution 6.1.1.2. a.. 6.1.1.3.. Advantages and Disadvantages. 195 195 196. Depressurization of an Expanded Liquid Organic Solution 6.1.1.3.a.. 6.2.. 193. Advantage and Disadvantages. 196 197. Experimental Techniques. 199. 6.2.1. Materials and Chemicals. 199. 6.2.1.1.. Materials. 199. 6.2.1.2.. Chemicals. 199. 6.2.2. Experimental Procedure. 199. 6.2.3. Design of a Novel Combined System: Extraction - Air Assisted Particle Formation. 203. 6.2.4. Analytical Procedures. 210. 6.3.. Results and Discussion. 211. 6.4.. Conclusion. 227. 6.5.. Acknowledgements. 230. 6.6.. References. 230. xv.

(18) Chapter 7: Summary and Conclusions 7.1.. Summary of the work. 236. 7.1.1. Supercritical Carbon dioxide (SCCO2) Extraction of Ganoderma lucidum. 236. 7.1.2. Recovery of Water-soluble Compounds from Ganoderma lucidum by Hydrothermal Treatment. 237. 7.1.3. Hydrothermal Treatment of Petit vert for the Extraction of Pectin and Minerals. 239. 7.1.4. Removal of Oligomers from PET (Polyethyleneterephthalate) Materials in Food Packaging Industry. 241. 7.1.5. Particle Generation with Air-assisted Sub-critical Water Extraction Technology 7.2.. Final Comments. 243 246. xvi.

(19) LIST OF TABLES Pages Chapter 1: Critical Fluid Technology Table 1.1.. Comparison of physical and transport properties of gases, liquids and SCFs.. 4. Table 1.2.. Critical conditions for various supercritical fluids.. 10. Table 1.3.. Evaluation of supercritical fluid technology.. 11. Table 1.4.. SFE applications in food products.. 25. Table 1.5.. Applications of SWE the extraction of flavors and fragrances from plant material and food.. Table 1.6.. 26. Applications of SWE to the analysis of pesticides from plant material and food.. 26. Chapter 2: Supercritical Carbon Dioxide Extraction of Ganoderma lucidum Table 2.1.. Table 2.2. Major bioactive constituents in Ganoderma species and their function.. 40. Comparison of extraction yields of various extraction methods.. 50. Chapter 3: Recovery of water-soluble Compounds from Ganoderma lucidum by Hydrothermal Treatment Table 3.1.. Experimental conditions for slurry solutions to feed spray drier after semi-continuous scale sub-critical water treatment. xvii. 79.

(20) Table 3.2.. Table 3.3.. Efficiency and yield values for batch-scale sub-critical water extraction.. 85. GPC calibration analysis conditions.. 94. Chapter 4: Hydrothermal Treatment of Petit vert for the extraction of Pectin and Minerals Table 4.1. Basic chemical components of Petit vert.. 112. Chapter 5: Removal of Oligomers from PET (Polyethyleneterephthalate) Materials in Food Packaging Industry Table 5.1.. A list of FDA food stimulants.. 150. Table 5.2.. A list of EEC food stimulants.. 150. Table 5.3.. Qualitative evaluation* of extracts depending on time at 200 °C.. 164. Chapter 6: Particle Generation with Air-Assisted Sub-critical Water Extraction Technology Table 6.1.. Summary of the particle formation technologies using supercritical fluids.. 193. xviii.

(21) LIST OF FIGURES Pages Chapter 1: Critical Fluid Technology Figure 1.1.. Phase diagram of SCFs.. 5. Figure 1.2.. Pressure-temperature phase diagram.. 5. Figure 1.3.. Phase diagram of CO2 with constant density lines (g/l).. 6. Figure 1.4.. Phase diagram of CO2/ethanol mixture. The curved lines represent the modeled values and the dots the respective experimental value.. 12. Figure 1.5.. Phase diagram of CO2.. 14. Figure 1.6.. Enthalpies of gaseous and liquid carbon dioxide as a function of temperature.. 16. Figure 1.7.. Comparison of physical properties of fluids.. 17. Figure 1.8.. The change from two definite phases to one supercritical phase.. 18. Figure 1.9.. Density isotherms of carbon dioxide.. 19. Figure 1.10. Dielectric constant and density of carbon dioxide as a function of pressure at isothermal temperature of +50°C.. 20. Figure 1.11. Principle of supercritical batch extraction.. 21. Figure 1.12. Principle of continuous counter-current supercritical extractor.. 23. Figure 1.13. Decaffeination of coffee.. 24. Figure 1.14. Result of contents search using "supercritical fluids" and "carbon dioxide" as search terms (1999-2000).. xix. 24.

(22) Figure 1.15. Schematic representation a) of the RESS process and b) of the SASP process.. 28. Chapter 2: Supercritical Carbon Dioxide Extraction of Ganoderma lucidum Figure 2.1.. Reishi with varnished surface.. 35. Figure 2.2.. Reishi hardwood inoculation stumps.. 36. Figure 2.3.. Chemical structure of ganoderma triterpenoids.. 38. Figure 2.4.. Diagram of the supercritical fluid extraction apparatus.. 43. Figure 2.5.. Supercritical carbon dioxide extraction apparatus.. 44. Figure 2.6.. High Performance Liquid Chromatography (HPLC) analysis equipment.. Figure 2.7.. 46. SCCO2 extraction efficiencies at 313-333 K, 10-40 MPa, and 0.4 mL/min ethanol flow rate.. 47. Figure 2.8.. Extraction efficiencies (%) at 313 K.. 48. Figure 2.9.. CO2 consumption rate (g) against co-solvent flow at 313 K.. 49. Figure 2.10. Specific yields at 313 K in comparison to hot water extraction and EtOH extraction.. 51. Figure 2.11. Chromatogram of triterpenoid components extracted by non-modified supercritical fluid extraction, 313 K, 20 MPa.. 53. Figure 2.12. Chromatogram of triterpenoid components extracted by modified supercritical fluid extraction at 333 K, 20 MPa, 0.4 ml/min EtOH flow rate.. 54. xx.

(23) Figure 2.13. HPLC chromatogram for ganoderic alcohols, 313 K, 20 MPa, 0.4 ml/min EtOH flow rate.. 55. Figure 2.14. HPLC result for ganoderic acids at 313 K, 20 MPa, 0.4 mL/min ethanol flow rate.. 57. Figure 2.15. Temperature and flow rate effects on extraction yield under various pressures.. 58. Figure 2.16. Extraction yields at 313 K.. 59. Figure 2.17. Extraction yields at 323 K.. 60. Figure 2.18. Extraction yields at 333 K.. 61. Figure 2.19. Effects of temperature, pressure and EtOH flow rate on extraction. 62. Chapter 3: Recovery of water-soluble Compounds from Ganoderma lucidum by Hydrothermal Treatment Figure 3.1.. Batch-mode experimental set-up.. 73. Figure 3.2.. Laboratory scale hydrothermal treatment system.. 75. Figure 3.3.. Subcritical water semi-continuous type extraction flow diagram.. Figure 3.4.. 76. Spray drying apparatus (Yamato, ADL 310, Yamato ScientificC Co., Ltd., Tokyo- Japan).. 77. Figure 3.5.. Flow diagram for spray drying process.. 78. Figure 3.6.. TOC calibration curve (D (+)-Glucose standard,. Figure 3.7.. 98.0 %, Wako Pure Chemical Industries, Ltd., Japan).. 80. Percent recovery of WSOC at various temperatures.. 82. xxi.

(24) Figure 3.8.. Relation between extraction efficiency and reaction temperature.. Figure 3.9.. Recovery yield of WSOC at various temperatures in batch mode hydrothermal treatment.. 83. 86. Figure 3.10. Water Insoluble Organic Carbon (WIOC) Extraction Efficiency (%).. 87. Figure 3.11. Effect of temperature on the extraction curve, 10 MPa, 1 ml/min water flow rate.. 88. Figure 3.12. Effect of temperature on WSOC extraction for semi-continuous hydrothermal extraction.. 90. Figure 3.13. Comparison of extraction efficiencies for batch- and. Fig. 3.14.. semi-continuous scale treatments, 473 K.. 91. Effect of water to feed ratio on extraction at 473 K.. 92. Figure. 3.15. GPC Calibration Curve and Analysis Conditions (same polystyrene standard concentrations- 0.05 %, 10 µl injection).93 Figure 3.16. GPC Chromatogram.. 95. Figure 3.17. Micrographs of particles of Ganoderma lucidum obtained by electronic microscopy after hydrothermal water treatment, (1: original sample, 2: 373 K, 3: 423 K, 4: 448 K, 5: 473 K).. 96. Figure 3.18. Effect of temperature on Ganoderma extracts for batch mode (left) and semi-continuous mode (right) hydrothermal treatments. 98 Figure 3.19. SEM images of spray dried powders of G. lucidum after hydrothermal extraction at different temperatures.. xxii. 99.

(25) Figure 3.20. Spray dried products.. 99. Figure 3.21. Powdered forms of Ganoderma lucidum after spray drying.. 101. Figure 3.22. MALDI-TOF mass spectrum of peaks from Ganoderma sample extracted for 130 min at 473 K.. 102. Chapter 4: Hydrothermal Treatment of Petit vert for the extraction of Pectin and Minerals Figure 4.1.. Petit vert is a hybrid vegetable.. 109. Figure 4.2.. Structure of Petit vert.. 110. Figure 4.3.. Comparison of nutritive values of various foods with Petit vert.. 111. Figure 4.4.. Chemical composition similarity of Petit vert with other vegetables.112. Figure 4.5.. Main chain of pectic substance.. 113. Figure 4.6.. Assumption structure of protopectin in plant.. 114. Figure 4.7.. Subcritical water extraction flow diagram.. 115. Figure 4.8.. Temperature profile for experiments at constant temperatures.. 117. Figure 4.9.. Temperature profile for experiments at gradually increased temperatures.. 118. Figure 4.10. Dimethylphenol method for pectin determination.. 119. Figure 4.11. Ca2+concentration for experiments in constant temperatures.. 121. Figure 4.12. Mg2+concentration for experiments in constant temperatures.. 122. Figure 4.13. K+ concentration for experiments in constant temperatures.. 123. Figure 4.14. Cumulative amount of Ca2+ for experiments at gradually increased temperatures.. 124 xxiii.

(26) Figure 4.15. Cumulative amount of Mg2+ for experiments at gradually increased temperatures.. 125. Figure 4.16. Cumulative amount of K+ for experiments at gradually increased temperatures.. 126. Figure 4.17. Effects of of extraction temperature and time fractionation on galactronic acid amount. Figure 4.18. Recovery rate for total galactronic acid.. 127 128. Figure 4.19. Comparison of galactronic acid concentrations for different temperatures for 10 minutes fractionation.. 129. Figure 4.20. Comparison of galactronic acid concentrations for different temperatures for 30 minutes fractionation.. 130. Figure 4.21. Relation between K+ and galactronic acid recovery for temperature up to 150 °C.. 131. Figure 4.22. Relation between K+ and galactronic acid recovery for temperature up to 200 °C.. 132. Figure 4.23. Elution behaviour of Ca2+ and Mg2+ ions for temperature up to 150 °C.. 133. Figure 4.24. Elution behaviour of Ca2+ and Mg2+ ions for temperature up to 200 °C.. 134. Figure 4.25. Effect of temperature on extract color.. 134. Figure 4.26. Gel filtration chromatogram of pectin molecules in comparison with standard samples.. xxiv. 135.

(27) Chapter 5: Removal of Oligomers from PET (Polyethyleneterephthalate) Materials in Food Packaging Industry Figure 5.1.. Schemes for the PET polymerization process.. 140. Figure 5.2.. PET pellets.. 144. Figure 5.3.. Formation of PET (polyethylene terephthalate).. 145. Figure 5.4.. Drawing of the bottom part of a PET soft drink bottle illustrating sorption, migration and permeation.. 146. Figure 5.5.. A summary of the disadvantages of “flavour scalping”.. 148. Figure 5.6.. Sorbate, polymer and external factors effecting sorption in PET.. 149. Figure 5.7.. Formation of acetaldehyde from PET.. 151. Figure 5.8.. Cyclic oligomers identified in PET.. 152. Figure 5.9.. Batch-mode experimental set-up, a) reactor, b) electrical furnace, c) superheated water phase, d) PET pellets.. 153. Figure 5.10. Sub-critical scale hydrothermal treatment system.. 155. Figure 5.11. Sub-critical water extraction flow diagram.. 156. Figure 5.12. Experimental procedure flow chart.. 157. Figure 5.13. TOC analysis equipment, TOC-V series, standalone standard model.. 159. Figure 5.14. TOC calibration curve (D (+)-Glucose standard, 98.0 %, Wako Pure Chemical Industries, Ltd., Japan).. 160. Figure 5.15. Effect of temperature on each sample (oligomer-rich pellets).. 162. Figure 5.16. Overall effect of temperature on samples (oligomer-rich pellets). xxv.

(28) for 200, 220, 240,…, 300 °C (from left to the right).. 163. Figure 5.17. Elution profile of water-soluble compounds at 200 °C.. 165. Figure 5.18. Effect of water flow rate on oligomer removal.. 166. Figure 5.19. Change in physical formation of sample materials at 200 °C, 1 mL/min water flow rate.. 167. Figure 5.20. Change in physical formation of sample materials at 200 °C, 2 mL/min water flow rate.. 168. Figure 5.21. Plug in effect of low water flow rate on reaction cell.. 169. Figure 5.22. Steam treated sample pellets.. 170. Figure 5.23. Amount of water-insoluble extract at 200 °C for 1 mL/min (a) and 2 mL/min (b) water flow rate.. 172. Figure 5.24. Amount of water-insoluble extract at 175 °C for 2 mL/min water flow rate.. 173. Figure 5.25. Effect of water flow rate on water slouble carbon ectraction for 1 mL/min (a) and 2 mL/min (b) flow rates.. 175. Figure 5.26. Extract recovery profile at 175 °C, 2 mL/min water flow rate.. 176. Figure 5.27. Extract recovery profile at 200 °C, 2 mL/min water flow rate.. 177. Figure 5.28. Elution rates for experiments at 175 °C (a), 190 °C (b) and 200 °C (c).. 180. Figure 5.29. Reaction velocity for experiments at 190 °C (a) and 200 °C (b).. xxvi. 182.

(29) Chapter 6: Particle Generation with Air-Assisted Sub-critical Water Extraction Technology Figure 6.1.. Schematic of particle production process.. 189. Figure 6.2.. Drawings of pre-designed drying unit.. 191. Figure 6.3.. Typical RESS process.. 194. Figure 6.4.. Schematic of PGSS process.. 195. Figure 6.5.. Schematic of DELOS process.. 198. Figure 6.6.. Schematic illustration of the RESS experimental apparatus.. 200. Figure 6.7.. Mechanism of counter flow particle formation system.. 201. Figure 6.8.. Inside structure of air-atomizing nozzle. The nozzle jets. Figure 6.9.. are kept free from blockages with a self-cleaning pin.. 202. Particle formation system.. 204. Figure 6.10. Illustrations of the novel system design; a), b), c) views of overall system, d) extraction unit, e) nozlle (outside view), f) nozzle (inside view).. 206. Figure 6.11. Spray pattern of air-atomizing nozzle.. 208. Figure 6.12. Schematic representation of the process and its operational principles.. 209. Figure 6.13. Thin film formation in counter flow system at 150 °C (a) and 200 °C (b).. 212. Figure 6.14. Slurry formation in counter flow system at 150 °C (a) and 200 °C (b).. 213. Figure 6.15. Particle formation in counter flow system at 150 °C (a). xxvii.

(30) and 200 °C (b).. 214. Figure 6.16. Particles around the nozzle edges.. 215. Figure 6.17. Ganoderma powders produced at 100 °C and 150 °C.. 216. Figure 6.18. Ganoderma powders produced at 150 °C and 200 °C.. 216. Figure 6.19. Electro-microscopy pictures for samples distributed in water and dry ones at 100 °C (a), 150 °C (b), 200 °C (c).. 218. Figure 6.20. SEM micrographs of the generated Ganoderma particles obtained at 10 MPa.. 219. Figure 6.21. SEM micrographs of the generated Ganoderma particles obtained at 5 MPa.. 219. Figure 6.22. Micrographs of generated particles after hydrothermal treatment at 100 °C.. 220. Figure 6.23. Micrographs of generated particles after hydrothermal treatment at 150 °C.. 221. Figure 6.24. Micrographs of generated particles after hydrothermal treatment at 200 °C.. 221. Figure 6.25. Average diameter distribution of the Ganoderma particles at 100 °C (a), 150 °C (b) and 200 °C (c).. 224. Figure 6.26. Ganoderic alcohols in extract.. 225. Figure 6.27. Ganoderic acids in extract.. 226. Figure 6.28. MALDI-TOF mass spectrum for molecular weight distribution profile of generated particles.. xxviii. 227.

(31) CHAPTER 1 CRITICAL FLUID TECHNOLOGY. 1.1. Introduction “Let food be your medicine and medicine be your food.” − Hippocrates 400 B.C.. Health food is food grown organically. It is consumed in its natural form or after it is transformed by using natural substances into a high-nutrition value product, such as natural concentrates. It is believed that there are at least 43 chemical components, called essential nutrients, which must be present in our food. Deficiency of any of them in our food creates imbalance in our systems and leads to disease or death. Luckily, “Mother Nature” provides us with all the essential nutrients (such as from seeds, nuts, grains, spices, fruits, and vegetables as well as algae) for human growth and health. However, as some of the nutrients are lost or depleted by thermal, natural, or bacteriological degradation between the time of harvesting and the time of ingestion, it becomes absolutely essential to supplement our foods with these nutrients, particularly vitamins and antioxidants. These nutrients ought to be close to their original form so that they create the least disturbance in body systems. Consequently it is preferable that they can be derived or concentrated from natural sources, in the form of natural extracts [1]. The removal of the components of natural products from their source, whether for analytical or processing purposes, is historically one of the oldest chemical problems for separation. Predating even the alchemists, the production of dyes, perfumes, and foods from natural products, as well as alcohol fermentation processes, exemplify the economic importance of natural products separations on the civilization of mankind [2].. 1.

(32) In the last decade, new trends have emerged in the food industry. These trends include an enhanced concern for the quality and safety of food products, increased preference for natural products over synthetic ones and have driven supercritical fluid technology to become the primary alternative to traditional solvent extraction for the extraction and fractionation of active compounds. Supercritical fluid extraction using carbon dioxide as a solvent has provided an excellent alternative to the traditional solvent extraction of natural products over the last two decades. Supercritical CO2 with its clean, safe, inexpensive, nonflammable, nontoxic, environment-friendly, nonpolluting solvent properties was found to be selective in the separation of desired compounds without leaving toxic residues in extracts and without the risk of thermal degradation of processed products. Besides, the energy costs associated with this novel extraction technique are lower than costs for traditional solvent extraction methods. Water is an interesting medium and reagent for various conventional chemical reactions, purification and separation processes, because it is non-toxic, non-flammable, cheap and easily available. At room temperature, water is too polar to dissolve non-polar organic compounds, but at elevated temperatures and pressures water becomes less polar making it an interesting and environmentally friendly alternative to many organic solvents. Similarly, the extraction and isolation of compounds from solid matrices can also be performed efficiently with water at elevated temperatures and pressures. Pressurized hot water can also be utilized in liquid chromatography and in combinations of extraction and chromatographic techniques. Extraction with supercritical fluids is a unit operation in chemical engineering that could be employed for a variety of applications including the extraction and fractionation of edible fats and oils, purification of solid matrices, separation of tocopherols and other antioxidants, clean-up of herb medicines and food products from pesticides, detoxification of shellfish and concentration of fermentation broth, fruit juices, among others [3-7]. Supercritical fluid extraction has proved effective in the. 2.

(33) separation of essential oils and its derivatives for use in the food, cosmetics, pharmaceutical and other related industries, producing high-quality essential oils with commercially more satisfactory compositions (lower monoterpenes) than obtained with conventional hydrodistillation [8-11]. The goal of this project was recovery of natural products using supercritical fluid technology. Supercritical fluids and their common applications for both carbon dioxide and water were briefly explained. The goal of this project was to establish new approaches in supercritical technology in food industry. Therefore, supercritical fluids and their common applications for both carbon dioxide and water were briefly explained. Furthermore, supercritical fluid technology was applied on basis for natural products from mushroom called Ganoderma lucidum and a new hybrid vegetable called Petit vert. In the following sections, a general overview of supercritical fluids, the importance and recent industrial applications of supercritical fluid technology, as well as fundamental aspects which were studied in this work was described in more detail. Moreover, as carrying crucial importance for food packaging industry, removal of oligomers from PET (polyethylene terephthalate) by hydrothermal treatment has been studied in following chapters. Finally, a novel system design for particle generation has been investigated in separate chapter. 1.2. Supercritical Fluids (SCFs) A fluid heated to above the critical temperature and compressed to above the critical pressure is known as a supercritical fluid. Frequently the term, compressed liquid, is used to indicate a supercritical fluid, a near-critical fluid, an expanded liquid or a highly compressed gas. The phenomena and behavior of supercritical fluids has been the subject of research right from 1800’s. A supercritical fluid (SCF) is any compound at a temperature and pressure above the critical values. The critical temperature of a compound is defined as the temperature above which a pure, gaseous component cannot be liquefied regardless of the pressure. 3.

(34) applied. The critical pressure is then defined as the vapor pressure of the gas at the critical temperature. The temperature and the pressure at which the gas and liquid phases become identical is the critical point. In the supercritical environment, only one phase exists. The fluid, as it is termed, is neither a gas nor a liquid and is best described as intermediate to the two extremes. This phase retains the solvent power common to liquids as well as the transport properties common to gases [12]. Table 1.1 displays the comparison of typical values for density, viscosity and diffusivity of the gases, liquids and SCFs. Table 1.1. Comparison of physical and transport properties of gases, liquids and SCFs. Property. Gas. SCF. Liquid. Density (g/mL). (0.6-2) x 10-3. 0.2-1.0. 0.6-1.6. Viscosity (g cm-1 s-1). (1-3) x 10-3. (1-9) x 10-3. (0.2-3) x 10-2. Diffusivity (cm2 s-1). 0.1-0.4. (2-7) x 10-4. (0.2-2) x 10-5. Surface Tension (dynes cm-1). 0. 0. 30-60. As shown in Table 1.1, supercritical fluids have densities on the same order as liquids, yet their diffusion rates tend to be an order of magnitude faster than liquids. Meanwhile, like gases, supercritical fluids do not have any surface tension and have similar viscosities. These properties combine to form a unique medium in which to perform natural product extraction processes [13]. 1.2.1. Supercritical Carbon Dioxide (SCCO2) Being one of the most commonly used supercritical fluid (SCF), SCCO2; the related pressure-temperature diagram for carbon dioxide is presented in Figs. 1.1 and 1.2 to illustrate the differences between the gases, liquid and supercritical states.. 4.

(35) Liquid Gas. Liquid and vapor Supercritical fluid. Pc T6. P. T5 T4 T3. Tc. T1 Vc. T2. V. Figure 1.1. Phase diagram of SCFs.. Pc. Liquid. Solid. Pressure. Supercritical Fluid. Critical Point Gas Tc Temperature. Figure 1.2. Pressure-temperature phase diagram.. 5.

(36) The critical point (CP) is marked at the end of the gas-liquid equilibrium curve, and the supercritical fluid region is indicated by the shaded area. It can be shown that by using a combination of isobaric changes in temperature with isothermal changes in pressure, it is possible to convert a pure component from liquid to gas (and vice versa) via the supercritical region without incurring a phase transition [14]. The consequence of this phenomenon is illustrated in Fig.1.3, in which the reduced pressure is plotted as a function of constant density. At temperatures below the critical temperature, a two phase region exists. The isotherms represent tie-lines; the intersections indicate. the. densities of the gas and liquid phases. in. equilibrium with each other. At reduced temperatures greater than or equal to unity, two Figure 1.3. Phase diagram of CO2 with constant density lines (g/l).. important. features may be observed. Firstly,. the reduced density increases continuously with increasing pressure. If, as a first approximation, we assume solvent power to be proportional to solvent density it is clear that the ability of a SCF to dissolve a solute can be varied over an extremely wide range by manipulating system pressure.. 6.

(37) 1.2.2. Supercritical Water (SCW) The trend towards using supercritical fluids in chemical practice intensified only at the beginning of the eighties. Technical processes using carbon dioxide as supercritical medium were developed (e.g. decaffeination and hop extraction). Furthermore, due primarily to the special solvent properties of supercritical fluids, supercritical fluid chromatography and the dissolution and subsequent precipitation of particles having adjustable particle size distributions (RESS, SAS) now form part of industrial practice. Supercritical water (SCW) has been growing in importance as a medium for chemical reactions for about ten years. This is attributable primarily to relatively recent targets set in waste water treatment, that is to say the elimination of poorly biodegradable substances. It is possible to convert these substances into less toxic and more readily degradable compounds. In that time there has been a marked rise in general interest in the use of the special pVT-dependent physical properties of SCW. Further, it is only very recently that the potential for synthesis which SCW chemistry affords has caught the eye of chemists. A key advantage of chemistry in SCW is the possibility of varying the properties of the reaction medium over a wide range solely by changing the pressure and temperature and of optimizing the reaction in this way without changing solvent. Furthermore, the reaction kinetics can be strongly affected in the supercritical region by varying the pressure (kinetic pressure effect). In addition, many non polar organic substances (e.g. cyclohexane) and gases (e.g. oxygen) are highly soluble in SCW so that mass transfer restrictions due to phase boundaries do not apply. In summary SCW has great potential with regard to the optimization of chemical syntheses. There are, however, drawbacks arising from working at high pressures (high investment costs), the problem of corrosion (expensive materials) and the lack of kinetic and thermodynamic data. F or these reasons applied research in the field of chemistry in. 7.

(38) SCW must not remain confined to synthetic chemistry, rather the issues concerning materials and thermodynamics must also be addressed in the research at the same time. To go through the properties of SCW, it can be expressed as in following; Water is an interesting medium and reagent for various conventional chemical reactions, purification and separation processes, because it is non-toxic, non-flammable, cheap and easily available. At room temperature, water is too polar to dissolve non-polar organic compounds, but at elevated temperatures and pressures water becomes less polar making it an interesting and environmentally friendly alternative to many organic solvents. Similarly, the extraction and isolation of compounds from solid matrices can also be performed efficiently with water at elevated temperatures and pressures. Pressurized hot water can also be utilized in liquid chromatography and in combinations of extraction and chromatographic techniques. Supercritical water, like any supercritical fluid, has properties similar to both liquid and vapor, and is often described as an intermediate form between them. The dissolving power of SCW is high, and solubility can be tuned through changes in temperature and pressure. Relative permittivity is a good measure of solvent-solute interactions and can be related to polarity: a high relative permittivity favors the solubility of high polarity or ionic compounds, whereas a low relative permittivity facilitates the solubility of low polarity compounds. At room temperature the relative permittivity of water is high (approx. 78.5), but this figure decreases dramatically with increase in temperature, making organic non-polar compounds soluble and inorganic compounds insoluble in water. The hydrogen bonding capability of water is also considerably reduced under supercritical conditions. Most organic reactions fail to occur, or only take place slowly, in water at room temperature. The high solvating power, compressibility and favorable mass transport properties of SCW make it an interesting medium for reactions [15]. Using SCW allows the manipulation of the reaction environment through adjustments to pressure and. 8.

(39) temperature. Pressurized hot water can be used in applications where moderate temperatures are sufficient to obtain the desired physico-chemical properties. For example, the mass transport properties and solvent-solute interactions in PHW (temperature 200 – 300°C) are often suitable for the extraction of compounds with low polarity and reasonable thermal stability. In summary, the behavior of a fluid in the supercritical state can be described as that of a very mobile liquid. The solubility behavior approaches that of the liquid phase while penetration into a solid matrix is facilitated by the gas-like properties (illustrated above how CO2 density affects the solvent power in different phases of it). As a consequence, the rates of extraction and phase separation can be significantly faster than for conventional extraction processes. Furthermore, the extraction conditions can be controlled to effect a selected separation. The critical properties of some commonly used SCFs are listed in Table 1.2. Further important characteristics of SCFs as a technology are presented in Table 1.3.. 9.

(40) Table 1.2. Critical conditions for various supercritical fluids. Fluid. Critical. Critical Pressure. Critical Density. Temperature ( C). (bar). (kg/m3). Ethylene. 9.3. 50.4. 220. Xenon. 16.6. 58.4. 120. Carbon Dioxide. 31.1. 73.8. 470. Ethane. 32.2. 48.8. 200. Acetylene. 35.3. 61.4. 231. Nitrous Oxide. 36.5. 71.7. 450. Propane. 95.7. 42.5. 220. Ammonia. 132.5. 112.8. 240. I- Propanol. 235.2. 47.6. 270. Methanol. 239.5. 81.0. 270. Water. 374.2. 220.5. 320. Toluene. 318.6. 41.1. 290. o. 10.

(41) Table 1.3. Evaluation of supercritical fluid technology. Advantages. Disadvantages. Dissolving power of SCF is controlled by. Elevated pressure required. pressure or temperature SCF easily recoverable from the extract. Compression costs. due to its volatility Non-toxic solvents leave no harmful. Relatively high capital investment for. residues. equipment. Separations not possible by traditional. -. methods can be effected Thermally labile compounds can be. -. extracted as low temperatures can be employed Relatively inexpensive solvents which can. -. be continuously recycled.. 1.3. Modification of Supercritical Fluids Although there exist many supercritical fluids with different solvent properties (Table 1.1), the majority of applications use carbon dioxide. As discussed previously, the polarity of carbon dioxide can be adjusted to some extent by changing pressure and temperature. Even so, CO2 is basically non-polar. Its solvent properties are comparable to hexane or chlorinated hydrocarbons such as dichloromethane. Therefore, CO2 dissolves mostly with lipophilic compounds. Even at high densities pure CO2 is still relatively non-polar. To enhance its polarity CO2 can be modified by adding, e.g. a short chain alcohol to the fluid. The most common modifiers are methanol, ethanol and 2propanol. The solubilities of polar compounds in CO2 increase drastically with modification, typically 1.2 orders in magnitude which would be impossible for a non11.

(42) modified fluid at any density. This enhancement in solvent power has been attributed to dipole-dipole, dipole-induced dipole hydrogen bonding and other polarity forces [16]. The effect of increasing the solvent density with modification has only a slight effect on increasing the solvent strength. Low molecular weight alcohols are very soluble in CO2 as indicated by the phase diagram of the CO2/ethanol system shown in Fig. 1.4. At high pressures ethanol and CO2 are miscible in practically all proportions, and the intersolubility is enhanced by lowering the temperature. On the other hand, EtOH is only slightly soluble in CO2 vapor at its vapor/liquid equilibrium pressure which restrains the modifier from recirculating within CO2 vapor at the solute recovery stage. Therefore, the product from a modified CO2 process is usually recovered as a modifier solution.. Figure 1.4. Phase diagram of CO2/ethanol mixture. The curved lines represent the modeled values and the dots the respective experimental value [17].. 12.

(43) In many natural extraction applications the sample contains water which also modifies CO2. Water forms carbonic acid with CO2 and changes its polarity. The solubility of water in CO2 at supercritical conditions is ca. 0.4.0.6% w/w [18]. In many extractive applications modification with water is inadvertent and the inherently lipophilic fluid becomes polar. Instead of the expected oily extract the product may be an aqueous product and at low yield. In supercritical fluid chromatography, modification not only increases the solubility of polar samples but also affects the chromatographic performance by modifying the stationary phase. 1.4. Other States of Fluids In this thesis, the term supercritical fluid is used when both fluid temperature and pressure exceed their respective critical values Tc and Pc. Fig. 1.5 presents the phase diagram of a typical fluid (carbon dioxide) where the fluid states are marked. A fluid at vapor/liquid equilibrium and in the temperature range approaching the critical point is called a near critical liquid. An industrially important near critical liquid is water whose hydrolytic properties change 21 drastically near the critical region [19]. A fluid whose temperature is below Tc but whose pressure is greater than the saturation pressure is simply a liquid but is often called a sub-critical fluid or sub-cooled fluid. Especially when the temperature of the sub-critical fluid is below the freezing point of water, i.e. 273.16 K, it is often called cryogenic. A gas whose pressure is below the vapor/liquid saturation line is superheated or thinned gas. This is important in steam engines and power plants where water vapor is superheated in order to increase thermodynamic efficiency. In the sub-critical range there is no point of discontinuity between liquid and supercritical phases when Tc is exceeded. The density and other properties of the liquid change monotonously. This has practical consequences in sub- and supercritical fluid chromatography since at high pressure the temperature can be freely adjusted across the Tc limit.. 13.

(44) Figure 1.5. Phase diagram of CO2. The sublimation, melting and saturation lines are equilibrium curves, i.e. two phases are present in the lines except at the triple point (T 56.6°C, P 5.2 bar) where three phases coexist and at the critical point (T +31.3°C, P 73.8 bar) where liquid and vapor phases become one phase. Elsewhere in the diagram there is one phase present. The nomenclature of different fluid states varies in the literature. 1.5. Basic Properties of SCFs Two researchers, Hannay and Hogarth, at a meeting of the Royal Society (London) in 1879, reported that supercritical fluids have a pressure-dependent dissolving power-the higher the pressure, the higher their dissolving power [15].. 14.

(45) They described their work and summarized their findings as follows: We have the phenomenon of a solid dissolving in a gas, and when the solid is precipitated by reducing the pressure, it is brought down as a 'snow' in the gas. The researchers referred to supercritical fluids as gases, which, in fact, they are. In the interest of brevity, the term 'gas', or the abbreviation 'SCF' for supercritical fluids, will be used liberally throughout this paper. The solubility behavior was not exploited until many, many years later, but it is of historical interest to relate some of the events surrounding their findings. There arose serious (but, as were the times, polite) controversy at the October 1879 society meeting: Some of the members who were present said, "Gases cannot dissolve solid compounds. The researchers must have erred and instead found solubility in superheated liquids." In other carefully planned and executed experiments the researchers did, however, substantiate their previous findings. Gases, in other words, supercritical fluids, could indeed dissolve many compounds. Fig. 1.1 shows isotherms and typical behavior of a real gas as it is subjected to different pressures and temperatures. It should be noted that there are no phase transitions above Tc. The isotherms shown in the figure are smooth; they have no tie lines. Tie lines are the horizontal portions of the isotherms, though they are really not really part of the isotherms. A substance is in supercritical state when its temperature and pressure exceed its liquid/vapor supercritical point. At this point the gas and vapor phases unite and their enthalpies of vaporization become zero as shown in Fig. 1.6. The solvent power of a supercritical fluid is related to the solvent density in the critical region. As the reduced density increases to values comparable to liquid, the supercritical fluid begins to act as a liquid solvent. The physical properties of CO2 and other supercritical fluids are summarized in Table 1.2 and Fig. 1.7.. 15.

(46) .. Figure 1.6. Enthalpies of gaseous and liquid carbon dioxide as a function of temperature. The vertical difference of the curves represents the enthalpy of vaporization at the respective temperature. The temperature ranges in supercritical fluid processes depend on the fluid used and reflect the respective critical values. The majority of supercritical work is done with CO2 which has a Tc of 31.3°C (304.6 K). For practical reasons water is often used as the heat transfer medium in CO2 work. This limits the maximum temperature of the process to near 100°C (373 K) which is quite feasible in most applications. At the lower limit, the temperature of CO2 is clearly limited by its triple point -56.6°C (216.6 K) where carbon dioxide fluid solidifies. The viscosity of liquid high-pressure CO2 is very low. 16.

(47) near down to cryogenic temperatures which make it a favorable solvent for low temperature chromatography. Working with CO2 at low temperatures is one of the main topics of this thesis.. Figure 1.7. Comparison of physical properties of fluids. In addition, Fig. 1.8 shows photos of the same system. From left to right, the temperature is increasing. In the upper-left photo, there are two phases present, liquid and gas, and the distinction between them is obvious. The following are near the critical temperature, so the separation of the two phases is becoming obscured. In the photo on the bottom-right, there is no phase distinction, so this is above the critical temperature and is a supercritical fluid.. 17.

(48) (1). (2). (3). (4). (5). (6). (7). (8). Figure 1.8. The change from two definite phases to one supercritical phase. Fig. 1.9 depicts the pressure/density isotherms of carbon dioxide. At the two phase region below the critical temperature, the fluid compressibility is infinite (isotherm is vertical) because the liquid and vapor phases are in equilibrium. When Tc is approached, the equilibrium densities of the gas and vapor phases approach each other. At the critical point the discontinuation in the density isotherm disappears. Above the critical temperature, the compressibility near Cp is high. Thus, a small change in pressure results in a large change in density. The fluid is gas-like. When pressure is further increased, the fluid compressibility gradually decreases and it becomes more liquid-like. At high pressures the densities of supercritical fluids (SCF) become similar to those of liquids and the fluid begins to act as a liquid solvent.. 18.

(49) Figure 1.9. Density isotherms of carbon dioxide. The isotherms are arranged in descending order of temperature. Below critical temperature there are two densities at the same pressure due to the two phases (liquid/vapor or solid/vapor) in equilibrium. Cp = critical point. Supercritical fluids possess a wide range of solvent characteristics which can be adjusted by the user. Since there is only one solvent phase present in the supercritical state both P and T can be adjusted. The density change vs. pressure change is greatest near the critical region where the solvent properties also change most rapidly. When the density increases the solubility of the less volatile components generally increase. Fig. 1.10 depicts how the density and dielectric constant which is a function of fluid polarity change in a concerted way with isothermal changes in pressure [20]. At high pressures the process economics begins to become an obstacle due to heavy investment costs. In industry, supercritical extraction processes can be categorized as either moderate pressure processes (<350 bars) or high pressure processes (350.600 bars). Pressures in 19.

(50) excess of 600 bars are rarely used in industry since the cost of increasing pressure exceeds the advantages obtained at higher pressures.. Figure 1.10. Dielectric constant and density of carbon dioxide as a function of pressure at isothermal temperature of +50°C. 1.6. Extraction with Supercritical Fluids Supercritical extraction has been applied to a large number of solid matrices. The desired product can be either the extract or the extracted solid itself. The advantage of using supercritical fluids in extraction is the ease of separation of the extracted solute from the supercritical fluid solvent by simple expansion. In addition, supercritical fluids have liquid like densities but superior mass transfer characteristics compared to liquid solvents due to their high diffusion and very low surface tension that enables easy penetration into the porous structure of the solid matrix to release the solute [21, 22]. 1.6.1. Batch Extraction. 20.

(51) Supercritical extraction can be a batch or continuous process. In a batch process, the raw material to be extracted is loaded in a pressure vessel(s) at atmospheric pressure as shown in Fig.1.11. The vessel is closed, pressurized, heated to operation temperature and the fluid is pumped through the extractor. During extraction the most soluble components dissolve first and hence the composition of the sample and extract change during the process. The fluid is directed through into a pressure reducing valve where its pressure is reduced to a predetermined level. There may be several pressure reduction stages in sequence but in the last stage the pressure is reduced to the fluid saturation pressure where in practice adiabatic flash expansion of the fluid splits it into liquid and vapor phase at equilibrium [23]. The expansion results in an aerosol where the liquid phase often forms a mist within the vapor phase. Eventually the heavier liquid phase forms a layer at the bottom of the vessel where most of the precipitated solute also remains [24].. Figure 1.11. Principle of supercritical batch extraction. There can be several separators arranged in series at decreasing pressures. The process in the figure contains only one. 21.

(52) separation stage where the depressurized fluid is led via an immersion pipe through the liquid CO2 pool at the separator bottom. This acts as a demisting device. In the separation vessel the solvent properties of liquid and vapor CO2 differ considerably. Solvent power of liquid CO2 is comparable to that of supercritical CO2 but the vaporous CO2 in the separator head-space has only limited solvent capacity. This is also evident from the binary phase diagram in Fig. 1.4. Therefore, the product accumulates within the liquid phase at the bottom of the vessel- either as a solution or as slurry. During extraction the liquid CO2 is gently boiling, the CO2 vapor is recirculated to the pumps to be repressurized and reheated. Only the most volatile solute components may recirculate within the vapor. If this is not desired, they can be trapped using e.g. an activated charcoal line filter. 1.6.2. Continuous Extraction Industry prefers continuous processing methods. In extraction this requires that the sample be pumpable, preferably in a counter-current manner. Usually this applies only to liquid raw materials, e.g. various oils. There is one application where dry matter is processed in a continuous way, i.e., the Maxwell House semi-continuous decaffeination process where beans are moved by shocks from the top to bottom of the vessel using lock hoppers [25]. In column extraction, the sample is pumped into a vertical column which contains fillings or a mechanical agitator to enhance the contact between sample and solvent [26]. The extracting solvent is pumped so that sample and solvent move in opposite directions. A typical counter-current extraction apparatus is depicted in Fig. 1.12.. 22.

(53) Figure 1.12. Principle of continuous counter-current supercritical extractor. In this example, the liquid feed is pumped in at the top while the CO2 solvent is pumped counter-currently from the bottom. The products are collected as the CO2 extract in the separator and the oily raffinate at the bottom of the column. This column employs an agitated rotating disc (ARD) mixer as the contactor [26]. 1.7. Applications and Commercial Processes of SCFs During the past 20 years Supercritical Fluid (SCF) processing has developed from a laboratory scale to commercial processes. Applications of analytical SFE are numerous and continue to focus on fossil fuels and environmental samples, foods, natural products, and polymers. Many of these applications have adopted the advances in SFE previously discussed. In reviewing the SFE application areas, we choose to classify the work by sample type rather than analyte type.. 23.

(54) The Caffeine content: 0.7~3%. relatively. new. processes include coffee decaffeination (Fig. 1.13), hops extraction, catalyst. 0.02% decreases below. regeneration, extraction. and of. organic. wastes from water and soil and SCF chromatography. Figure 1.13. Decaffeination of coffee.. As a result of the increasing scientific interest in the therapeutic role of essential fatty acids, there is considerable economic incentive to develop a process for the extraction of these materials from natural sources such as fish oils. Supercritical fluid extraction technology has been regarded as an ideal method for this purpose [27]. By the end. of. the. 1990’s,. according to a review presented by Rozzi and Singh [28], there was a significant. increase. in. studies of applications of supercritical CO2 in the chromatography, Figure 1.14. Result of contents search using "supercritical fluids" and "carbon dioxide" as search terms (1999-2000) [28].. petrochemical. and. pharmaceutical arenas, as shown. in. Fig.. 1.14.. Supercritical Fluid Chromatography, Fractionation, Reactions, Applications in the material and polymer industry, Food applications, Pharmaceutical and environmental applications are some supercritical fluids processes that are commercialized [29].. 24.

(55) 1.7.1. Food Applications The food industry is always looking for the best separation technology to obtain natural compounds of high purity, healthy products of excellent quality with several industrial applications. Research into energetically less costly technologies with respect to the environment is required. A summary of commercial applications and examples of recent developments illustrate the different possibilities that SFE has in industrial food processes. One of the first commercial applications of SCF technology was the decaffeination of coffee. As a result of the increasing in the therapeutic role of essential fatty acids, there is considerable economic incentive to develop a process for the extraction of these materials from natural sources such as fish oils. SFE technology has been regarded as an ideal method for this purpose. SFE, especially using CO2 is today a popular technology for rapid, contamination-free extraction in the food and pharmaceutical industries. Table 1.4 summarizes some of the known applications and newer applications of the SFE technique. Table 1.4. SFE applications in food products. Paprika color (oleoresin) extraction from meats, pickles Extraction of vegetable oils and fats Flavors, fragrances, aromas, and parfumes Antioxidants from plant materials Stabilization of fruit juices Deoiling of fast foods Thyme oil extraction from meat and pharmaceutical products. Decaffeination of coffee and tea Extraction of herbal medicines Food colors from botanicals Denicotinization of tobacco Hops extraction for bitter Essential oil extraction. Supercritical water extraction (SWE) is another process that have been applied in food industry. Studies on plant materials have concentrated on two areas, the extraction of naturally occurring plant products, principally essential oils (Table 1.5), and secondly on the determination of pesticide residues (Table 1.6). A recent review of methods for essential oils from plants was compared in case of SFE, SWE, microwave extraction and steam distillation [30].. 25.

(56) Table 1.5. Applications of SWE the extraction of flavors and fragrances from plant material and food.. Table 1.6. Applications of SWE to the analysis of pesticides from plant material and food.. SWE thus frequently provides a viable alternative to the proposal that SFE should be seriously considered as a good method for the extraction of herbs and natural. 26.

(57) products. Both provide a clean solvent free method, acceptable as a food product, with few side effects and may even provide alter native mixtures with different compositions. 1.8. A New Application: Supercritical Particle Formation In the 1970s and 1980s, supercritical processes were mainly focused on extraction and separation but, within the last 10 years, interest in supercritical fluid processing has also arisen in other areas. Although extensive R&D investigations have been made, only few supercritical fluids’ industrial applications have been developed. Most companies consider that supercritical fluid technology is too expensive because of the very high investment costs in comparison with classical low pressure-equipments and that it should be restricted to high-added-value products. This is not true when large volumes of materials are treated as shown in the case of the extraction of the caffeine from the coffee beans. At present time, the most important markets are related to natural product processing for applications in food and neutraceutical/pharmaceutical/cosmetic industries. The recrystallization of materials by supercritical fluid processing enables the manufacture of special structured products of significant high quality and function that simply cannot be produced with conventional methods. This is due to the versatile operating conditions that are possible with SCFs and their mixtures and that provide the flexibility in controlling the size of the particles (from microns to nanometers). Two methods have been developed to produce ultrafine particles and several reviews are available on polymer particles processing using supercritical fluids [31-37]: . Rapid expansion of supercritical fluid solutions, RESS process. . Supercritical anti-solvent precipitation, SASP process. The coating or encapsulation of nanoparticles finds particular interest for the controlled release of drugs, genes and other bioactive agents. For example, the production of polymeric microspheres for controlled drug delivery is an area where RESS process is used. This process allows the production of thin film coatings, polymer 27.

(58) fibers, and fine particles of a sub-micrometer size. The RESS process is unique, as RESS products are generated “dry”, meaning little or no residual solvent. The process works by exploiting the property changes brought about by density changes. In this process, materials are dissolved in a suitable supercritical fluid. The mixture is then sprayed through a heated nozzle as shown in Fig. 1.15a. This rapid expansion of the homogeneous supercritical solution is accompanied by a temperature and a pressure drop leading to phase separation. The nozzle diameter and length are among the parameters that influence the particle size generated.. Figure 1.15. Schematic representation a) of the RESS process and b) of the SASP process [29]. The SASP process involves the mixing of a polymer solution with a supercritical fluid that functions as an anti-solvent, i.e. the polymeric compound is not soluble in the supercritical phase. This process is also known as precipitation of a. 28.

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