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The effect of molecular structure and operating conditions on the solubility of triglycerides in supercritical… Cheok, Nai Tin 1986

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THE EFFECT OF MOLECULAR STRUCTURE AND OPERATING CONDITIONS ON THE SOLUBILITY OF TRIGLYCERIDES IN SUPERCRITICAL C 0 2 by CHEOK, NAI TIN B.Sc ( E n g . ) , U n i v e r s i t y of Guel p h , 1984. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE i n FACULTY OF GRADUATE STUDIES Department of B i o - R e s o u r c e E n g i n e e r i n g We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d , THE UNI^E^SITY OF BRITISH COLUMBIA May,1986 © Cheok, N a i T i n , May,1986 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the THE UNIVERSITY OF BRITISH COLUMBIA, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree that p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of Bio-Resource E n g i n e e r i n g THE UNIVERSITY OF BRITISH COLUMBIA 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date: May,1986 A b s t r a c t In order to assess the f e a s i b i l i t y of using s u p e r c r i t i c a l f l u i d as a s o l v e n t f o r o i l e x t r a c t i o n i n the o i l and f a t i n d u s t r y , b a s i c i n f o r m a t i o n i s r e q u i r e d on o i l s o l u b i l i t y as a f u n c t i o n of v a r i o u s system parameters. Such in f o r m a t i o n would be u s e f u l i n the design of e x t r a c t i o n systems. T h i s r e s e a r c h work s t u d i e d the e f f e c t s of temperature and pressure on the e q u i l i b r i u m s o l u b i l i t i e s of t r i g l y c e r i d e s in s u p e r c r i t i c a l carbon d i o x i d e which i s c o n s i d e r e d one of the best s o l v e n t s f o r s u p e r c r i t i c a l f l u i d e x t r a c t i o n . A s e r i e s of experiments was c a r r i e d out using a m o d i f i e d l i q u i d chromatograph. Samples t e s t e d i n c l u d e d pure simple t r i g l y c e r i d e s that are s a t u r a t e d and unsaturated, and t r i g l y c e r i d e mixtures. E x t r a c t i o n experiments were extended to cocoa b u t t e r , palm k e r n e l o i l and t h e i r mixtures with v a r i o u s weight, r a t i o s . The e f f e c t of sample water content on the o i l s o l u b i l i t y was i n v e s t i g a t e d using cocoa butter/water mixtures i n d i f f e r e n t p r o p o r t i o n s . The s o l u b i l i t i e s of simple t r i g l y c e r i d e s were found to depend s t r o n g l y on pressure and temperature. T r i g l y c e r i d e s with a longer carbon chain (C18) e x h i b i t lower s o l u b i l i t i e s over the range of temperatures and pressures s t u d i e d . Unsaturated t r i g l y c e r i d e s were more s o l u b l e than t h e i r s a t u r a t e d c o u n t e r p a r t s . Furthermore, f o r the s a t u r a t e d t r i g l y c e r i d e s , the s o l u b i l i t i e s v a r i e d i n v e r s e l y as t h e i r molecular weights. i i S i g n i f i c a n t f r a c t i o n a t i o n occured d u r i n g the e x t r a c t i o n of simple t r i g l y c e r i d e mixtures at 36 MPa and 55 °C. R e s u l t s of t e s t s on cocoa b u t t e r and palm ke r n e l o i l s e p a r a t e l y i n d i c a t e d t h a t no f r a c t i o n a t i o n had taken p l a c e d u r i n g the e x t r a c t i o n p r o c e s s . However, s i g n i f i c a n t f r a c t i o n a t i o n was again observed when the two o i l s were mixed. Sample water content up to 50% by weight had n e g l i g i b l e e f f e c t on the o i l e x t r a c t a b i l i t y and i t s e q u i l i b r i u m s o l u b i l i t y i n C0 2 . Table of Contents A b s t r a c t . . . i i LIST OF FIGURES v i i . LIST OF TABLES x i ACKNOWLEDGEMENT '. X i i i 1 . INTRODUCTION 1 1 .1 General 1 1 . 2 O b j e c t i v e s 5 2. LITERATURE REVIEW 6 2.1 S u p e r c r i t i c a l F l u i d 6 2.2 Previous Works 12 2.3 E f f e c t of Pressure and Temperature 16 2.4 E f f e c t of Water Content 21 2.5 SCFE of T r i g l y c e r i d e s 22 2.5.1 O i l s and Fa t s 22 2.5.2 C0 2 E x t r a c t i o n T r i g l y c e r i d e s 23 2.5.3 S u p e r c r i t i c a l F r a c t i o n a t i o n 29 3. METHODS AND MATERIALS 38 3.1 Experimental Equipment 38 3.1.1 Hewlett-Packard L i q u i d Chromatograph 38 3.1.2 E x t r a c t i o n V e s s e l s -...39 3.1.3 Flow R e s t r i c t e r '..41 3.1.4 Sampling U n i t 42 3.2 Solvent Flowpath 45 3.3 Experimental E x t r a c t i o n Procedures 47 3.3.1 Operating C o n d i t i o n s 47 3.3.1.1 Pressure 47 3.3.1.2 Temperature 47 i v 3.3.1.3 C0 2 Flowrate 48 3.3.2 V e s s e l Loading Procedure 48 3.3.2.1 L i q u i d Samples 48 3.3.2.2 S o l i d Samples 49 3.3.3 P r e - e x t r a c t i o n C l e a n i n g 50 3.3.4 Equipment S t a r t u p 50 3.3.5 E x t r a c t Sampling 51 3.3.6 Equipment Shutdown 52 3.3.7 S o l u b i l i t y Determination 52 3.4 M a t e r i a l s 54 3.4.1 Carbon Dioxide 54 3.4.2 Pure T r i g l y c e r i d e s 54 3.4.3 O i l Samples 55 3.5 F a t t y A c i d A n a l y s i s 55 3.5.1 T r a n s e s t e r i f i c a t i o n 56 3.5.2 Gas Chromatographic Procedure 57 3.5.2.1 Gas Chromatographic C o n d i t i o n s ....57 3.5.2.2 Peak I d e n t i f i c a t i o n and Q u a n t i f i c a t i o n Procedure 58 4. RESULTS AND DISCUSSIONS 60 4.1 C0 2 E x t r a c t i o n of Pure T r i g l y c e r i d e 60 4.1.1 T r i g l y c e r i d e S o l u b i l i t y as a Function of Temperature and Pressure 60 4.1.2 T r i g l y c e r i d e S o l u b i l i t y as a Function- of Molecular Weight (or Carbon Chain Length) .71 4.1.3 E f f e c t of Degree of F a t t y A c i d s S a t u r a t i o n on the S o l u b i l i t y of Pure T r i g l y c e r i d e s 75 4.2 C0 2 E x t r a c t i o n of T r i g l y c e r i d e Mixtures 79 4.3 E x t r a c t i o n of Cocoa Butter and Palm Kernel O i l ..85 v 4.3.1 F a t t y A c i d Composition of Palm Kernel O i l and Cocoa Butter 85 4.3.2 C0 2 E x t r a c t i o n of Pure O i l s 87 4.3.3 C0 2 E x t r a c t i o n of O i l Mixtures 95 4.4 E f f e c t of Water Content on O i l S o l u b i l i t i e s ....100 5. CONCLUSIONS 105 6. RECOMMENDATIONS 107 REFERENCES 108 APPENDIX I 112 v i LIST OF FIGURES F i g u r e 2-1: Phase diagram f o r carbon d i o x i d e showing the r e l a t i o n s h i p of the s u p e r c r i t i c a l s t a t e to the s o l i d , l i q u i d and vapour s t a t e s . The c r i t i c a l p o i n t i s d e s i g n a t e d as C and the t r i p l e p o i n t as TP 7 F i g u r e 2-2: Reduced p r e s s u r e - d e n s i t y diagram f o r carbon d i o x i d e . S u p e r c r i t i c a l f l u i d (SCF) and n e a r - c r i t i c a l l i q u i d regions are i n d i c a t e d (Giddings et al.,1969) 8 F i g u r e 2-3: The l a r g e i n c r e a s e i n the s o l u b i l i t y of p.iodochlorobenzene i n the s u p e r c r i t i c a l e t hylene ( Tc = 282 K, Pc = 5 MPa ) at 298 K brought about at e l e v a t e d p r e s s u r e s (Williams,1981) 17 F i g u r e 2-4: The gas phase c o n c e n t r a t i o n of phenaphthelene i n v a r i o u s s u p e r c r i t i c a l gases at 313 K and 40 MPa, showing the importance of a c l o s e correspondence between the e x t r a c t i o n temperature and the c r i t i c a l temperature of the gas (Williams,1981) 18 F i g u r e 2-5: S o l u b i l i t y of naphthalene i n s u p e r c r i t i c a l e t h ylene as a f u n c t i o n of temperature at d i f f e r e n t p r e s s u r e s (Ethylene : Tc = 282 K, Pc = 5 MPa) (Williams,1981) 20 F i g u r e 2-6 : S o l u b i l i t y of Canola o i l in C0 2 as a f u n c t i o n of pressure at v a r i o u s temperature ( F a t t o r i , 1 9 8 6 ) 25 F i g u r e 2-7: E f f e c t s of temperature and pressure on the s o l u b i l i t y of soybean o i l i n s u p e r c r i t i c a l C0 2 ( F r i e d r i c h and,Pryde,1984) 26 F i g u r e 2-8: S o l u b i l i t y of Rapeseed and Soybean o i l s i n C0 2 as a f u n c t i o n of pressure at 20°C and 40°C ( S t a h l et al . , 1 980; Bunzenberger et al.,1984) 27 F i g u r e 2-9: Density of carbon d i o x i d e as a f u n c t i o n of p r e s s u r e at d i f f e r e n t temperatures. The c r i t i c a l p o i n t i s d e s i g n a t e d as CP ( V u k a l o v i c h and Altunin,1968) 28 v i i F i g u r e 2-10: E x t r a c t i o n curves f o r pure t r i p a l m i t o l e i n (C16:1), t r i o l e i n (C18:1) and t r i - 1 1 - e i c o s e n o i n (C20:1). E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C ( F a t t o r i , 1 9 8 6 ) ...30 F i g u r e 2-11: S a p o n i f i c a t i o n number and i o d i n e number of the s u p e r c r i t i c a l e x t r a c t s of c o d - l i v e r o i l at d i f f e r e n t stages of an e x t r a c t i o n (Zosel,l978) 32 F i g u r e 2-12: Mass f r a c t i o n of the s u p e r c r i t i c a l C0 2 e x t r a c t s of a mixture of three simple t r i g l y c e r i d e s at d i f f e r e n t stages of the e x t r a c t i o n . E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C ( F a t t o r i , 1 986) 36 F i g u r e 3-1: Cross s e c t i o n a l view of e x t r a c t i o n v e s s e l #1. A l l measurements shown are i n cm, except were i n d i c a t e d 40 F i g u r e 3-2: Exploded view of Model 7037 pressure r e l i e f v a l v e 43 F i g u r e 3-3: Cross s e c t i o n a l view of the sampling u n i t ....44 Fi g u r e 3-4: Schematic diagram of the experimental s u p e r c r i t i c a l f l u i d e x t r a c t i o n system 46 F i g u r e 3-5: E x t r a c t i o n curve of cocoa b u t t e r f a t using carbon d i o x i d e . E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C ..53 F i g u r e 4-1: S o l u b i l i t y of v a r i o u s simple t r i g l y c e r i d e s in C0 2 as a f u n c t i o n of pressure at 75°C 61 F i g u r e 4-2: S o l u b i l i t y of t r i o l e i n (C18:1) i n C0 2 as a f u n c t i o n of pressure at four d i f f e r e n t temperatures 62 F i g u r e 4-3: S o l u b i l i t y of t r i l i n o l e i n (C18:2) in C0 2 as a f u n c t i o n of pressure at four d i f f e r e n t temperatures ....63 Fi g u r e 4-4: S o l u b i l i t y of t r i o l e i n (C18:1) i n C0 2 as a f u n c t i o n of d e n s i t y of C0 2 at v a r i o u s temperatures 65 F i g u r e 4-5: S o l u b i l i t y of t r i l i n o l e i n (C18:2) i n C0 2 as a funcion of d e n s i t y at of C0 2 at v a r i o u s temperatures ...66 v i i i F i g u r e 4-6: S o l u b i l i t y of t r i o l e i n (C18:1) i n C0 2 as a f u n c t i o n of temperature at four C0 2 d e n s i t i e s 67 Fi g u r e 4-7 a f u n c t i o n S o l u b i l i t y of t r i l i n o l e i n (C18:2) i n C0 2 of temperature at four C0 2 d e n s i t i e s .... as 68 Fi g u r e 4-8: D e n s i t y of carbon d i o x i d e as a f u n c t i o n of pressure at d i f f e r e n t temperatures. The c r i t i c a l p o i n t i s d e s i g n a t e d as CP. (Newritt et al.,1956;Vukalovich and A l t u n i n , 1 968) 70 F i g u r e 4-9: S o l u b i l i t y of t r i m y r i s t i n (C14:0), t r i p a l m i t i n (C16:0) and t r i s t e a r i n (C18:0) i n C0 2 as a f u n c t i o n of pressure at 75°C 72 F i g u r e 4-10: The negative l o g a r i t h m of the s o l u b i l i t y of the three t r i g l y c e r i d e s i n C0 2 as a f u n c t i o n of t h e i r molecular weights 74 F i g u r e 4-11: The negative l o g a r i t h m of the s o l u b i l i t y of two groups of t r i g l y c e r i d e s i n C0 2 as a f u n c t i o n of t h e i r molecular weights 76 Fi g u r e 4-12: S o l u b i l i t y of t r i s t e a r i n (C18:0), t r i o l e i n (C18:1) and t r i l i n o l e i n (C18:2) i n C0 2 as a f u n c t i o n of press u r e s at 75°C 77 F i g u r e 4-13: E x t r a c t i o n curve f o r the mixture of t r i m y r i s t i n (C14:0) and t r i o l e i n (C18:1) i n the weight r a t i o of 75:25. The e x t r a c t i o n was performed at 36 MPa and 75°C 80 F i g u r e 4-14: E x t r a c t i o n curve f o r the mixture of t r i m y r i s t i n (C14:0) and t r i o l e i n (C18:1) i n the weight r a t i o of 50:50. The e x t r a c t i o n was performed at 36 MPa and 75°C 81 F i g u r e 4-15: E x t r a c t i o n curve f o r the mixture of t r i m y r i s t i n (C14:0) and t r i o l e i n (C18:1) i n the weight r a t i o of 25:75. The e x t r a c t i o n was performed at 36 MPa and 75°C 82 F i g u r e 4-16: S o l u b i l i t y of cocoa b u t t e r i n C0 2 f u n c t i o n of pressure at v a r i o u s temperatures .... as .88 ix F i g u r e 4-17: S o l u b i l i t y of palm ke r n e l o i l i n C0 2 as a f u n c t i o n of pressure at v a r i o u s temperatures 89 F i g u r e 4-18: E x t r a c t i o n curve f o r pure cocoa b u t t e r . E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C 91 F i g u r e 4-19: E x t r a c t i o n curve f o r pure palm k e r n e l o i l . E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C 92 F i g u r e 4-20: E x t r a c t i o n curve f o r the mixture of cocoa b u t t e r and palm kernel o i l i n the weight r a t i o of 75:25. E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C 96 F i g u r e 4-21: E x t r a c t i o n curve f o r the mixture of cocoa b u t t e r and palm ker n e l o i l i n the weight r a t i o of 50:50. E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C 97 F i g u r e 4-22: E x t r a c t i o n curve f o r the mixture of cocoa b u t t e r and palm kernel o i l i n the weight r a t i o of 25:75. E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C 98 F i g u r e 4-23: E x t r a c t i o n curve f o r a mixture of 50:50 (wt %) of cocoa butter/water mixture. E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C 103 x LIST OF TABLES Table 2-1: T y p i c a l values of v i s c o s i t y , d e n s i t y and d i f f i s i v i t y f o r l i q u i d , gaseous and SC-C0 2 10 Table 2-2: P h y s i c a l constants of some compound used as s u p e r c r i t i c a l f l u i d 11 Table 3-1: Dimensions of the e x t r a c t i o n v e s s e l s 39 Table 3-2: S p e c i f i c a t i o n s of commercial siphon grade carbon d i o x i d e 54 Table 3-3: S p e c i f i c a t i o n s of the t r i g l y c e r i d e samples used f o r the experiments 55 Table 3-4: Gas chromatographic parameters fo r the f a t t y a c i d methyl e s t e r analyses 57 Table 3-5: Retention times and response f a c t o r s f o r f a t t y a c i d methyl e s t e r s r e l a t i v e to methyl p a l m i t a t e (C16:0) ". 59 Table 4-1: F a t t y a c i d composition of the C0 2 e x t r a c t s of t r i m y r i s t i n (C14:0) and t r i o l e i n (C18:1) mixtures i n the weight r a t i o of 75:25 (A), 50:50 (B) and 25:75 (C) 83 Table 4-2: F a t t y a c i d composition of cocoa b u t t e r 86 Table 4-3: F a t t y a c i d composition of palm k e r n e l o i l 86 Table 4-4: F a t t y a c i d composition of C0 2 e x t r a c t s of cocoa b u t t e r . E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C 90 Table 4-5: F a t t y a c i d composition of C0 2 e x t r a c t s of palm k e r n e l o i l . E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C ..90 Table 4-6: T r i g l y c e r i d e composition of cocoa b u t t e r 93 x i Table 4-7: T r i g l y c e r i d e composition of palm k e r n e l o i l ...94 Table 4-8: F a t t y a c i d composition of the C0 2 e x t r a c t s of the mixture of cocoa b u t t e r and palm ker n e l o i l i n the weight r a t i o of 75:25 (A), 50:50 (B) and 25:75 (C). E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C 99 Table 4-9: S o l u b i l i t y of cocoa b u t t e r in C0 2 at v a r i o u s water c o n t e n t s . E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C ..102 x i i ACKNOWLEDGEMENT I wish to express my s i n c e r e a p p r e c i a t i o n and g r a t i t u d e to Dr. N.R. B u l l e y f o r h i s s u p e r v i s i o n and guidance of my M.A.Sc. program, and f o r h i s v a l u a b l e encouragement, c r i t i c i s m s and pat i e n c e through the p r o d u c t i o n of t h i s t h e s i s . I would a l s o l i k e to thank Dr. K.V. Lo and P r o f e s s o r L.M. S t a l e y f o r s e r v i n g as the committee members. S p e c i a l thanks are due to Dr. J.M. F a t t o r i who set up the experimental u n i t and k i n d l y p r o v i d e d a d v i c e on many o c c a s i o n s . F i n a l l y , my s i n c e r e thanks go to my f a m i l y and f r i e n d s f o r t h e i r encouragement, companionship and u n f a i l i n g support. T h i s r e s e a r c h was funded by N a t u r a l Science and En g i n e e r i n g Research C o u n c i l . x i i i Chapter 1 INTRODUCTION 1.1 GENERAL The s e p a r a t i o n of o i l s or l i p i d s and other l i q u i d hydrocarbons from p l a n t m a t e r i a l s i s a w e l l developed technology. In the o i l s and f a t s i n d u s t r y , s o l v e n t e x t r a c t i o n using n-hexane, a p a r a f f i n i c petroleum f r a c t i o n , i s the most e x t e n s i v e l y used technique f o r r e c o v e r i n g food grade o i l from o i l s e e d s such as soybeans, cottonseeds, canolas and sunflowerseeds. A s e r i o u s disadvantage of n-hexane e x t r a c t i o n i s i t s extreme f l a m m a b i l i t y . Rather e l a b o r a t e p r e c a u t i o n s have been developed to a v o i d f i r e and e x p l o s i o n , but danger of severe a c c i d e n t s remains. In a d d i t i o n , i t s i n c r e a s i n g c o s t and p o t e n t i a l h e a l t h problems has made i t d e s i r a b l e f o r the i n d u s t r y to search f o r a l t e r n a t i v e e x t r a c t i o n methods. Recently, s u p e r c r i t i c a l f l u i d e x t r a c t i o n (SCFE), a novel kind of s e p a r a t i o n technique which does not use organic s o l v e n t s has generated a great d e a l of i n t e r e s t . I t has been shown that s u p e r c r i t i c a l C0 2 can be e f f i c i e n t at e x t r a c t i n g o i l from d i f f e r e n t o i l sources ( S t a h l et al.,1980; Hubert and Vitzthum,1978; d e F i l l i p i , 1 9 8 2 , Mangold,1982 ; F r i e d r i c h and Li s t , 1 9 8 2 and F a t t o r i , 1 9 8 6 ) . The use of SCFE .for a c h i e v i n g f r a c t i o n a t i o n of o i l s has a l s o been r e p o r t e d (Anon,1981; Zosel,l978 ; F r i e d r i c h and Pryde,l984; S t a h l et al. , 1 9 8 0 ) . Although the technique i s 1 2 q u i t e complicated and r e q u i r e s s o p h i s t i c a t e d high pressure equipment, rese a r c h i n t o i t s use has grown almost e x p o n e n t i a l l y over the l a s t decade. S u p e r c r i t i c a l f l u i d e x t r a c t i o n i s a technique that e x p l o i t s the s o l v e n t power of s u p e r c r i t i c a l f l u i d s at temperatures and p r e s s u r e s near the c r i t i c a l p o i n t . In t h i s r e g i o n , s l i g h t changes i n temperature and p ressure can cause l a r g e changes in s o l v e n t d e n s i t y and thus d i s s o l v i n g power (Humphrey et al.,1984). In the b a s i c process of s u p e r c r i t i c a l e x t r a c t i o n , a s u b s t r a t e i s brought i n t o c o n t a c t with a s u p e r c r i t i c a l f l u i d , the o i l / s u p e r c r i t i c a l phase i s i s o l a t e d , and f i n a l l y the i s o l a t e d f l u i d phase i s decompressed to the p o i n t where the s o l v e n t power of the gas i s reduced and the d i s s o l v e d m a t e r i a l condenses as a s o l i d or l i q u i d . An extension of t h i s process i s the s e p a r a t i o n by s u c c e s s i v e e x t r a c t i o n s of a mixture of m a t e r i a l s , u s i n g m i l d c o n d i t i o n s to e x t r a c t f i r s t the more v o l a t i l e m a t e r i a l s and then more severe c o n d i t i o n s to e x t r a c t the l e s s v o l a t i l e m a t e r i a l s . The extent of an e x t r a c t i o n can be c o n t r o l l e d by the s e l e c t i o n of an a p p r o p r i a t e gas, by a d j u s t i n g the temperature and p r essure of the e x t r a c t i o n , and by a l t e r i n g the r a t i o of s u b s t r a t e / g a s in the e x t r a c t i o n v e s s e l . SCFE i s a l s o known as 'dense gas e x t r a c t i o n ' , ' s u p e r c r i t i c a l gas e x t r a c t i o n ' or ' d e s t r a c t i o n ' . I t combines f e a t u r e s of both d i s t i l l a t i o n and l i q u i d e x t r a c t i o n . I t i s p a r t i c u l a r l y e f f e c t i v e f o r the i s o l a t i o n of substances of 3 r e l a t i v e l y high molecular weight and r e l a t i v e l y low p o l a r i t y . The technique i s s i m i l a r to c o n v e n t i o n a l s o l v e n t e x t r a c t i o n i n that the m a t e r i a l to be e x t r a c t e d i s 'washed' from the s u b s t r a t e using a s u i t a b l e s o l v e n t . , and yet d i s t i n c t from the c o n v e n t i o n a l method i n that the s o l v e n t i s not a l i q u i d , but ra t h e r a f l u i d above i t s c r i t i c a l p o i n t . In comparision with l i q u i d s o l v e n t s the s u p e r c r i t i c a l f l u i d has high d i f f u s i v i t y , but low d e n s i t y and v i s c o s i t y , thus a l l o w i n g r a p i d e x t r a c t i o n and phase s e p a r a t i o n (Williams,1981). Moreover, when compared with the l i q u i d - l i q u i d processes, there i s a grea t e r f l e x i b i l i t y i n the s e l e c t i o n of the o p e r a t i n g parameters of temperature and pr e s s u r e . One of the p r i n c i p a l advantage over d i s t i l l a t i o n i s that s e p a r a t i o n can be accomplished at moderate temperatures and as a consequence the process can be a p p l i e d to the recovery of h e a t - l a b i l e substances of low v o l a t i l i t y . In simple terms, t h i s technique can be c h a r a c t e r i z e d by the f o l l o w i n g unique f e a t u r e s which make i t one of the most promising t e c h n o l o g i e s f o r f u t u r e use i n the f a t s and o i l s i n d u s t r y ( F r i e d r i c h and Pryde, 1984). 1. three s u p e r c r i t i c a l f l u i d parameters, d e n s i t y , temperature and composition can be e a s i l y v a r i e d , 2. temperatures are u s u a l l y c l o s e to the c r i t i c a l temperatures. High b o i l i n g p o i n t and/or heat s e n s i t i v e components may be taken i n t o the e x t r a c t i n g phase at r e l a t i v e l y low temperatures. 3. e s s e n t i a l l y complete s e p a r a t i o n of s o l v e n t / s o l u t e with 4 high s o l v e n t recovery can be accomplished by isothermal decompression 4. compounds can be s e l e c t i v e l y d i s s o l v e d by changing the d e n s i t y of the f l u i d s , 5. A great range of s o l v e n t s may be employed, f o r example, carbon d i o x i d e , ethane, ethylene and propane. The s o l v e n t power of an a p p r o p r i a t e gas may be improved, i f r e q u i r e d , by a d d i t i o n of a t h i r d component c a l l e d an e n t r a i n e r , thus extending f u r t h e r the range of s o l v e n t c h a r a c t e r i s t i c s a v a i l a b l e . 6. the s o l u t e s can be f r a c t i o n a t e d d u r i n g the s o l v e n t / s o l u t e s e p a r a t i o n . In order to assess the f e a s i b i l i t y and m e r i t s of using s u p e r c r i t i c a l f l u i d as a s o l v e n t i n the o i l s and f a t s i n d u s t r y , b a s i c i n f o r m a t i o n i s r e q u i r e d on o i l s o l u b i l i t y as a f u n c t i o n of v a r i o u s system parameters. Since t r i g l y c e r i d e s are the major components of food grade o i l s , r e s u l t s obtained from experiments u s i n g pure t r i g l y c e r i d e s should l e a d to a b e t t e r uderstanding of the more complex mixtures of o i l s . I t has been shown that d i f f e r e n t t r i g l y c e r i d e s e x h i b i t d i f f e r e n t s o l u b i l i t i e s i n s u p e r c r i t i c a l carbon d i o x i d e ( F a t t o r i , 1 9 8 6 ) . Research on the r e l a t i o n s h i p between a t r i g l y c e r i d e molecular s t r u c t u r e and i t s e q u i l i b r i u m s o l u b i l i t y would pr o v i d e a f u r t h e r view on the p r o c e s s . A l s o , changes i n s o l u b i l i t y and e x t r a c t composition f o r t r i g l y c e r i d e mixtures would p r o v i d e b a s i c i n f o r m a t i o n f o r use i n the design of e x t r a c t i o n / f r a c t i o n a t i o n systems. 5 The e f f e c t of water content in the o i l source m a t e r i a l s a l s o r e q u i r e s study. Since water content i s an a l t e r a b l e parameter, understanding of i t s b a s i c e f f e c t s on the k i n e t i c s of e x t r a c t i o n i s important. 1.2 OBJECTIVES The o b j e c t i v e s of t h i s r e s e a r c h a r e : 1. To determine the e q u i l i b r i u m s o l u b i l i t i e s of pure simple t r i g l y c e r i d e s i n s u p e r c r i t i c a l C0 2 as a f u n c t i o n of : a. temperature b. p r essure 2. To determine the e f f e c t of t r i g l y c e r i d e molecular s t r u c t u r e on i t s s o l u b i l i t y i n s u p e r c r i t i c a l C0 2 with emphasis on : a. carbon chain l e n g t h of the f a t t y a c i d s b. carbon chain s a t u r a t i o n of the f a t t y a c i d s 3. To determine the change of t r i g l y c e r i d e f a t t y " a c i d composition as a f u n c t i o n of e x t r a c t i o n time for simple two component t r i g l y c e r i d e mixtures. 4. To e v a l u a t e the p o t e n t i a l use of the e f f e c t of changes in p r essure and/or temperature on f r a c t i o n a t i o n of the s u p e r c r i t i c a l t r i g l y c e r i d e e x t r a c t s . 5. To determine the e f f e c t of sample water content on the s o l u b i l i t y of t r i g l y c e r i d e s i n C0 2. 6. E x t r a c t i n g of palm k e r n e l o i l , cocoa b u t t e r and the mixture of both as the t e s t m a t e r i a l s to assess p o t e n t i a l f o r e x t r a c t i o n / f r a c t i o n a t i o n . Chapter 2 LITERATURE REVIEW 2. 1 SUPERCRITICAL FLUID An understanding of the term s u p e r c r i t i c a l f l u i d can be obtained by r e f e r r i n g to the pressure-temperature phase diagram of a pure substance ( F i g u r e 2-1). When a gas such as C0 2 or ethylene i s compressed, some d i s t i n c t changes in the p h y s i c a l p r o p e r t i e s and behavior are observed below a c e r t a i n temperature T, whereby s a t u r a t e d l i q u i d and vapour can e x i s t t o g e t h e r . Below a c e r t a i n volume f o r a given pressure the m a t e r i a l must be a l i q u i d , and i f i t i s allowed to expand, a l l the given mass w i l l be g a s i f i e d at a p a r t i c u l a r volume. At temperature above T, i t i s not p o s s i b l e to l i q u e f y the gas no matter what pressure i s a p p l i e d . The temperature,T , i s termed the c r i t i c a l temperature of the gas. F i g u r e 2-2 i s p l o t t e d u s ing the reduced v a r i a b l e s T P r and p r . The s u p e r c r i t i c a l f l u i d region f o r a pure compound i s s t r i c t l y d e f i n e d as that r e g i o n of temperatures and p r e s s u r e s g r e a t e r than or equal to the c r i t i c a l temperature and c r i t i c a l p r e s s u r e , r e s p e c t i v e l y , of the compound ( i . e , reduced p r e s s u r e s and temperatures g r e a t e r than or equal to u n i t y ) . T y p i c a l l y , The SCF region of i n t e r e s t i s d e f i n e d at c o n d i t i o n s bounded approximately by 0.95 < T r < 1.4 and P r > 1.0. In t h i s r e g i o n , the f l u i d i s h i g h l y c o m p r e s s i b l e . For example, at a constant T r of 1.10, i n c r e a s i n g p r e s s u r e from 6 7 CO CO (Oca 5 -L x J o c e o ZD ~~ CO V — Q_ oo CN 1 1 1 , 1 Q I 1 1 I I 1 I I h ' " T " " 1 " \ \ \ \ \ \ -supercrrticaf I - liquid ~ solid - vapor T P -i A t ^ i i I I I I I I I l I i i i i i i i i -100.0 -80.0 -60.0 -40.0 -20.0 0.0 20.0 40.0 60.0 80.0 100.0 120.0 TEMPERATURE (°C) F i g u r e 2-1: Phase diagram f o r carbon d i o x i d e showing the r e l a t i o n s h i p of the s u p e r c r i t i c a l s t a t e to the s o l i d , l i q u i d and vapour s t a t e s . The c r i t i c a l p o i n t i s d e s i g n a t e d as C and the t r i p l e p o i n t as TP. 8 F i g u r e 2-2: Reduced p r e s s u r e - d e n s i t y diagram f o r carbon d i o x i d e . S u p e r c r i t i c a l f l u i d (SCF) and n e a r - c r i t i c a l l i q u i d r e g i o n s are i n d i c a t e d . (Giddings et a l . , 1 9 6 9 ) . 9 P =1.0 to P > 1.0 s i g n i f i c a n t l y i n c r e a s e s the d e n s i t y from r e l a t i v e l y low values to l i q u i d - l i k e d e n s i t i e s . At higher T r s , the pressure i n c r e a s e to produce an e q u i v a l e n t d e n s i t y i n c r e a s e become g r e a t e r . T h i s c o n s i d e r a t i o n s e t s the upper bound on temperature. At constant P r of 1.50, d e c r e a s i n g temperatures has a s i m i l a r e f f e c t on d e n s i t y , and at higher reduced p r e s s u r e s , the d e n s i t y i s l e s s s e n s i t i v e to temperature changes. In the v i c i n i t y of the c r i t i c a l p o i n t , l a r g e d e n s i t y changes can be produced with e i t h e r r e l a t i v e l y small p r e s s u r e or temperature changes. In a d d i t i o n to the unique s o l u b i l i t y behavior of s u p e r c r i t i c a l f l u i d , there are c e r t a i n d e s i r a b l e p h y s i c o - c h e m i c a l p r o p e r t i e s which make i t a good s o l v e n t . As shown in Table 2-1, while s u p e r c r i t i c a l f l u i d s have a l i q u i d - l i k e d e n s i t y and hence s o l v e n t l o a d i n g comparable to a l i q u i d , the d i f f u s i v i t y and v i s c o s i t y are intermediate to that of a l i q u i d and gas. T h e r e f o r e , the dense gas phase w i l l manifest s o l u b i l i t i e s approaching those of the l i q u i d phase, yet w i l l penetrate f a s t e r and deeper i n t o a s o l i d matrix of n a t u r a l substances to be e x t r a c t e d or would progress f a s t e r through a densely packed f i x e d bed or column. 10 TABLE 2-1 : TYPICAL VALUES OF VISCOSITY, DENSITY AND DIFFUCIVITY FOR LIQUID, GASEOUS AND SC"C0 2 (Newitt et al., 1 9 5 6 ) . Property Gas L i q u i d S u p e r c r i t i c a l F l u i d D e n s i t y (kg nr 3 ) 0 . 001 1 .0 0 . 1 -0.8 V i s c o s i t y (cP) 0.01 0.5-0.1 0.05-0.10 D i f f u s i v i t y (mm2 s" 1) 10 0.001 0.01-0.1 In g e n e r a l terms, i t i s suggested that the c l o s e p r o x i m i t y of molecules i n the l i q u i d phase imparts to the l i q u i d c e r t a i n s o l v e n t powers through the a c t i o n of i n t e r m o l e c u l a r f o r c e s . Solvent e x t r a c t i o n can t h e r e f o r e be r e l a t e d to the d e n s i t y of the f l u i d . S u p e r c r i t i c a l s o l v e n t s c o v e r i n g a wide range of e x t r a c t i o n temperatures and v a r y i n g c o n s i d e r a b l y i n s i z e and p o l a r i t y are a v a i l a b l e . Many of these are r e l a t i v e l y inexpensive and abundant. Table 2-2 l i s t s the c r i t i c a l p r e s s u r e s , c r i t i c a l temperatures and c r i t i c a l d e n s i t i e s of these substances. 11 TABLE 2-2 : PHYSICAL CONSTANTS OF SOME COMPOUNDS USED AS SUPERCRITICAL FLUID. (Williams,1981; Hubert and Vitzthum, 1978). Compound C r i t i c a l C r i t i c a l Cr i t i i temperature pressure densi (K) (MPa) (g cm Methane 191 4.60 0. 1 62 Ethylene 282 5.03 0.218 Carbon d i o x i d e 304 7.38 0.468 Ethane 305 4.88 0.203 Propylene 365 4.62 0.233 Propane 370 4.62 0.217 Ammonia 406 11.3 0.235 D i e t h y l ether 467 3.64 0.265 n-Pentane 470 3.37 0.237 Acetone 508 4.70 0.278 Methanol 513 8.09 0.272 Benzene 562 4.89 0.302 Toluene 592 4.11 0.292 P y r i d i n e 620 5.63 0.312 Water 647 22.0 0.322 C0 2 i s co n s i d e r e d one of the best s o l v e n t s f o r s u p e r c r i t i c a l f l u i d e x t r a c t i o n f o r a number of reasons: In a d d i t i o n to i t s s o l v e n t p r o p e r t i e s and low c r i t i c a l temperature, C0 2 has the advantages of being n e i t h e r flammable nor t o x i c , and i s c o n s i d e r e d n o n - c o r r o s i v e when i n combination with moisture to the m a t e r i a l s used i n p r o c e s s i n g n a t u r a l p r o d u c t s . I t i s a l s o inexpensive and r e a d i l y a v a i l a b l e i n l a r g e q u a n t i t i e s and high p u r i t y (Hubert and Vitzthum,1978). The r e s t r i c t i o n of us i n g a s u p e r c r i t i c a l gas alone as the s o l v e n t i s not necessary. Brunner and Peter (1982) 1 2 suggested that by using a s u i t a b l e e n t r a i n e r , the s e p a r a t i o n f a c t o r can be enhanced. 2.2 PREVIOUS WORKS The phenomenon of s u p e r c r i t i c a l f l u i d e x t r a c t i o n was recog n i z e d over 100 years ago when the e f f e c t s of pressure on the s o l u b i l i t i e s of potassium i o d i d e i n ethanol were observed (Hannay and Hogarth,1879). L a t e r , i t was r e a l i z e d that the s o l v e n t power of s u p e r c r i t i c a l gases c o u l d be i n v o l v e d i n g e o l o g i c a l processes through the i n f l u e n c e of water on rock formation, and of methane i n petroluem formation and m i g r a t i o n . In power s t a t i o n s the adoption of s u p e r c r i t i c a l steam p r e s s u r e s l e d to the d e p o s i t i o n of s i l i c a on the blades of steam t u r b i n e s . A c c o r d i n g to W i l l i a m s (1981),the f i r s t p r o posal f o r the p r a c t i c a l a p p l i c a t i o n of s u p e r c r i t i c a l e x t r a c t i o n was made in 1943 f o r the d e a s p h a l t i n g of petroluem o i l s . Zhude (1958) i n the U.S.S.R. d e s c r i b e d a s i m i l a r scheme u s i n g s u p e r c r i t i c a l propane, and demonstrated the f r a c t i o n a t i o n of crude o i l with s u p e r c r i t i c a l methane, the e x t r a c t i o n of o z o c e r i t e wax from ores, and the e x t r a c t i o n of l a n o l i n from wool grease. Fundamental s t u d i e s on SCFE were i n i t i a t e d by Zosel as e a r l y as 1962 in Germany . His work i n c l u d e s the e x t r a c t i n g of l i p i d s and other n a t u r a l products with s u p e r c r i t i c a l f l u i d s (Zosel,1976). In 1970, he d i s c o v e r e d the d e c a f f e i n a t i o n of green c o f f e e with the p h a r m a c o l o g i c a l l y 1 3 completely a c c e p t a b l e carbon d i o x i d e which then has been developed i n t o commercial s c a l e (Zosel,1978). Using t h i s technique, the c a f f e i n e content of the bean i s decreased from an i n i t i a l value l y i n g between 3% and 0.7% to 0.02%. I t i s found that the c a f f e i n e i s s e l e c t i v e l y removed by the C0 2, no substances c o n t r i b u t i n g to the aroma being l o s t . Hubert and Vitzthum (1978) have demonstrated that l i p i d s can be e x t r a c t e d from copra, sunflower seeds, soybeans and s h e l l peanuts with C0 2 at p r e s s u r e s ranging from 28 to 35 MPa. Paul and Wise (1971) p u b l i s h e d a comprehensive review of the p r i n c i p l e s of gas e x t r a c t i o n . They d i s c u s s e d the p h y s i c a l b a s i s i n r e l a t i o n to d i s t i l l a t i o n and e x t r a c t i o n , and suggested p o s s i b l e areas of a p p l i c a t i o n . A review of the a v a i l a b l e data f o r a number of s u p e r c r i t i c a l f l u i d s o l v e n t s was made by I r a n i and Funk (1977). They a l s o compared the energy requirement f o r d i s t i l l a t i o n versus s u p e r c r i t i c a l f l u i d e x t r a c t i o n . ,and concluded that energy savings are r e a l i z e d with s u p e r c r i t i c a l f l u i d e x t r a c t i o n i f the process i s operated at low gas compression r a t i o s . The f i r s t symposium devoted e n t i r e l y to e x t r a c t i o n with s u p e r c r i t i c a l gases was h e l d in Germany in 1978 (WiIke,1978). At t h i s symposium, the pioneer r e s e a r c h e r s from Germany presented papers on d i f f e r e n t aspects of s u p e r c r i t i c a l f l u i d e x t r a c t i o n . Zosel (1978) and Hubert and Vitzthum (1978) d i s c u s s e d the general p r i n c i p l e s of 1 4 s u p e r c r i t i c a l f l u i d e x t r a c t i o n and t h e i r p r a c t i c a l a s p e c t s , i n c l u d i n g e x t e n s i v e experimental i n f o r m a t i o n on the e x t r a c t i o n of n a t u r a l products such as h o p s , c a f f e i n e , t o b a c c o , and f l a v o r s , with a number of d i f f e r e n t s u p e r c r i t i c a l s o l v e n t s such as ethane,ethylene, C0 2 and H 20. Although numerous s o l v e n t s are presented, carbon d i o x i d e i s , by f a r , the most e x t e n s i v e l y used s u p e r c r i t i c a l f l u i d s o l v e n t . Other t o p i c s d i s c u s s e d i n c l u d e the t h e o r e t i c a l aspects of s u p e r c r i t i c a l f l u i d e x t r a c t i o n (Schneider,1978) , e m p i r i c a l methods f o r determining the s o l u b i l i t i e s of compounds i n s u p e r c r i t i c a l f l u i d s ( S t a h l et a l . , ( l 9 7 8 ) ) and c r i t e r i a f o r the design of a f u l l - s c a l e s u p e r c r i t i c a l f l u i d e x t r a c t i o n p l a n t (Eggers,1978). A f t e r t h i s symposium, many a d d i t i o n a l papers have appeared on both the p r a c t i c a l and t h e o r e t i c a l aspects of s u p e r c r i t i c a l f l u i d e x t r a c t i o n . The review by W i l l i a m s (1981) giv e s an i n d i c a t i o n of the breadth of s e p a r a t i o n problems to which s u p e r c r i t i c a l f l u i d e x t r a c t i o n has been a p p l i e d . These problems i n c l u d e the d e c a f f e i n a t i o n of c o f f e e , e x t r a c t i o n of hops, s p i c e s , and tobacco, f r a c t i o n a t i o n of high b o i l i n g mixtures, d e a s p h a l t i n g of heavy petroleum f r a c t i o n s , e x t r a c t i o n of m i n eral d e p o s i t s , and t e r t i a r y o i l recovery with s u p e r c r i t i c a l f l u i d e x t r a c t i o n , e s p e c i a l l y with s u p e r c r i t i c a l C0 2. P a u l a i t i s et a l (1982) d e s c r i b e d a number of p r a c t i c a l a p p l i c a t i o n s , and gave an e x t e n s i v e account of experimental 15 s t u d i e s which i n c l u d e the area of t r a n s p o r t p r o p e r t i e s of s u p e r c r i t i c a l f l u i d s o l v e n t s . The recent review paper by McHugh et a l . ( l 9 8 5 ) p r o v i d e d the most up to date review of the t h e o r e t i c a l a s p e c t s and data r e l a t e d to SCFE, as w e l l as the l a t e s t a p p l i c a t i o n s . One of the most e x t e n s i v e s t u d i e s was c a r r i e d out in Germany by by S t a h l and co-workers (1978,1980,1983). They have developed a m i c r o e x t r a c t i o n apparatus which they d i r e c t l y coupled to a t h i n l a y e r chromatography f o r q u i c k l y s c r e e n i n g the s o l u b i l i t i e s of a wide range of n a t u r a l products i n s e v e r a l s u p e r c r i t i c a l f l u i d s o l v e n t s . In the study, they e s t a b l i s h e d a number of v a r i a b l e s which c o n t r o l the s o l u b i l i t y of n a t u r a l products i n s u p e r c r i t i c a l C0 2. They found t h a t : f r a c t i o n a t i o n of condensed phases i s p o s s i b l e i f the mixture c o n s t i t u e n t s e x h i b i t l a r g e d i f f e r e n c e s i n vapour pr e s s u r e , mass, or p o l a r i t y . low molar mass hydrocarbons and l i p o p h i l i c organic compounds such as e s t e r s , e t h e r s , and l a c t o n e s are e a s i l y e x t r a c t a b l e ; h y d r o x y l and c a r b o x y l groups on compounds make t h e i r e x t r a c t i o n extremely d i f f i c u l t ; sugars and amino a c i d s are not e x t r a c t a b l e . As the knowledge of s u p e r c r i t i c a l f l u i d e x t r a c t i o n i n c r e a s e s , i t s a p p l i c a t i o n s are becoming more d i v e r s e . Apart from the a p p l i c a t i o n s mentioned p r e v i u o s l y , other areas of p o t e n t i a l a p p l i c a t i o n i n c l u d e : e x t r a c t i o n i n drug 1 6 manufacturing (McHugh et al.,1985), removing o r g a n i c s from waste water (Worthy,1981), e x t r a c t i o n of v o l a t i l e aromatics from flowers and p l a n t s (Calame and S t e i n e r , 1 9 8 2 ) , and d i f f e r e n t kinds of s p i c e s (Hubert and Vitzthum,,1978). 2.3 EFFECT OF PRESSURE AND TEMPERATURE The system ethylene-^.iodochlorobenzene, F i g u r e 2-3 i l l u s t r a t e s the l a r g e enhancement in s o l u b i l i t y that occurs at s u p e r c r i t i c a l c o n d i t i o n s (Williams,1981). Measurements were made at a temperature of 298 K, c l o s e to the c r i t i c a l temperature of ethylene, 282 K. At low p r e s s u r e s the gas phase c o n c e n t r a t i o n of the i n v o l a t i l e s o l i d was extremely s m a l l . I n c r e a s i n g the p r e s s u r e to above 5 MPa,the c r i t i c a l p r e ssure of ethylene, the s o l u b i l i t y of the s o l i d i n c r e a s e d by three orders of magnitude. E x t r a c t i o n p r e s s u r e s i n the range of 10-40 MPa are g e n e r a l l y necessary to achieve adequate d e n s i t i e s . T h i s p r essure range has so f a r been i n v e s t i g a t e d most f r e q u e n t l y . The occurrence of f u r t h e r d i s s o l u t i o n phenomena at higher p r e s s u r e s cannot be excluded, but t h e i r use would probably be r e j e c t e d f o r economic reasons. The p r e f e r e d temperature range fo r e x t r a c t i o n with C0 2 i s approximately 35 - 80 °C. The importance of c a r r y i n g out the e x t r a c t i o n near the c r i t i c a l temperature of the gas i s i l l u s t r a t e d i n F i g u r e s 2-4, which show r e s u l t s obtained when phenaphthelene was c o n t a c t e d at 313 K and 40 MPa with gases of d i f f e r e n t c r i t i c a l temperatures (Williams,1981). The gases having 1 7 0.01 Pressure, MPa F i g u r e 2-3: The l a r g e i n c r e a s e i n the s o l u b i l i t y of p.iodochlorobenzene i n the s u p e r c r i t i c a l e t hylene ( Tc = 282 K , Pc = 5 MPa ) at 298 K brought about at e l e v a t e d p r e s s u r e s ( W i l l i a m s , 1 9 8 1 ) . 18 1 1 1 1 1 1 1 1 r CD CO _ LO —I e=> \ CD _ en •<* C r i t i c a l temperature of gas (K) F i g u r e 2-4: The gas phase c o n c e n t r a t i o n of phenaphthelene i n v a r i o u s s u p e r c r i t i c a l gases at 313 K and 40 MPa, showing the importance of a c l o s e correspondence between the e x t r a c t i o n temperature and the c r i t i c a l temperature of the gas (Williams,1981). 1 9 c r i t i c a l temperatures much below the e x t r a c t i o n temperature, i n c l u d i n g n i t r o g e n (116 K), methane (191 K) and carbon t e t r a f l u o r i d e (222 K), d i d not e x t r a c t the phenanthrene, whereas those with c r i t i c a l temperatures near the e x t r a c t i o n temperature, namely ethylene (283 K), carbon d i o x i d e (304 K) and ethane (305 K), proved e f f e c t i v e s o l v e n t s . Tsekhanskaya et a l . ( l 9 6 4 ) r e p o r t e d that the s o l u b i l i t y of a substance i n a s u p e r c r i t i c a l f l u i d (e.g. naphthalene i n compressed ethylene T c = 9.21°C; P c = 5 MPa) changes with pressure at d i f f e r e n t temperatures, as seen i n F i g u r e 2-5. At moderate p r e s s u r e s , a r i s e i n temperature caused a decrease in the s o l u b i l i t y of the s o l i d component. At higher p r e s s u r e s , a r i s e i n temperature causes an i n c r e a s e i n the s o l u b i l i t y . T h i s behavior can be e x p l a i n e d i n terms of the d e n s i t y of the gas. A r i s e i n temperature at constant pressure l e a d s , on the one hand, to a decrease i n gas d e n s i t y and on the other, to an e x p o n e n t i a l i n c r e a s e i n the vapour pressure of the s o l i d . At the region s l i g h t l y above the c r i t i c a l temperature, gas d e n s i t y at moderate pr e s s u r e s i s lowered by a temperature r i s e to such an extent that the c o n c e n t r a t i o n of the s o l u t e i n the s u p e r c r i t i c a l phase decreases c o n s i d e r a b l y . At h i g h p r e s s u r e s the decrease i n d e n s i t y caused by the temperature r i s e i s so small that the i n c r e a s e in vapour pressure of the s o l i d leads to a higher c o n c e n t r a t i o n i n the s u p e r c r i t i c a l phase. In a separate study by Brogle (1982), s i m i l a r phenomena were observed. Hence the author suggested two g e n e r a l r u l e s f o r 20 30 UhPal 12 7 MPa 101 MPa 81 MPa 61 MPa 2fl0 290 300 310 320 330 Temperature , K F i g u r e 2-5: S o l u b i l i t y of naphthalene i n s u p e r c r i t i c a l e t h y l e n e as a f u n c t i o n of temperature at d i f f e r e n t p r e s s u r e s ( E t h y l e n e : Tc = 282 K , Pc = 5 MPa). (Williams,1981). 21 s u p e r c r i t i c a l s o l v e n t s : Solvent power of a s u p e r c r i t i c a l s o l v e n t i n c r e a s e s with d e n s i t y at a given temperature. Solvent power of a s u p e r c r i t i c a l s o l v e n t i n c r e a s e s with temperature at a given d e n s i t y . 2.4 EFFECT OF WATER CONTENT L i t t l e work has been r e p o r t e d on the e f f e c t of water content on the s u p e r c r i t i c a l f l u i d e x t r a c t i o n process. I t i s an important parameter to be c o n s i d e r e d , as water i s present in almost a l l n a t u r a l substances. The presence of water i s e s s e n t i a l i n the e x t r a c t i o n of n i c o t i n e . However, at the normal moisture content (10-13%. by weight) of tobacco, s u p e r c r i t i c a l C0 2 i s a poor s o l v e n t f o r n i c o t i n e and tends to remove the aroma i n s t e a d (Hubert and Vitzthum,1978). For the e x t r a c t i o n of n i c o t i n e i n a s i n g l e stage process, they found that i t was necessary to i n c r e a s e the water content to at l e a s t 25%. Zosel (1978) s t a t e d that c o f f e e beans should be soaked using water p r i o r to d e c a f f e i n a t i o n with s u p e r c r i t i c a l carbon d i o x i d e . During the e x t r a c t i o n of soybean o i l , F r i e d r i c h and Pryde (1984) r e p o r t e d that moisture contents between 3 and 15 % had no s i g n i f i c a n t e f f e c t on o i l e x t r a c t a b i 1 i t y and composition. 22 2.5 SCFE OF TRIGLYCERIDES 2.5.1 OILS AND FATS Fats and o i l s c o n s i s t predominantly of g l y c e r y l e s t e r s of f a t t y a c i d s ( t y p i c a l l y above 85% by weight), s o - c a l l e d t r i g l y c e r i d e s a l s o r e f e r r e d to as t r i a c y l g l y c e r o l s . N a t u r a l l y , t r i g l y c e r i d e s e x i s t i n many d i f f e r e n t forms, with t h e i r f a t t y a c i d s e r i e s ranging from C1 to C22 and h i g h e r . It i s recognized that the t r i g l y c e r i d e s t r u c t u r e of the f a t s and o i l s i s i n t i m a t e l y r e l a t e d to the type and d i s t r i b u t i o n of t h e i r f a t t y a c i d s , and they are mainly mixtures of mixed r a t h e r than simple t r i g l y c e r i d e s (Sonntag,1979). A number of f a t s are p r i z e d f o r t h e i r i n d i v i d u a l f l a v o r s and s t r u c t u r e s , and are used with l i t t l e or no p u r i f i c a t i o n . For example, cocoa b u t t e r has a s p e c i f i c arrangment of the f a t t y a c i d c o n s t i t i u e n t s i n the g l y c e r i d e s that give i t the unique f l a v o r , t e x t u r e and m e l t i n g c h a r a c t e r i s t i c s d e s i r a b l e f o r use i n c o n f e c t i o n a r y products, e s p e c i a l l y c h o c o l a t e (Gutcho,1979). Since cocoa b u t t e r i s a r e l a t i v e l y expensive n a t u r a l product, f o r many years, attempts have been made to provide a product as i t s s u b s t i t u t e . I t has been suggested that s e l e c t i v e f r a c t i o n a t i o n of other cheaper o i l s (such as palm k e r n e l and palm o i l ) c o u l d produce some products that have s i m i l a r p r o p e r t i e s of the s p e c i a l t y f a t s . C o n v e n t i o n a l l y , f r a c t i o n a t i o n of o i l s can be achi e v e d by expensive vacuum d i s t i l l a t i o n at f a i r l y high 23 temperatures, or by c r y s t a l i z a t i o n methods where petroluem s o l v e n t s are i n v o l v e d (Pike 1980). 2.5.2 CO 2 EXTRACTION TRIGLYCERIDES Although no commercial s u p e r c r i t i c a l ~ C 0 2 o i l e x t r a c t i o n p l a n t s are known to be in o p e r a t i o n , recent i n t e n s i v e s t u d i e s of t h i s technique on v a r i o u s o i l s e e d s show i t s p o t e n t i a l . One of the f i r s t i n t e n s i v e s t u d i e s d e a l i n g with o i l s e e d e x t r a c t i o n using SC-C0 2 was undertaken by S t a h l et a l . (1980). The authors have d e s c r i b e d a semi-batch e x t r a c t i o n system and a method of measuring the e q u i l i b r i u m s o l u b i l i t y of o i l i n C0 2. V a r i o u s parameters i n f l u e n c i g the e x t r a c t i o n process were s t u d i e d . These i n c l u d e : temperature, p r e s s u r e , s o l v e n t f l o w r a t e and o i l s e e d s pre-treatment. The p h y s i c a l p r o p e r t i e s (such as c o l o r , t a s t e and odour) and chemical p r o p e r t i e s (such as major and minor const i t i u e n t s and o x i d a t i v e s t a b i l i t y ) were a l s o s t u d i e d . S i m i l a r s t u d i e s were a l s o c a r r i e d out by some re s e a r c h e r s i n North America. F r i e d r i c h and co-workers ( F r i e d r i c h and L i s t , 1982; L i s t and F r i e d r i c h , 1985; C h r i s t i a n s o n et a l , 1984, F r i e d r i c h and Pryde, 1984), worked on soybeans, cottonseeds and corn germ. F a t t o r i (1986) s t u d i e d the e x t r a c t i o n of c a n o l a o i l . In most cases, C0 2 was used as the s o l v e n t . The s o l u b i l i t y of o i l , as i n f l u e n c e d by o p e r a t i n g parameters and o i l composition are the main concerns of t h i s 24 work. F i g u r e 2-6,2-7 and 2-8, show the s o l u b i l i t i e s of v a r i o u s seed o i l s as a f u n c t i o n of temperature and pressure as determined by d i f f e r e n t authors. As can be seen, i n a l l cases, the s o l u b i l i t y v a r i e s with temperature and p r e s s u r e . In g e n e r a l , the s o l u b i l i t y i n c r e a s e s with i n c r e a s i n g p r e s s u r e . For example, canola o i l (Figure 2-6), at 55 °C the s o l u b i l i t y i n c r e a s e s from 2 mg/g C0 2 at 20 MPa to 12 mg/g C0 2 at 36 MPa. However, the change i n s o l u b i l i t y with temperature i s d i f f e r e n t at d i f f e r e n t p r e ssure l e v e l s f o r most o i l s . T y p i c a l l y , the s o l u b i l i t y isotherms c r o s s at the int e r m e d i a t e p r e s s u r e s . T h i s c r o s s o v e r of s o l u b i l i t y curves has been e x p l a i n e d i n terms of the i n t e r a c t i o n between the d e n s i t i e s of C0 2 and the vapour pressure of the o i l s (Peter and Brunner,1978; F r i e d r i c h and Pryde,l984; Brogle,l982 and F a t t o r i , 1 9 8 6 ) . G e n e r a l l y , a n i n c r e a s e i n temperature causes an i n c r e a s e i n vapor pressure of the s o l u t e which means an in c r e a s e of s o l u t e s o l u b i l i t y i n s o l v e n t . On the other hand, the temperature i n c r e a s e w i l l a l s o cause a decrease i n C0 2 d e n s i t y which means a decrease of i t s s o l v e n t c a p a c i t y . As can be seen i n F i g u r e 2-9, the d e n s i t y of C0 2 changes most r a p i d l y with temperature at p r e s s u r e s near the c r i t i c a l r e g i o n . Hence, i n t h i s r e g i o n , the d e n s i t y e f f e c t due to temperature changes i s much more pronounced than that of the vapor pressure e f f e c t , whereas at higher p r e s s u r e s , the density/temperature change i s r e l a t i v e l y s m a l l . Hence, the vapor pressure e f f e c t predominates. 25 CvJ — O (_) £-3 I—I R O ° >-I— I—I 00 CO CD O CO CD Osl 10.0 14.0 18.0 22.0 26.0 30.0 34.0 38.0 P R E S S U R E (MPa) n f 9 o r L o 2 " 6 : S o l V b i l i t y of Canola o i l i n C0 2 as a f u n c t i o n of p r e s s u r e at v a r i o u s temperature ( F a t t o r i , 1 9 8 6 ) . r U n C t l o n 26 20.0 30.0 40.0 50.0 60.0 Pressure, Mpa F i g u r e 2-7: E f f e c t s of temperature and s o l u b i l i t y of soybean o i l i n s u p e r c r i t i c a l and Pryde,1984). p r e s s u r e on the C0 2 ( F r i e d r i c h 27 I I I I I I I I I I I I I I I I I I I 1 1 0.0 4.0 8.0 12.0 16.0 20.0 24.0 28.0 32.0 36.0 40.0 PRESSURE (MPa) F i g u r e 2-8: S o l u b i l i t y of Rapeseed and Soybean o i l s i n C0 2 as a f u n c t i o n of p r e s s u r e at 20°C and 40°C ( S t a h l et al.,1980; Bunzenberger et a l . , 198-4). 28 CO m Ol M«°. 0 0 ° • d o r <_)° CO t I i i i i i i i — i — i — i — i — i — i — i — i — r j i i » J i i i i J i i i i 0.0 4.0 8.0 12.0 16.0 20.0 24.0 28.0 32.0 36.0 40.0 PRESSURE (MPa) F i g u r e 2 - 9 : D e n s i t y of carbon d i o x i d e as a f u n c t i o n of p r e s s u r e at d i f f e r e n t temperatures. The c r i t i c a l p o i n t i s d e s i g n a t e d as CP ( V u k a l o v i c h and Altunin,1968) 29 It i s o f t e n not an easy task to p r e d i c t which e f f e c t predominates under c e r t a i n c o n d i t i o n f o r d i f f e r e n t substances without experimetal data. As can be seen from F i g u r e s 2-6 to 2-8, the c r o s s o v e r occurs at d i f f e r e n t p r e s s u r e s and temperatures f o r d i f f e r e n t o i l s . The range of s o l u b i l i t y data v a r i e s among the d i f f e r e n t o i l s . For example, F i g u r e 2-8 shows that soybean o i l i s more s o l u b l e than rapeseed o i l under s i m i l a r e x t r a c t i o n c o n d i t i o n s . T h i s d i f f e r e n c e c o u l d be due to the d i f f e r e n c e i n the components of the o i l s . Rapeseed o i l c o n t a i n s a higher percentage of e r u c i c a c i d (C22:1) than soybean o i l . As suggested by Peter and Brunner (1978), an o i l having t r i g l y c e r i d e s of higher molecular weight would be expected to e x h i b i t a lower s o l u b i l i t y i n C0 2. F a t t o r i (1986) has a l s o r e p o r t e d s i m i l a r r e s u l t s f o r pure t r i g l y c e r i d e s ( F i g u r e 2-10). 2.5.3 SUPERCRITICAL FRACTIONATION Many hig h molecular weight o i l s c o n s i s t s of a wide range of homologous members. Because the o i l s e x h i b i t low vapor p r e s s u r e c h a r a c t e r i s t i c s , they are d i f f i c u l t to f r a c t i o n a t e e i t h e r by high vacuum d i s t i l l a t i o n or by s o l v e n t c r y s t a l l i z a t i o n . Moreover, these o i l s possess components that resemble d i f f e r e n t s t r u c t u r e s or f u n c t i o n a l i t i e s but e x h i b i t the same, or n e a r l y the same, vapor p r e s s u r e . Thus d i s t i l l a t i o n can not p u r i f y such m a t e r i a l s . In a d d i t i o n , l i q u i d s o l v e n t e x t r a c t i o n i s g e n e r a l l y i n e f f e c t i v e f o r 30 I I I I t I I I I I I I I I I I I I I I I I L 0.0 16.0 32.0 48.0 64.0 80.0 96.0 112.0 128.0 144.0 160.0 178.0 192.0 TOTAL C02 PASSED THROUGH BED (g) F i g u r e ^ - l O : E x t r a c t i o n c u r v e s f o r pure t r i p a l m i t o l e i n (C16:1), t r i o l e i n (C18.-1) and t r i - 1 1 - e i c o s e n o i n (C20:1). E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C. ( F a t t o r i , 1 9 8 6 ) . 31 removing such i m p u r i t i e s or f o r f r a c t i o n a t i n g the o i l s because the so l v e n t power of a l i q u i d which can d i s s o l v e such an o i l i s so great that i t can not be r e a d i l y d i f f e r e n t i a t e d by molecular weight. Because the s o l v a t i o n power of s u p e r c r i t i c a l f l u i d s can be c o n t r o l l e d to a c l o s e degree by a p p r o p r i a t e s e l e c t i o n of p r e s s u r e , and because many high-molecular o i l s can be d i s s o l v e d i n a v a r i e t y of common s u p e r c r i t i c a l f l u i d s , f r a c t i o n a t i o n of these o i l s by hig h - p r e s s u r e gases has been accomplished (Zosel,1978; S t a h l and Quirin,1983; Brunner and Peter,1982). In g e n e r a l , i f s u i t a b l e P/T combinations are s e l e c t e d i t i s p o s s i b l e to achieve some degree of f r a c t i o n a t i o n d u r i n g the s e p a r a t i o n (Hubert and Vit z t h u m l 9 7 8 ) . The phenomenon of f r a c t i o n a t i o n was f i r s t r e p o r t e d by Zosel (1978), who d e s c r i b e d a procedure to separate a mixture of p - o l e f i n s . The i n i t i a l mixture was made up of C 1 6 , C 1 8 and C 2o o l e f i n s with 17,17 and 34 % volume r e s p e c t i v e l y . A n a l y s i s of the v a r i o u s f r a c t i o n s of the e x t r a c t showed that s e p a r a t i o n had been accomplished.He re p o r t e d that one f r a c t i o n c o n t a i n i n g about 25 % of the C 1 6 o l e f i n had a p u r i t y of over 95 %. He a p p l i e d the procedure to separate c o d - l i v e r o i l which c o n s i s t s of a multicomponent mixture of t r i g l y c e r i d e s that are too i n v o l a t i l e to separate by c o n v e n t i o a l d i s t i l l a t i o n methods. F i g u r e 2-11 shows the changes i n s a p o n i f i c a t i o n values and i o d i n e numbers of each e x t r a c t f r a c t i o n d u r i n g the course of e x t r a c t i o n . The s a p o n i f i c a t i o n value and i o d i n e number are a s s o c i a t e d with 32 I I I i i j — 1 : — r — 1 1 1 0 20 AO 60 80'. 100 EX.TRfiCT [ w t . - % ] — -F i g u r e 2-11: S a p o n i f i c a t i o n number and iodine number of the s u p e r c r i t i c a l e x t r a c t s of c o d - l i v e r o i l at d i f f e r e n t stages of an e x t r a c t i o n (Zosel,1978). 33 the molecular weight and degree of u n s a t u r a t i o n respect i v e l y . L a t e r , Peter and Brunner (1978) demonstrated the s e p a r a t i o n of the monoglycerides from a mixture of o l e i c a c i d g l y c e r i d e s using a c o u n t e r c u r r e n t process with C0 2 as the s o l v e n t . They managed to concentrate the monoglyceride content of the e x t r a c t to 95-100% from a mixture c o n t a i n i n g about 45 % monoglycerides. Panzner et a l . (1980) has r e p o r t e d some data on s e p a r a t i o n of g l y c e r i d e s using the s u p e r c r i t i c a l e x t r a c t i o n technique with an e n t r a i n e r . E x t r a c t i o n s were c a r r i e d out at low p r e s s u r e s ( 8 - 1 0 MPa), with e n t r a i n e r s such as carbon t e t r a c h l o r i d e and hexane being used to improve the s o l u b i l i t y . His r e s u l t s showed that when u s i n g mixtures of carbon d i o x i d e and carbon t e t r a c h l o r i d e to separate mixtures of g l y c e r i d e s (mono-oleate 40 %,. d i o l e a t e 50 % and t r i o l e a t e 10 % ), the t r i o l e a t e made up 75-99 % by weight of the e x t r a c t . With hexane as an e n t r a i n e r , he was able to separate t r i o l e a t e of 98-99 % p u r i t y from d i o l e a t e and mono-oleate. He concluded that the s o l u b i l i t y of t r i o l e a t e in C0 2 can be i n c r e a s e d s u b s t a n t i a l l y by s u i t a b l e adjustment of the c o n c e n t r a t i o n of an e n t r a i n e r such as hexane in the C0 2 . I t has a l s o been found that b u t t e r f a t can be f r a c t i o n a t e d using s u p e r c r i t i c a l C0 2 (Mangold, 1982). T h i s process y i e l d s a f r a c t i o n that c o n t a i n s twice as much t r i g l y c e r i d e of s h o r t - c h a i n f a t t y a c i d s as the s t a r t i n g 34 m a t e r i a l s . S i m i l a r r e s u l t s have been repo r t e d (Anon, 1981) that s u p e r c r i t i c a l C0 2 was extremely e f f e c t i v e as a s p e c i f i c f r a c t i o n a t i o n s o l v e n t f o r coconut o i l and cocoa b u t t e r . In the coconut o i l t r i a l , 20 grams of o i l were separated i n t o f i v e f r a c t i o n s , each of which was analysed by sensory e v a l u a t i o n and gas chromatography. Lactones, low molecular weight f a t t y a c i d s which give coconut o i l i t s f a m i l i a r f l a v o r , were e f f e c t i v e l y i s o l a t e d i n the f i r s t two f r a c t i o n s . The same trends were observed with cocoa b u t t e r . The cocoa f l a v o r and aromatic v o l a t i l e s were i s o l a t e d i n the e a r l y f r a c t i o n s . No i n f o r m a t i o n on the f a t t y a c i d composition of the e x t r a c t e d o i l s was given by the author. S t a h l and co-workers (1980,1983) have r e p o r t e d e x t e n s i v e l y on the study of s u p e r c r i t i c a l f r a c t i o n a t i o n . I t i s i n t e r e s t i n g to p o i n t out the two d i f f e r e n t f i n d i n g s from t h e i r s t u d i e s . In t h e i r f i r s t paper,they s t a t e d that f r a c t i o n a t i o n of condensed phases i s p o s s i b l e i f the mixture c o n s t i t u e n t s e x h i b i t l a r g e d i f f e r e n c e s i n vapor p r e s s u r e , mass, or p o l a r i t y ( S t a h l et al.,1980). In t h e i r l a t e r s t u d i e s , S t a h l and Q u i r i n (1983) measured the s o l u b i l i t i e s of t e t r a c y c l i c s t e r i o d s which d i f f e r e d in molecular s t r u c t u r e s but had p r a c t i c a l l y the same vapour pressure at the system o p e r a t i n g temperature. They found that c a r b o x y l groups on the s t e r i o d s , such as b i l e a c i d s , rendered the s t e r i o d s v i r t u a l l y i n s o l u b l e i n the s u p e r c r i t i c a l C0 2, while c a r b o n y l groups had l i t t l e e f f e c t on s t e r i o d s o l u b i l i t i e s . In t h i s study, however, the d i f f e r e n c e s i n masses and m e l t i n g p o i n t s 35 of the s t e r i o d s had no d i r e c t i n f l u e n c e on the s o l u b i l i t y behavior. Hence, they argued that s u p e r c r i t i c a l f l u i d e x t r a c t i o n c o n s t i t u t e s a new s e p a r a t i o n and f r a c t i o n a t i o n technique which does not depend on d i f f e r e n c e s i n the mixture components' vapour pressure and which e x h i b i t s s e l e c t i v e l y f o r c e r t a i n c l a s s e s of compounds. Brogle (1982) has proposed a few p r a c t i c a l examples f o r f r a c t i o n a t e d e x t r a c t i o n : f r a c t i o n a t i o n of s p i c e e x t r a c t i o n i n t o f l a v o r f r a c t i o n ( e s s e n t i a l l y o i l ) and t a s t e f r a c t i o n . f r a c t i o n a t i o n of f a t t y o i l s i n t o f i r s t f r a c t i o n r i c h i n f l a v o r and fr e e f a t t y a c i d s , a second f r a c t i o n of mostly g l y c e r i d e s and a t h i r d f r a c t i o n r i c h i n waxes and pigments. f r a c t i o n a t i o n of f a t t y o i l s i n t o f r a c t i o n s r i c h i n mono-, d i - or t r i g l y c e r i d e s r e s p e c t i v e l y . In a recent study by F a t t o r i (1986), the author found that the degree of f r a c t i o n a t i o n of canol a o i l s u s ing s u p e r c r i t i c a l C0 2 was not as pronounced as p r e v i o u s l y r e p o r t e d (Anon, 1981). However, he was able to f r a c t i o n a t e a mixture of pure simple t r i g l y c e r i d e s (C16:1, C18:1 and C2 0 : l ) , w i t h the l i g h t e r f r a c t i o n appearing i n the e a r l y p e r i o d s of the process, and the he a v i e r f r a c t i o n was conce n t r a t e d in the l a t e r stages ( F i g u r e 2-12). He suggested that the canol a o i l was made up of a pool of homologous mixed t r i g l y c e r i d e s which were very s i m i l a r i n molecular mass, even though the d i s t r i b u t i o n of f a t t y a c i d s among them 36 t i i i I i i i i i i i i r~~i i i i i i i i i r • C16:"| t r i p a l m i t o l e i n • C20:1 t r i e i c o s e n o i n t t t t t t l t f t l t t t t I t 1 I ! L _ J L 0.0 8.0 16.0 24.0 32.0 40.0 48.0 56.0 64.0 72.0 80.0 88.0 96.0 PERCENT EXTRACTED IX) F i g u r e 2-12: Mass f r a c t i o n of the s u p e r c r i t i c a l C0 2 e x t r a c t s of a mixture of three simple t r i g l y c e r i d e s at d i f f e r e n t s t a g e s of the e x t r a c t i o n . E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C. ( F a t t o r i , 1 9 8 6 ) . 37 might be d i f f e r e n t . Hence, these t r i g l y c e r i d e s might have approximately the same s o l u b i l i t y i n s u p e r c r i t i c a l C0 2 and might e x p l a i n the r e l a t i v e l y constant s o l u b i l i t y observed throughout the e n t i r e e x t r a c t i o n p r o c e s s . For the mixture of pure simple t r i g l y c e r i d e s , each component has a d i f f e r e n t molecular mass and thus a d i s t i n c t s o l u b i l i t y b e havior. T h i s r e s u l t e d in changes i n the o v e r a l l s o l u b i l i t y r a t e depending on the composition of the mixture. Chapter 3 METHODS AND MATERIALS 3.1 EXPERIMENTAL EQUIPMENT In t h i s r e s e a r c h , a l l e x t r a c t i o n experiments were c a r r i e d out using a m o d i f i e d Hewlett-Packard 1081B High Performance L i q u i d Chromatograph (HPLC). 1 B a s i c a l l y , the system c o n s i s t s of four major components: the HPLC, e x t r a c t i o n v e s s e l s , f l o w r e s t r i c t o r and sampling u n i t . D e t a i l s on the m o d i f i c a t i o n s to the HPLC are r e p o r t e d by F a t t o r i (1986). 3.1.1 HEWLETT-PACKARD LIQUID CHROMATOGRAPH The HPLC i s a microprocessor c o n t r o l l e d instrument i n c o r p o r a t i n g a r e c i p r o c a l diaphragm pump and a s o l v e n t flow system. Flow r a t e s can be s e l e c t e d between 0 to 9.9 ml/min in increments of 0.01 ml/min. Pressure can be s e l e c t e d from 0 to 40 MPa,with 0.1 MPa increments. The chromatograph a l s o i n c o r p o r a t e s an oven with o p e r a t i n g temperatures v a r y i n g from 25°C to 99°C (with 0.1°C increments). The d i f f e r e n t process parameters can be p r e c i s e l y c o n t r o l l e d w i t h i n the a l l o w a b l e v a r i a t i o n l i m i t s f o r t h i s work: ±0.03 ml/min f o r the f l o w r a t e ; ±0.2 MPa f o r the pressure and ±1.0°C f o r the oven temperature. There i s no d i r e c t manual c o n t r o l of these parameters, as they can only be set through the m i c r o p r o c e s s o r . The 'Sources f o r a l l equipments and s u p p l i e r s are d e t a i l e d i n Appendix I. 38 39 system w i l l a u t o m a t i c a l l y shut o f f the pump i f the maximum pressure i s exceeded. Again, F a t t o r i (1986) g i v e s a d e t a i l e d d e s c r i p t i o n on the c o n t r o l mechanism of pressure and f l o w r a t e . 3.1.2 EXTRACTION VESSELS E x t r a c t i o n v e s s e l s (autoclaves) were used to accommodate the m a t e r i a l s to be e x t r a c t e d . These v e s s e l s , made of 316 s t a i n l e s s s t e e l , can withstand the very high o p e r a t i n g pressure o f t e n encountered. Three e x t r a c t i o n v e s s e l s of d i f f e r e n t s i z e s were used f o r t h i s work . The s i z e s p e c i f i c a t i o n s f o r each of the e x t r a c t i o n v e s s e l s are l i s t e d i n Table 3-1. TABLE 3-1: DIMENSIONS OF THE EXTRACTION VESSELS. V e s s e l number 1 2 3 Inside Diameter ( cm ) 1 .27 1 .27 2.54 Insi d e Length ( cm ) 8.2 11.4 8.2 V e s s e l Volume ( cm 3 ) 10.4 14.4 41.6 V e s s e l Wall Thic kness ( cm ) 0.6 0.6 0.8 Fi g u r e 3-1 i s a machine drawing of v e s s e l no.1. The e x t r a c t i o n v e s s e l (autoclave) resembles a t h i c k w a l l e d tube with a f l a n g e d top. The cap of each v e s s e l i s a separate p i e c e which can be removed upon l o a d i n g the m a t e r i a l , and i s t i g h t l y secured to the body of the v e s s e l by e i g h t s t a i n l e s s s t e e l cap screws .after f i l l i n g . The cap was f a b r i c a t e d with 40 Swagelok tube f i t t i n g - 1/16" tube to 1/8" NPT r / 1 1 ' , 1 1 1 , / 1 / s -IT" ,- , i / « i / . ' 1 / 0.16-A A V 1/8 National Pipe Thread (NPT) 0.3J / / 'e-32 Un i f ied National Coarse thread --inch 2J4 • — 1/8 Swagelok tube f i t t i n g ^ as above " National Pipe Thread F i g u r e 3-1: Cross s e c t i o n a l view of e x t r a c t i o n v e s s e l #1. A l l measurements shown are i n cm, except were i n d i c a t e d . 41 a machined s e a l i n g r i d g e which w i l l s i t on the f l a t s e a l i n g s u r f a c e of the v e s s e l . The s e a l s were designed to be leak p r o o f . The b o l t s i z e f o r v e s s e l 1 i s 10.6 mm, and 6.4 mm f o r the l a r g e r v e s s e l s . Each of the 10.6mm b o l t s was t i g h t e n e d to 60 cm-kgs. The 6.4mm b o l t s were t i g h t e n e d to 120 cm-kgs. The bottom and top of the v e s s e l s are f i t t e d with 3.2 x 1.6 mm Swagelok NPT f i t t i n g s . Solvent f l o w - l i n e connections to and from the v e s s e l s are made v i a these f i t t i n g s . 3.1.3 FLOW RESTRICTER The r e s t r i c t e r i s l o c a t e d i n the HPLC oven and mounted in a heated aluminum block to prevent f r e e z i n g up durin g the r a p i d expansion of the d e p r e s s u r i n g C 0 2 / s o l u t e mixture. The op e r a t i o n of the e x t r a c t i o n system i s a f f e c t e d by the c h a r a c t e r i s t i c s of the flow r e s t r i c t e r which serves to maintain the pressure i n the e x t r a c t o r at the d e s i r e d v a l u e . A v a r i e t y of flow r e s t r i c t e r s were assessed. The Parker MV-200 metering v a l v e , used i n the e a r l i e r work, c o u l d adequately c o n t r o l the C0 2 f l o w r a t e and maintain the d e s i r e d p r e s s u r e . However, the performance d r i f t e d a f t e r a number of op e r a t i o n s because the s e a l g r a d u a l l y wore o f f . Another r e s t r i c t e r t e s t e d was a Nupro 'R3A' s e r i e s e x t e r n a l l y a d j u s t a b l e pressure r e l i e f v a l v e , and i t was found to have a l a r g e dead volume i n a d d i t i o n to the leak problem. E v e n t u a l l y a Rheodyne Model 7037 pressure r e l i e f v a l v e was found to be the most s t a b l e f o r t h i s type of m a t e r i a l . I t has a very small i n t e r n a l volume and allows p r e s s u r e s e t t i n g 42 to be a d j u s t e d over a range of 7 to 48 MPa . The flow stream only comes i n contact with 316 s t a i n l e s s s t e e l . F i g u r e 3-2 shows an exploded view of the valve . I t c o n s i s t s of a s p r i n g - l o a d e d diaphragm which i s s e a l e d a g a i n s t the cap around i t s p e r i p h e r y and which seats a g a i n s t a p o l i s h e d c e n t r a l seat machined in the cap. T h i s geometry forms an annular channel surrounding the c e n t r a l s e a t . The volume contained i n the annular channel i s approximately 6 y l . Four s p r i n g washers are used to exert a f o r c e on the diaphragm to s e a l i t a g a i n s t the vent seat. The adjustment of s p r i n g f o r c e i s accomplished by a d i f f e r e n t i a l thread arrangement. When the f l u i d pressure exceeds the r e l i e f s e t t i n g , the f l u i d l i f t s the diaphragm o f f the seat and exposes the vent post where the f l u i d i s c o n t i n u o u s l y f l u s h e d out. 3.1.4 SAMPLING UNIT A f t e r p a s s i n g through the r e s t r i c t e r , carbon d i o x i d e w i t h i n the system changes from the s u p e r c r i t i c a l to the gaseous s t a t e . Accompanying t h i s change of s t a t e i s a l a r g e decrease in the s o l v a t i o n c a p a c i t y of the carbon d i o x i d e . Compounds such as o i l s , that are s o l u b l e i n the C0 2 on the upstream s i d e of the r e s t r i c t e r , p r e c i p i t a t e downstream. The sampling u n i t allowed the s e p a r a t i o n and c o l l e c t i o n of the o i l d r o p l e t s thus formed. The sampling system enables s e q u e n t i a l o i l samples to be c o l l e c t e d and the carbon d i o x i d e to be measured by a wet t e s t meter. The system (Figure 3-3) , c o n s i s t e d of a 30 mm 43 P i n P l a t e Screws (3) Cap Screws (3)-J A d j u s t i n g Screw - Hex P l a t e Inner Screw <5> J 2 } s LT n Body Spring Washers (4) .Spring Pad - Plunger - Seal P l a t e C e n t e r i n g Ring Diaphragm Cap F i g u r e . 3 - 2 : Exploded view of Model 7037 p r e s s u r e r e l i e f v a l ve, 44 F i g u r e 3-3: Cro s s s e c t i o n a l view of the sampling u n i t . A l l measurements shown are i n mm, except were i n d i c a t e d . 45 long s e c t i o n of 1.6 mm O.D. s t a i n l e s s s t e e l tubing connected to the r e s t r i c t e r v a l v e using a Swagelock adaptor. A 50 mm s e c t i o n of 1.6mm O.D. s t a i n l e s s s t e e l tubing was connected to the op p o s i t e end of the union and d i r e c t e d through a s e p a r a t o r . The separator resembled a 25 mm long x 15 mm O.D. brass c y l i n d e r with a 1.5mm diameter channel f o r the i n l e t / o u t l e t tubing down the c e n t r e , a 6.0 mm channel f o r gas e x i t at one s i d e and a threaded f i t t i n g f o r the sample v i a l at the bottom. An O-ring was used f o r the sample v i a l f i t t i n g , and Swagelok f i t t i n g s were used f o r the other connections to ensure a s e a l . The two-phase gas and e n t r a i n e d o i l mixture flowed through the i n l e t t u b i ng i n t o the sample v i a l where the o i l was d e p o s i t e d . The o i l - f r e e C0 2 then passed from the v i a l i n t o the gas e x i t channel along a 1 m rubber t u b i n g , 6.4mm i n diameter. The tubing was channeled through the oven w a l l and then connected to the wet t e s t meter where the flow r a t e of o i l - f r e e C0 2 was measured and the C0 2 vented. 3.2 SOLVENT FLOWPATH In b r i e f the o p e r a t i o n of the system ( s c h e m a t i c a l l y shown i n F i g u r e 3-4) , was as f o l l o w s . L i q u i d carbon d i o x i d e from a storage c y l i n d e r passes through the s h u t o f f v a l v e , a Nupro 7 jum s i n t e r e d f i l t e r and then i n t o the co o l e d diaphragm pump head. The c o o l e d l i q u i d C0 2 then flows through a pr e s s u r e - f l o w monitoring device i n t o the HPLC oven. Here i t s temperature i s brought to the 46 s h u t - o f f v a l v e (C02) oven ..!:.?.?.P?.J?.a..^ ..y.?i.?....9.P.n.t'.rol l e d r e s t r i c t e r v a l v e lvv4 temperature e q u i l i b r a t i o n ^ c o i l hC 3fi.lt er e x t r a c t i q n i v e s s e l g j sample c o l l e c t ion v e s s e l flow meter F i g u r e 3-4: s u p e r c r i t i c a l Schematic diagram of f l u i d e x t r a c t i o n system. the experimental 47 d e s i r e d value by passing through a 7 m s t a i n l e s s s t e e l (1.6mm diameter) tube. At t h i s stage, carbon d i o x i d e i n i t s s u p e r c r i t i c a l s t a t e i s ready to enter the e x t r a c t i o n v e s s e l where s o l u t e i s d i s s o l v e d . The C0 2 l e a v i n g the v e s s e l t r a v e l s through two 2^m f r i t s to a flow r e s t r i c t e r where pressure i s reduced to near atmospheric. The C0 2 and o i l s separate i n t o two phases, and flow i n t o the sampling u n i t where the o i l i s c o l l e c t e d . The gaseous C0 2 i s d i r e c t e d through a wet gas-meter and f i n a l l y vented to the atmosphere. A l l of the tubing w i t h i n the system i s 0.1 mm ID s t a i n l e s s s t e e l and the connections were made using 1.6mm s t a i n l e s s s t e e l Swagelok f i t t i n g s . 3.3 EXPERIMENTAL EXTRACTION PROCEDURES 3.3.1 OPERATING CONDITIONS 3.3.1.1 Pressure Four l e v e l s were s e l e c t e d at 15,25,30 and 36 MPa. They range between the c r i t i c a l p r e s s u r e of C0 2 (7.3 MPa) and the maximum o p e r a t i n g pressure of the HPLC. Densit y of C0 2 (and hence i t s s o l v e n t power) changes r a p i d l y w i t h i n t h i s p r e s s u r e range. 3.3.1.2 Temperature Four l e v e l s were s e l e c t e d at 25, 35, 55 and 75 °C. The minimum o p e r a t i n g temperature of the HPLC oven i s 48 25°C and i s below the c r i t i c a l temperature of C0 2, while the other temperatures chosen are above the c r i t i c a l temperature. 3.3.1.3 C0 2 Flowrate I t has been shown that the e q u i l i b r i u m s o l u b i l i t y of o i l i n C0 2 i s independent of the f l o w r a t e of C0 2 p a s s i n g through the e x t r a c t i o n bed ( F a t t o r i , 1 9 8 6 ; S t a h l et a l , l 9 8 0 ) . Hence, the f l o w r a t e of l i q u i d C0 2 p a s s i n g through the pump head was set at 1.0 ml/min f o r a l l experiments i n t h i s work. 3.3.2 VESSEL LOADING PROCEDURE 3.3.2.1 L i q u i d Samples The v e s s e l s were f i r s t p a r t i a l l y f i l l e d with f i n e (0.5.mm) g l a s s beads. The l i q u i d t r i g l y c e r i d e samples were then d e p o s i t e d on the g l a s s beads. T h i s procedure was used f o r two reasons: the l i q u i d on a matrix of g l a s s beads presented a l a r g e r s u r f a c e area a v a i l a b l e f o r mass t r a n s f e r than would an equal mass of l i q u i d i n the empty v e s s e l ; d i s s o l v e d C0 2 c o u l d be r e l e a s e d more e f f e c t i v e l y d u r i n g the decompression stage of the e x t r a c t i o n experiments thereby reducing the r i s k of the l i q u i d foaming out of the v e s s e l . D e t a i l s of the v e s s e l l o a d i n g procedure are o u t l i n e d below: Gla s s beads - enough to approximately 49 h a l f - f i l l each v e s s e l - were pl a c e d i n the autoclave on top of a small p l u g of g l a s s wool. The l i q u i d sample, e q u i v a l e n t to about 7% of the mass of the beads, was then p i p e t t e d on top of the beads and allowed to 'sink i n ' over a p e r i o d of ten minutes. By performing the procedure in a g l a s s t e s t - t u b e , i t was e s t a b l i s h e d that the method r e s u l t s in a r e l a t i v e l y even d i s t r i b u t i o n of o i l throughout the bed. The mass of l i q u i d was determined to the nearest 0.001 g by weighing the p i p e t t e before and a f t e r the l i q u i d o i l t r a n s f e r . Before f i t t i n g the top of the v e s s e l , another plug of g l a s s wool was i n s e r t e d . During e x t r a c t i o n C0 2 flowed through the v e s s e l from bottom to top. 3.3.2.2 S o l i d Samples The v e s s e l was f i r s t f i l l e d with f i n e (0.5 mm) g l a s s beads up to 1/3 of i t s volume. A weighed amount of s o l i d f a t (about 2 to 4 g) was p l a c e d i n the v e s s e l on top of the bead bed. Fine-spun g l a s s wool was p l a c e d at both ends of the v e s s e l f o r the same purpose mentioned be f o r e . The s e a l i n g s u r f a c e s were then cleaned with a chloroform-wetted t i s s u e . A f t e r s e a l i n g , the autoclave was p l a c e d i n the oven at a temperature above the m e l t i n g p o i n t of the f a t f o r 1/2 hour to ensure that a l l the s o l i d f a t melted and flowed i n t o the matrix of the g l a s s beads. A l l of the s o l i d f a t s were e x t r a c t e d at temperatures above t h e i r m e l t i n g p o i n t s . 50 3.3.3 PRE-EXTRACTION CLEANING Before each e x t r a c t i o n was performed, the flow system downstream of the e x t r a c t i o n v e s s e l i n c l u d i n g the f i l t e r s , the r e s t r i c t e r v a l v e and the sampling u n i t , was f l u s h e d with 1:1 chloroform/methanol s o l u t i o n , to cl e a n up any m a t e r i a l s l e f t behind from the pr e v i o u s run. The HPLC was always on i t s standby mode when not i n o p e r a t i o n . The c o o l e r was switched on approximately two hours p r i o r to the s t a r t of an experiment. T y p i c a l l y , i t was set at -20°C. The p r e - e x t r a c t i o n c l e a n i n g r o u t i n e began when the pump head temperature shown on the HPLC d i s p l a y board s t a b i l i z e d at around -5°C. The system was assembled with the e x t r a c t i o n v e s s e l f i l l e d with approximately 1:1 chloroform/methanol s o l u t i o n . The s h u t - o f f v a l v e of the C0 2 c y l i n d e r was opened and the r e s t r i c t e r v a l v e was set to allow the C02. to flow through f r e e l y . The s o l u t i o n i n the v e s s e l was f i r s t f o r c e d through the downstream l i n e and di s c h a r g e d . Thence, the C0 2 was allowed to f l u s h through the system c o n t i n u o u s l y f o r one hour. F i n a l l y , the C0 2 was shut o f f and the emptied v e s s e l was removed. 3.3.4 EQUIPMENT STARTUP F o l l o w i n g the c l e a n i n g procedure,the system was reassembled with another e x t r a c t i o n v e s s e l f i l l e d with the e x t r a c t i n g m a t e r i a l s ( t y p i c a l l y o i l and beads f o r t h i s work). The oven temperature was f i r s t set to the d e s i r e d value (the o p e r a t i n g temperature) and the heater on the 51 r e s t r i c t e r v a l v e set at 75°C. The system was allowed to s t a b i l i z e f o r about 30 minutes a f t e r which time the set values of fl o w r a t e ( t y p i c a l l y 1.0 ml/min) and pressure (40 MPa) were entered. The pump was a c t i v a t e d by p r e s s i n g the 'pre-run' button on the HPLC. Within 5 to 10 seconds, the flow reached the set v a l u e , while the pressure continued to b u i l d up w i t h i n the system. By a d j u s t i n g the r e s t r i c t e r v a l v e , the pressure e v e n t u a l l y reached and s t a b i l i z e d at the d e s i r e d o p e r a t i n g v a l u e . 3.3.5 EXTRACT SAMPLING The sampling procedure normally began immediately a f t e r the system pressure s t a b i l i z e d , i . e . about 5 to 10 minutes a f t e r a c t i v a t i n g the pump. Before the e x t r a c t i o n began,10 to 30 empty 1.8 ml g l a s s v i a l s were weighed. The number of v i a l s used depended on the number of data p o i n t s needed. During the sampling procedure, a v i a l of known weight was f i t t e d to the sampling head and o i l c o l l e c t e d f o r set p e r i o d s of time. The v i a l and i t s contents were removed and re-weighed, and a new pre-weighed v i a l a t t a c h e d to the c o l l e c t o r . The q u a n t i t y of gaseous C0 2 that passed through the v i a l d u r i n g that sampling p e r i o d was determined using the wet t e s t gas meter and the corr e s p o n d i n g mass of C0 2 c a l c u l a t e d from i t s molar volume with r e f e r e n c e to the temperature i n d i c a t e d on the gas meter. The exact sampling time c o u l d be obtained from the 52 ch a r t r e c o r d e r . With t h i s i n f o r m a t i o n , the f l o w r a t e of C0 2 p a s s i n g through the e x t r a c t i o n v e s s e l was c a l c u l a t e d . 3.3.6 EQUIPMENT SHUTDOWN At the completion of an experiment, the pump was turned o f f by s e t t i n g the system back to the standby mode. The C0 2 supply was shut o f f , the heaters were turned o f f , and the system allowed to d e p r e s s u r i z e . Normally, the system pressure would drop to 0.1 MPa w i t h i n 1 to 2 hours, a f t e r which the v e s s e l was removed from the system. 3.3.7 SOLUBILITY DETERMINATION The average o i l c o n c e n t r a t i o n i n C0 2 at any i n t e r v a l d u r i n g the e x t r a c t i o n was determined from the mass of o i l c o l l e c t e d and the mass of C0 2 that passed through d u r i n g that i n t e r v a l . If the accummulative mass of o i l i s p l o t t e d a g a i n s t the corresponding mass of C0 2 at c e r t a i n o p e r a t i n g c o n d i t i o n s , an e x t r a c t i o n curve as shown i n F i g u r e 3-5 can be obt a i n e d . The slope of the i n i t i a l l i n e a r p o r t i o n of the curve r e p r e s e n t s the s a t u r a t e d c o n c e n t r a t i o n of o i l i n C0 2 ( e q u i l i b r i u m s o l u b i l i t y ) at the o p e r a t i n g c o n d i t i o n s . A l l the experiments were performed at l e a s t twice under the same c o n d i t i o n s (pressure,temperature and C0 2 f l o w r a t e ) . 53 Vj^t, J~5-* E x t ™ c t i ° n . curve of cocoa butter fat using carbon d i o x i d e . E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C. 54 3.4 MATERIALS 3.4.1 CARBON DIOXIDE Carbon d i o x i d e was obtained from p r e s s u r i z e d s t e e l c y l i n d e r s s u p p l i e d by Medigas P a c i f i c . Each c y l i n d e r h e l d 30 kg of carbon d i o x i d e which c o u l d be withdrawn i n l i q u i d form through an eductor tube w i t h i n the c y l i n d e r . The amount of carbon d i o x i d e remaining i n the c y l i n d e r at any time was determined by s u b t r a c t i n g the t a r e weight of the c y l i n d e r from i t s a c t u a l weight. 200-300 hours of system run time c o u l d be obtained with each c y l i n d e r . S p e c i f i c a t i o n s of commercial siphon grade carbon d i o x i d e are l i s t e d i n Table 3-2. TABLE 3-2: SPECIFICATIONS OF COMMERCIAL SIPHON GRADE CARBON DIOXIDE. 99% PURE <10 ppm CO <1 ppm H 2S <5 ppm NO2 <0.3 ppm C0C1 2 (phosgene) <5 ppm SO 2 <25 ppm H 20 3.4.2 PURE TRIGLYCERIDES T r i g l y c e r i d e s are g l y c e r o l e s t e r s d e r i v e d from s e v e r a l d i f f e r e n t c a r b o x y l i c a c i d s . Many t r i g l y c e r i d e s are commercially a v a i l a b l e . F i v e were obtained f o r t h i s r e s e a r c h p r o j e c t . They were s e l e c t e d to represent many of the 55 commonly o c c u r r i n g t r i g l y c e r i d e s i n vegetable o i l s (Sonntag,1979). A l l of the t r i g l y c e r i d e s were obtained from Sigma Chemical Co.Ltd. The s p e c i f i c a t i o n s f o r each are given below (Table 3-3). When not i n use, the t r i g l y c e r i d e s were s t o r e d at -20°C. TABLE 3-3: SPECIFICATIONS OF THE TRIGLYCERIDE SAMPLES USED DURING THE EXPERIMENTS. Name Carbon Melt ing Molecular M o l e c u l a r P u r i t y Number Point Formula Weight T r i m y r i s t i n C14:0 56.5 C 4 5 H 8 606 723. 19 99 % T r i p a l m i t i n C16:0 66.4 C 5 1 H 9 8 0 6 807.35 99 % T r i s t e a r i n C18:0 71.5 C5 7 H 1 1O O G 891 .51 99 % T r i o l e i n C18: 1 4.9 C 5 7^ 1 0 <l00 885.47 99 % T r i l i n o l e i n C18:2 12.9 C57H9 806 879.50 99 % 3.4.3 OIL SAMPLES Cocoa b u t t e r and palm k e r n e l o i l were used i n t h i s work. Both o i l s were p r o v i d e d by the Weston Research Centre. When not i n use, they were a l s o kept r e f r i g e r a t e d at -20°C. 3.5 FATTY ACID ANALYSIS Two steps were i n v o l v e d . In the f i r s t s tep the f a t t y a c i d c h a i n s were c l e a v e d from the t r i g l y c e r i d e s and simultaneously converted to t h e i r methyl e s t e r s . In the second step, the methyl e s t e r s were i d e n t i f i e d and q u a n t i f i e d using a gas chromatograph. 56 3.5.1 TRANSESTERIFICATION The method adopted was based on that developed by Shehata et a l . ( 1 9 7 0 ) . The e s t e r i f y i n g reagents are made up of 12.5 ml of 0.5N sodium methoxide i n methanol s o l u t i o n , 8.5 ml of anhydrous d i e t h y l ether and 5.0 ml of petroleum ether, which formed a s i n g l e phase at room temperature. The 0.5N s o l u t i o n of sodium methoxide in methanol was prepared by adding 0.675 g of the anhydrous sodium methoxide powder to 5 ml of abs o l u t e methanol. T h i s reagent was s t o r e d at -20 °C i n a cap f l a s k , and the maximum storage time was two weeks. A l l of the chemicals were obtained from BDH Chemicals. A small amount ( u s u a l l y 1 to 3 drops) of o i l , melted i f necessary, was t r a n s f e r r e d to a 1.8 ml b o r o s i l i c a t e - g l a s s screw capped v i a l equipped with T e f l o n s e p t o r . Approximately 1 ml of the e s t e r i f y i n g reagent was added to the v i a l using a d i s p o s a b l e pasteur p i p e t t e . The v i a l was capped, then r o t a t e d and shaken g e n t l y to ensure complete mixing. The r e a c t i o n mixture was allowed to stand at room temperature fo r two minutes, a f t e r which the mixture was d i l u t e d with about 0.5 ml of petroleum ether, and separated i n t o two phases when a drop of water was in t r o d u c e d . The r e s u l t i n g mixture was shaken f o r about 30 seconds to f a c i l i t a t e the t r a n s f e r of the methyl e s t e r s i n t o the petroleum ether phase. The v i a l and i t s contents were then c e n t r i f u g e d f o r 10 minutes i n order to remove any suspended sodium methoxide. T h e r e a f t e r , the top mm of the petroleum ether 57 l a y e r , which c o n t a i n e d the methyl e s t e r s , was t r a n s f e r r e d by a p i p e t t e to another 1.8 ml v i a l . The s o l u t i o n was ready f o r i n j e c t i o n i n t o a gas chromatograph. 3.5.2 GAS CHROMATOGRAPHIC PROCEDURE 3.5.2.1 Gas Chromatographic C o n d i t i o n s The f a t t y a c i d e s t e r s o l u t i o n was analysed using a Perkin-Elmer (PE) Sigma gas chromatography (GC), connected to a SP4290 I n t e g r a t o r . The GC was equipped with a Hydrogen Flame I o n i z a t i o n Detector (FID) and f i t t e d w ith a 1.83 m x 3.2 mm s t a i n l e s s s t e e l column packed with SP-2330 on 100/120 mesh Chromosorb WAW. The column was obtained pre-packed from Supelco Corp. A l l GC analyses were performed i n accordance with the c o n d i t i o n s shown i n Table 3-4. TABLE 3-4: GAS CHROMATROGRAPHIC PARAMETERS FOR THE FATTY ACID METHYL ESTER ANALYSES. Column SP-2330 on 100/120 mesh Detector Detector gas C a r r i e r gas C a r r i e r gas flow I n i t i a l temperature I n i t i a l time I n i t i a l r a t e F i n a l temperature F i n a l time Sample s i z e Chromosorb WAW Flame i o n i z a t i o n A i r and Hydrogen h e l i urn 20 cm 3/min 160 °C 1.5 s 20°C/s 200 °C 5 s 1 Ml 58 3.5.2.2 Peak I d e n t i f i c a t i o n and Q u a n t i f i c a t i o n Procedure The standard f a t t y a c i d methyl e s t e r s used f o r the r e t e n t i o n time and response f a c t o r i n f o r m a t i o n were obtained from Sigma Chemical Co. L t d . The methyl e s t e r s were i d e n t i f i e d by t h e i r corresponding r e t e n t i o n time they stay i n the column before e l u t e d . Two standard mixtures of pure methyl e s t e r s were used to o b t a i n r e t e n t i o n time i n f o r m a t i o n . The standards were d i l u t e d to a c o n c e n t r a t i o n of I00mg/25ml petroleum e t h e r , before i n j e c t i n g i n t o the GC which was set at the c o n d i t i o n s mentioned above. The r e t e n t i o n times obtained f o r the v a r i o u s methyl e s t e r s are shown i n Table 3-5. Weight response f a c t o r (WRF) was used to c o r r e c t the d i s c r e p a n c y between the peak areas on the chromatogram ( i . e . the response of FID) and the a c t u a l weight percents (Ackmans and S i p o s , l 9 6 4 ) . The values of WRF are device dependent. Hence, c a l i b r a t i o n of WRFs was performed. The i n t e g r a t o r SP4905 used i n t h i s work has a programme to c a l c u l a t e the WRF. Table 3-5 shows the Hydrogen FID weight response f a c t o r s f o r v a r i o u s f a t t y a c i d methyl e s t e r s used i n t h i s r e s e a r c h . The response f a c t o r s were c a l c u l a t e d with r e f e r e n c e to methyl p a l m i t a t e (C16:0). Each value was an average of v a l u e s obtained from ten separate i n j e c t i o n s . 59 TABLE 3-5: RETENTION TIMES AND RESPONSE FACTORS FOR FATTY ACID METHYL ESTERS RELATIVE TO METHYL PALMITATE (C16:0). F a t t y Methyl Ac i d E s t e r Carbon Number Response F a c t o r Retent ion Time (s) Methyl C a p r y l a t e C8:0 0 .872 0. 80 Methyl Caprate CI 0:0 0 .873 1 . 25 Methyl Laurate C1 2:0 0 .910 2. 08 Methyl M y r i s t a t e C14:0 0 .997 3. 10 Methyl P a l m i t a t e C1 6:0 1 .000 4. 10 Methyl S t e a r a t e C18:0 1 .083 5. 35 Methyl Oleate C1 8 : 1 1 .011 5. 75 Methyl L i n o l e a t e C18:2 1 .117 6. 45 Methyl L i n o l e n a t e C18:3 1 .229 7. 45 Methyl A r a c h i d a t e C20:0 1 .092 7. 1 5 Chapter 4 RESULTS AND DISCUSSIONS 4.1 CO 2 EXTRACTION OF PURE TRIGLYCERIDE In t h i s study, f i v e pure simple t r i g l y c e r i d e s were e x t r a c t e d u s i n g s u p e r c r i t i c a l C0 2 at d i f f e r e n t l e v e l s of pressure and temperature. In each case, the s o l u b i l i t y of t r i g l y c e r i d e in C0 2 was determined from the corresponding e x t r a c t i o n curve using the procedure o u t l i n e d i n S e c t i o n 3.3.7. The general s o l u b i l i t y c h a r a c t e r i s t i c s of t r i g l y c e r i d e s as a f u n c t i o n of pressure are shown i n F i g u r e 4-1. The s o l u b i l i t i e s f o r a l l of the t r i g l y c e r i d e s i n c r e a s e with i n c r e a s i n g p r essure at 75 °C. T r i m y r i s t i n (C14:0),the l i g h t e s t compound, has the h i g h e s t s o l u b i l i t y , f o l l o w e d by t r i p a l m i t i n (C16:0), the next l i g h t e s t , and then the C18 compounds. The e f f e c t of f a t t y a c i d u n s a t u r a t i o n on s o l u b i l i t y ( i . e . C18:0, C18:1 and C18:2) i s a l s o shown and w i l l be d i s c u s s e d i n more d e t a i l i n the f o l l o w i n g s e c t i o n s . 4.1.1 TRIGLYCERIDE SOLUBILITY AS A FUNCTION OF TEMPERATURE  AND PRESSURE F i g u r e 4-2 and Fi g u r e 4-3 show the e f f e c t of temperature and pressure on the e q u i l i b r i u m s o l u b i l i t i e s of t r i o l e i n (C18:1) and t r i l i n o l e i n (C18:2) r e s p e c t i v e l y . At constant temperature, the s o l u b i l i t y of both t r i g l y c e r i d e s i n c r e a s e s with i n c r e a s i n g p r essure over the range s t u d i e d . 60 61 F i g u r e 4-1: S o l u b i l i t y of v a r i o u s simple t r i g l y c e r i d e s C0 2 as a f u n c t i o n of p r e s s u r e at 75°C. 62 CM U (M O o ; cn. O CD cm 00 CO CO CO 3 CM 14.0 18.0 22.0 26.0 30.0 PRESSURE (MPa) 34.0 38.0 F i g u r e 4-2: S o l u b i l i t y of t r i o l e i n (C18-1) i n r n =^ f u n c t i o n of p r e s s u r e at four d i f f e r e n t temperatures. 2 3 63 CD CN O o 01 O CD 01 00* t= >-\— I—I <=> _ J CO (—1 GO ZD I O o CO 3 CD CM* 14.0 18.0 22.0 26.0 30.0 PRESSURE (MPa) 34.0 38.0 F i g u r e 4-3: S o l u b i l i t y o f t r i l i n o l e i n (C18:2) i n CO, as a f u n c t i o n of p r e s s u r e at four d i f f e r e n t temperatures. 64 However, t h i s i n c r e a s e i s d i f f e r e n t at d i f f e r e n t temperatures. At the lower temperatures,25°C and 35°C, the s o l u b i l i t y i n c r e a s e s almost l i n e a r l y with p r e s s u r e , and at 55°C and 75°C, the r e l a t i o n s h i p becomes n o n - l i n e a r , whereby the slope of the s o l u b i l i t y curve i s seen to i n c r e a s e with p r e s s u r e . Hence, at low p r e s s u r e , the o i l s o l u b i l i t y i s higher at lower temperatures, whereas at higher p r e s s u r e s , the reverse phenomenon was observed. For example, at 15 MPa, the s o l u b i l i t y of t r i o l e i n (C18:1) (F i g u r e 4-2) i s 2.0 mg/g C0 2 at 25°C and 0.3 mg/g C0 2 at 55°C. As pressure i n c r e a s e s , the s o l u b i l i t y i n c r e a s e s , however, the s o l u b i l i t y / p r e s s u r e g r a d i e n t at 55°C i s l a r g e r than that at 25°C. The s o l u b i l i t y isotherms c r o s s when the pressure reaches 29 MPa. For any pressure g r e a t e r than t h i s v a l u e , the s o l u b i l i t y at higher temperature i s g r e a t e r than that at lower temperature. T h i s c r o s s o v e r of the s o l u b i l i t y curve at d i f f e r e n t temperatures has been r e p o r t e d by other authors ( Z o s e l , 1*978; Peter and Brunner,1978; d e F i l l i p i , 1 9 8 2 ; F r i e d r i c h and Pryde, 1984 and F a t t o r i , 1986). If the s o l u b i l i t y i s expressed as a f u n c t i o n of the d e n s i t y of C0 2 (which i n turn i s a f u n c t i o n of temperature and p r e s s u r e ) , a simpler r e l a t i o n s h i p can be obtained (Figure 4-4, 4-5 and F i g u r e 4-6, 4-7). For any d e n s i t y , the s o l u b i l i t y i n c r e a s e s with temperature;likewise at any temperature the s o l u b i l i t y i n c r e a s e s with i n c r e a s i n g d e n s i t y . These phenomena f o l l o w the two general r u l e s proposed by Brogle (1982), which have been b r i e f l y d i s c u s s e d 65 6 6 CM O <-> C D 00 >-CO CD ZD I O CO CD CM C D C D ©- •o T=25*.C -+ T=35'C -» T=55'C •* T=75T 0.4 0.7 DENSITY, kg m-3 f S n . ^ ' e n ^ ^ ; 1 ^ c ° f t r i l i n o l e i n (C18:2, i n C0 2 as a c y a c o t c ° 2 at v a r i o u s temperatures. 67 C N — C\l 8 5 O — cn \ _ J I—I O c n co >-I— es H to* CD ° 5 CO C N 20.0 30.0 40.0 50.0 60.0 TEMPERATURE ( C ) 70.0 80.0 F i g u r e 4-6: S o l u b i l i t y of t r i o l e i n (C18:1) i n C0 2 as a f u n c t i o n of temperature a t four C0 2 d e n s i t i e s . 68 C M — CM O cn CO CO cn e >-^— i — i i j — i CD CO CD CM 20.0 30.0 40.0 50.0 60.0 TEMPERATURE ( eC ) 70.0 80.0 F i g u r e 4-7: S o l u b i l i t y of t r i l i n o l e i n (C18:2) i n CO, as a f u n c t i o n of temperature a t four C0 2 d e n s i t i e s . 69 in S e c t i o n 2-2. The complex r e l a t i o n s h i p between s o l u b i l i t y and pressure at d i f f e r e n t temperatures can be e x p l a i n e d by r e f e r r i n g to F i g u r e 4-8. In g e n e r a l , a r i s e i n temperature at constant pressure leads to a decrease i n C0 2 d e n s i t y . On the other hand, a r i s e in temperature a l s o leads to an e x p o n e n t i a l i n c r e a s e i n the vapour pressure of the o i l s i n the s u p e r c r i t i c a l phase. An i n c r e a s e of the vapour pressure of the s o l u t e i n the s o l v e n t phase r e s u l t s i n an i n c r e a s e i n the c o n c e n t r a t i o n of the s o l u t e i n the s o l v e n t (Brunner and Peter,1978). Near the c r i t i c a l p o i n t of C0 2, the d e n s i t y changes r a p i d l y with temperature below 80 °C. A s m a l l change i n t h i s r e g ion may l e a d to a l a r g e change in C0 2 d e n s i t y and a commensurate change in o i l s o l u b i l i t y . At higher p r e s s u r e s , however, the same temperature change has a s m a l l e r e f f e c t on the f l u i d d e n s i t y . In t h i s case, the i n c r e a s e i n vapour pressure of the s o l u t e may more than o f f s e t the decrease i n s o l v a t i o n c a p a c i t y of the f l u i d due to i t s decreased d e n s i t y . The net e f f e c t i s an o v e r a l l i n c r e a s e i n s o l u b i l i t y . In g e n e r a l , the e x t r a c t i o n of most t r i g l y c e r i d e s should be c a r r i e d out at a pressure g r e a t e r than 15 MPa. For the t r i g l y c e r i d e s s t u d i e d , the s o l u b i l i t i e s are r e l a t i v e l y low at pressure below 15 MPa, ranging from 0.1 mg/g C0 2 to 2.0 mg/g C0 2. The steep curves between 15 and 36 MPa show that more e f f i c i e n t s e p a r a t i o n can be achieved by decompression. For example, f o r an e x t r a c t i o n of t r i o l e i n , a p r e s s u r e drop from 36 MPa ( s o l u b i l i t y * 10.5 mg/g C0 2) to 15 MPa 70 i—i—i—i—i—i—i—r i—i—i—i—i—i—i—i—r <n CO CO cn >- L • d CM co CM 0.0 4.0 8.0 12.0 16.0 20.0 24.0 28 .0 32 .0 36 .0 40.0 PRESSURE (MPa) F i g u r e 4-8: D e n s i t y of carbon d i o x i d e as a f u n c t i o n of pr e s s u r e at d i f f e r e n t temperatures. The c r i t i c a l p o i n t i s d e s i g n a t e d as CP. (Newritt,1956;Vukalovich and A l t u n i n , 1 9 6 8 ) . 71 ( s o l u b i l i t y = 0.5 mg/g C0 2) at 55 °C w i l l y i e l d about 95 % of the d i s s o l v e d o i l . Separ a t i o n can a l s o be achieved by a temperature change while h o l d i n g pressure c o n s t a n t . However, t h i s method i s more d i f f i c u l t due to the n o n - l i n e a r v a r i a t i o n of s o l u b i l i t y with temperature at d i f f e r e n t p r e s s u r e s . Under c e r t a i n c o n d i t i o n s , a temperature r i s e i s r e q u i r e d but other cases may c a l l f o r the opposite a c t i o n . C o n s i d e r i n g t r i l i n o l e i n ( F i g ure 4-3), with a temperature r i s e from 25 °C ( 6.5 mg/g C0 2 ) to 75 °C ( 1.5 mg/g C0 2 ) at 15 MPa, about 75 % of the d i s s o l v e d o i l can be recovered. I f the same temperature change i s a p p l i e d at 36 MPa, v i r t u a l l y no o i l can be c o l l e c t e d . 4.1.2 TRIGLYCERIDE SOLUBILITY AS A FUNCTION OF MOLECULAR  WEIGHT (OR CARBON CHAIN LENGTH) S o l u b i l i t y curves of three s a t u r a t e d simple t r i g l y c e r i d e s ; t r i m y r i s t i n (C14:0), t r i p a l m i t i n (C16:0) and t r i s t e a r i n (C18:0) as determined at 75 °C and v a r i o u s pressures (15,25,30 and 36 MPa) are presented i n F i g u r e 4-9. At the lower pressure of 15 MPa, the three compounds e x h i b i t e s s e n t i a l l y the same s o l u b i l i t y i n C0 2. As pressure r i s e s , the s o l u b i l i t y of each compound has i n c r e a s e d s u b s t a n t i a l l y . T r i m y r i s t i n (C14:0), l i g h t e s t among the group, e x h i b i t s the g r e a t e s t i n c r e a s e i n s o l u b i l i t y over the range of p r e s s u r e s s t u d i e d . When pressure reached 36 MPa, the s o l u b i l i t y of t r i m y r i s t i n i s 40 mg/g C0 2, compared to 72 C N CO 00 C\l O <=> cn O "3-CN cn > - C D I- C D CO ZD CO C N t — CO 14.0 18.0 22.0 26.0 30.0 PRESSURE ( NPa ) 34.0 38.0 F i g u r e 4-9: S o l u b i l i t y of t r i m y r i s t i n (C14:0), t r i p a l m i t i n (C16:0) and t r i s t e a r i n (C18:0) i n C0 2 as a f u n c t i o n of p r e s s u r e at 75°C. 73 16.5 mg/g C0 2 f o r t r i p a l m i t i n and 7.5 mg/g C0 2 f o r t r i s t e a r i n . I t i s apparent that t r i g l y c e r i d e s o l u b i l i t y v a r i e s i n v e r s e l y with molecular weight. T h i s e f f e c t was expected as these t r i g l y c e r i d e s belong to a homologous s e r i e s . They possess s i m i l a r chemical p r o p e r t i e s such as p o l a r i t y and molecular c o n f i g u r a t i o n , but t h e i r v o l a t i l i t y i n the s o l v e n t phase decreases with i n c r e a s i n g molecular weight (Sonntag,1979; A r n o l d et al.,1963). As shown, the l i g h t e s t t r i g l y c e r i d e has the higher s o l u b i l i t y i n the s u p e r c r i t i c a l f l u i d and t h i s supports the f i n d i n g s of Brunner and Peter (1978) . The r e l a t i v e l y l a r g e v a r i a t i o n i n s o l u b i l i t y between these t r i g l y c e r i d e s i n d i c a t e s that f r a c t i o n a t i o n might be used to f a c i l i t a t e s e p a r a t i o n of a mixture d u r i n g the e x t r a c t i o n ( S t a h l and Q u i r i n , 1 9 8 3 ) . When the s o l u b i l i t i e s of these pure t r i g l y c e r i d e s (C14:0, C16:0 and C18:0) are p l o t t e d as a f u n c t i o n of t h e i r r e s p e c t i v e molecular weights on a s e m i - l o g a r i t h m i c s c a l e ( Figure 4-10), a s t r a i g h t l i n e r e l a t i o n s h i p i s observed when the pressure i s h e l d c o n s t a n t . T h i s i m p l i e s that the same percentage i n c r e a s e i n s o l u b i l i t y r e s u l t s from an equal i n c r e a s e i n molecular weight. F a t t o r i (1986) r e p o r t e d on a s e r i e s of t r i g l y c e r i d e s having a s i n g l e bond: t r i p a l m i t o l e i n (C16:1), t r i o l e i n (C18:1) and t r i - 1 1 - e i c o s e n o i n (C20:1). The s o l u b i l t y of each was determined at 36 MPa and 55 °C, and are shown i n F i g u r e 7 4 C14:0 _. 1 I I I I I I I I I I I I I 700.0 728.0 756.0 784.0 812.0 840.0 868.0 896.0 MOLECULAR WEIGHT (amy) F i g u r e 4-10: The negative logarithm of the s o l u b i l i t y of the three t r i g l y c e r i d e s i n C0 2 as a f u n c t i o n of t h e i r molecular weights. 75 4-11 f o r comparison. The d i f f e r e n c e i n slo p e s f o r the two l i n e s c o u l d be due to the d i f f e r e n c e i n o p e r a t i n g temperatures, 55 °C verses 75 °C, or to the d i f f e r e n c e i n degree of u n s a t u r a t i o n f o r the f a t t y a c i d s w i t h i n the two groups of t r i g l y c e r i d e s , one being s a t u r a t e d and the other having one s i n g l e bond. S i m i l a r s o l u b i l i t y e f f e c t has a l s o been observed by P l a t t n e r et a l . ( l 9 7 7 ) . The authors found that when a homologous s e r i e s of s a t u r a t e d t r i g l y c e r i d e s were e l u t e d through an HPLC column, a l i n e a r r e l a t i o n s h i p e x i s t e d between the number of carbon atoms i n the molecule and the l o g a r i t h m of the volume of s o l v e n t r e q u i r e d f o r the passage of the t r i g l y c e r i d e through the column. I t i s t h e r e f o r e p o s s i b l e to p r e d i c t the s o l u b i l i t y of other s a t u r a t e d t r i g l y c e r i d e s (such as t r i l a u r i n and t r c a p r i n ) from the r e l a t i o n s h i p e s t a b l i s h e d f o r the c o n d i t i o n s s t u d i e d . 4.1.3 EFFECT OF DEGREE OF FATTY ACIDS SATURATION ON THE  SOLUBILITY OF PURE TRIGLYCERIDES F i g u r e 4-12 shows the change i n e q u i l i b r i u m s o l u b i l i t y with pressure at 75°C f o r three simple t r i g l y c e r i d e s namely, t r i s t r e a r i n (C18:0), t r i o l e i n (C18:1) and t r i l i n o l e n (C18:2), having the same number of carbons and d i f f e r e n t degrees of u n s a t u r a t i o n . The a d d i t i o n of one double bond to each of the f a t t y a c i d s r e s u l t e d i n a 40% i n c r e a s e i n the e q u i l i b r i u m s o l u b i l i t y at 30 MPa and a 38 % i n c r e a s e at 36 76 i i i i i i i i i r -© (FattoriJ986)T=55'C :P=36 MPa -+ (This work) T=75'C;P=36 MPa CO CO ©-t— CM CO o ™ CO o o - J 00 C20: 1, C14:0 _L _L _L 700.0 740.0 780.0 820.0 860.0 900.0 MOLECULAR WEIGHT ( amu ) 940.0 980.0 F i g u r e 4-11: The negative logarithm of the s o l u b i l i t y of two groups of t r i g l y c e r i d e s in C0 2 as a f u n c t i o n of t h e i r molecular weights. 77 F i g u r e 4-12: S o l u b i l i t y of t r i s t e a r i n (C18:0) , t r i o l e i n (C18:1) and t r i l i n o l e i n (C18:2) i n C0 2 as a f u n c t i o n of p r e s s u r e s a t 75°C. 78 MPa. However with the second double bond i n c o r p o r a t e d in each of the f a t t y a c i d s , there was no s i g n i f i c a n t d i f f e r e n c e i n the e q u i l i b r i u m s o l u b i l i t i e s f o r p r e s s u r e s from 15 to 36 MPa. The c l o s e n e s s between the s o l u b i l i t i e s of t r i o e l i n and t r i l i n o l e i n shows that s e p a r a t i o n of these t r i g l y c e r i d e s using s u p e r c r i t i c a l f l u i d e x t r a c t i o n technique can h a r d l y be achieved. The l i n e a r r e l a t i o n s h i p developed e a r l i e r between the molecular weight and the negative l o g a r i t h m of the s o l u b i l i t y does not apply i n t h i s case. The t r i g l y c e r i d e s w i l l experience a s i g n i f i c a n t change in both the chemical and p h y s i c a l p r o p e r t i e s (Formo,l979) when a double bond i s added to each of the f a t t y a c i d c h a i n s . The molecular c o n f i g u r a t i o n of the f a t t y a c i d chains i s d i f f e r e n t and there i s an i n c r e a s e i n the p o l a r i t y around the c h a i n (Arnold et al.,1963). I t has been r e p o r t e d that p o l a r compounds such as p h o s p h o l i p i d s are v i r t u a l l y i n s o l u b l e i n s u p e r c r i t i c a l C0 2 ( F r i e d r i c h and L i s t , 1 9 8 2 ; F a t t o r i , 1 9 8 6 ) . However, i n t h i s study, the more p o l a r monosaturated t r i o l e i n was found to be more s o l u b l e i n C0 2 than the l e s s p o l a r t r i s t e a r i n . The e f f e c t of the molecular c o n f i g u r a t i o n change due to the double bonds on the s o l u b i l i t y c o u l d not be i n f e r r e d from the l i m i t e d o b s e r v a t i o n s d e r i v e d from t h i s study. 79 4.2 CO; EXTRACTION OF TRIGLYCERIDE MIXTURES E x t r a c t i o n s were performed on the mixture of t r i m y r i s t i n (C14:0) and t r i o l e i n (C18:1) i n the approximate weight r a t i o of 25:75,50:50 and 75:25. The e x t r a c t i o n curves shown i n F i g u r e s 4-13,4-14 and 4-15, i l l u s t r a t e that the amount of o i l removed per u n i t mass of C0 2 passed through the bed changed c o n t i n u o u s l y d u r i n g the course of the e x t r a c t i o n . I t i s p o s s i b l e that s a t u r a t i o n had been achieved w i t h i n the e x t r a c t o r d u r i n g the i n i t i a l p art of the e x t r a c t i o n s i n c e only a small f r a c t i o n of t r i g l y c e r i d e s had been e x t r a c t e d at t h i s stage. The the continuous change of the e x t r a c t i o n r a t e (or the e q u i l i b r i u m s o l u b i l i t y ) c o u l d p o s s i b l y r e s u l t from the changing composition of the o i l i n the v e s s e l . Since t r i m y r i s t i n (C14:0) and t r i o l e i n (C18:1) are r e l a t i v e l y d i f f e r e n t i n s o l u b i l i t y at t h i s p r e s s u r e and temperature (40 mg/g C0 2 and 10 mg/g C0 2 r e s p e c t i v e l y ) , i t i s very l i k e l y that f r a c t i o n a t i o n was o c c u r r i n g d u r i n g the e x t r a c t i o n . To ev a l u a t e t h i s o b s e r v a t i o n , the e x t r a c t s were c o l l e c t e d f o r f a t t y a c i d a n a l y s i s . The f a t t y a c i d p r o f i l e s from these experiments are shown i n Table 4-1. 80 0.0 8.0 16.0 24.0 32.0 40.0 48.0 56.0 TOTAL C02 PASSED THROUGH BED (g) F i g u r e 4-13: E x t r a c t i o n curve f o r the mixture of t r i m y r i s t i n (C14:0) and t r i o l e i n (C18:1) i n the weight r a t i o of 75:25 The e x t r a c t i o n was performed at 36 MPa and 75°C. 81 TOTAL C02 PASSED THROUGH BED (g) F i g u r e 4-14: E x t r a c t i o n curve f o r the mixture of t r i m y r i s t i n (C14:0) and t r i o l e i n (C18:1) i n the weight r a t i o of 50:50. The e x t r a c t i o n was performed at 36 MPa and 75°C. 82 0.0 8.0 16.0 24.0 32.0 40.0 48.0 56.0 64.0 TOTAL C02 PASSED THROUGH (g) F i g u r e 4-15: E x t r a c t i o n curve f o r the mixture of t r i m y r i s t i n (C14:0) and t r i o l e i n (C18:1) i n the weight r a t i o of 25:75. The e x t r a c t i o n was performed at 36 MPa and 75°C. 83 TABLE 4-1 : FATTY ACID COMPOSITION OF THE C0 2 EXTRACTS OF TRIMYRISTIN (C14:0) AND TRIOLEIN (C18:0) MIXTURES IN THE APPROXIMATE WEIGHT RATIO OF 75:25 (A), 50:50 (B) AND 25:75 (C) . (A) Mass F r a c t i o n F a t t y A c i d C14:0 C18:1 I n i t i a l Mixture 72.7 27.3 1 89.8 10.2 E x t r a c t i o n Number 2 64.7 35.3 (see F i g u r e 4-13) 3 34.5 65.5 4 19.2 80.8 (B) Mass F r a c t i o n F a t t y A c i d C14:0 C18:1 I n i t i a l Mixture 53.5 46.5 1 69.5 30.5 E x t r a c t i o n Number 2 34.0 66.0 (see F i g u r e 4-14) 3 17.2 82.8 4 7.8 92.2 84 (C) Mass F r a c t i o n F a t t y A c i d CI 4:0 C18:1 I n i t i a l Mixture 32.1 67.9 1 46.1 53.9 E x t r a c t i o n Number 2 34.0 65.9 (see F i g u r e 4-15) 3 17.2 82.8 4 4.7 95.3 It can be seen that f r a c t i o n a t i o n d i d occur d u r i n g the e x t r a c t i o n . In a l l cases, the i n i t i a l e x t r a c t s were s i g n i f i c a n t l y r i c h e r i n C14:0,the l i g h t e r t r i g l y c e r i d e , and the l a t e r e x t r a c t s c o n s i s t predominantly of t r i o l e i n (C18:1). F a t t o r i (1986) has r e p o r t e d that the molecular i n t e r a c t i o n s between a homologous s e r i e s of monounsaturated t r i g l y c e r i d e s are very s i m i l a r to those between the molecules of a pure substance, and hence the mixture of these t r i g l y c e r i d e s represented an i d e a l s o l u t i o n . The author was able to p r e d i c t the s o l u b i l i t y of the mixture using a m o d i f i e d v e r s i o n of R o u l t ' s equation : S o l m i x = Z m o l i x s o l i where S o l • i s the s o l u b i l i t y of the mixture i n the mix J s u p e r c r i t i c a l C0 2 phase; s o l ^ i s s o l u b i l i t y of the pure substance i n the s u p e r c r i t i c a l C0 2 phase and mol^ i s mole f r a c t i o n of the pure substance i n the mixture. In t h i s study, however, the mixture of t r i m y r i s r i n (C14:0) and t r i o l e i n (C18:1) d i d not appear to behave l i k e 85 the i d e a l s o l u t i o n mentioned. T h i s r e s u l t was not unexpected s i n c e the molecular i n t e r a c t i o n between the s a t u r a t e d t r i g l y c e r i d e and monounsaturated t r i g l y c e r i d e c o u l d be s i g n i f i c a n t due to t h e i r d i f f e r e n c e s in p o l a r i t y . 4.3 EXTRACTION OF COCOA BUTTER AND PALM KERNEL OIL The C0 2 e x t r a c t i o n of simple t r i g l y c e r i d e s have been d i s c u s s e d so f a r . S i m i l a r e x t r a c t i o n experiments were extended to the more complex mixtures of t r i g l y c e r i d e s such as those found in palm k e r n e l o i l and cocoa b u t t e r . In t h i s s e c t i o n , the f a t t y a c i d composition of each o i l was f i r s t a nalysed, then the r e s u l t s of the C0 2 e x t r a c t i o n experiments are d i s c u s s e d . 4.3.1 FATTY ACID COMPOSITION OF PALM KERNEL OIL AND COCOA  BUTTER From f a t t y a c i d a n a l y s i s experiments, the f a t t y a c i d compositions of cocoa b u t t e r and palm k e r n e l o i l were obtained and are t a b u l a t e d i n Tables 4-2 and 4-3. They compare f a v o r a b l y with values r e p o r t e d by Sonntag (1979). 86 TABLE 4-2: (Sonntag,1979) FATTY ACID COMPOSTION OF COCOA BUTTER Weight Percent F a t t y A c i d Cheok (1986) Sonntag (1979) C1 4:0 0.3 t r . C1 6:0 23.2 24.4 C18:0 35.3 35.4 C18:1 36.8 38. 1 C18:2 2.7 2. 1 C18:3 0.5 t r . C20:0 0.4 t r . TABLE 4-3: FATTY ACID COMPOSITION OF PALM KERNEL OIL (Sonntag,1979). Weight Percent F a t t y A c i d Cheok (1986) Sonntag (1979) C8:0 3.0 3 - 5 C1 0:0 2.9 3 - 7 C1 2 :0 42.7 40 - 52 C1 4:0 15.5 14 -17 C16:0 10.8 7 - 9 C18:0 6.1 1 -3 C18: 1 15.8 13 - 19 C18:2 2.8 0.5 - 2 C20:1 0.3 t r . - 1 87 4.3.2 CO 2 EXTRACTION OF PURE OILS F i g u r e 4-16 demonstrates that the s o l u b i l i t y of cocoa b u t t e r i n C0 2 as a f u n c t i o n of temperature and pressure f o l l o w e d a s i m i l a r p a t t e r n as that r e p o r t e d p r e v i o u s l y f o r pure C18 t r i g l y c e r i d e s . The s o l u b i l i t y ranges from a t r a c e q u a n t i t y of 0.2 mg/g C0 2 (at 15 MPa and 75 °C) to the maximum of 10 mg/g C0 2 (at 36 MPa and 55 °C). T h i s s o l u b i l i t y range i s c l o s e to that of the pure C18 t r i g l y c e r i d e s s t u d i e d and i s not s u r p r i s i n g s i n c e cocoa b u t t e r c o n t a i n s a high p r o p o r t i o n of C18 f a t t y a c i d s . In another set of experiments, the s o l u b i l i t y of palm ke r n e l o i l i n C0 2 was found to be higher than that of cocoa b u t t e r (Figure 4-17). As palm k e r n e l o i l c o n t a i n s a high p r o p o r t i o n of t r i g l y c e r i d e s t h a t c o n t a i n C12:0 and C14:0, the marked i n c r e a s e i n s o l u b i l i t y was to be expected. In order to examine whether f r a c t i o n a t i o n would occur duri n g the e x t r a c t i o n of a mixture of t r i g l y c e r i d e s such as those found i n palm k e r n e l o i l and cocoa b u t t e r , s e l e c t e d e x t r a c t f r a c t i o n s of each o i l were c o l l e c t e d f o r f a t t y a c i d a n a l y s i s . 88 C M O C J a 01 >-I— l — l 00 CO CP L O o C O «t CD C N 14.0 18.0 22.0 26.0 30.0 PRESSURE (MPa) 34.0 38.0 F i g u r e 4-16: S o l u b i l i t y of cocoa b u t t e r i n CO, as a f u n c t i o n of p r e s s u r e at v a r i o u s temperatures. ^u n c t i o n 89 CO LO CO CN O C D O R CM cn cn >-» ~ C D I CM CO O C D CO co CO 14.0 18.0 22.0 26.0 30.0 PRESSURE (MPa) 34.0 38.0 F i g u r e 4-17: S o l u b i l i t y of palm k e r n e l o i l i n CO-t u n c t i o n of p r e s s u r e at v a r i o u s temperatures. as a 90 TABLE 4-4: FATTY ACID COMPOSITION OF C0 2 EXTRACTS OF COCOA BUTTER. EXTRATION CONDITIONS: 36 MPa AND 55 °C. E x t r a c t i o n Number (Refer to F i g u r e 4-18) R e s i d u a l o i l l e f t F a t t y A c i d 1 2 3 i n v e s s e l C14:0 C16:0 C18:0 C1 8: 1 C18:2 C18:3 C20:0 0.27 26. 1 34.4 36.4 2.1 0.27 0.46 0.14 24. 1 35.4 35.8 3.7 0.96 0.95 0.02 23.4 36.0 35. 1 3.3 1.18 1.01 0.01 23.6 35.4 35.2 3.3 1 .23 1.21 TABLE 4-5: FATTY ACID COMPOSITION OF C0 2 EXTRACTS OF PALM KERNEL OIL. EXTRATION CONDITIONS: 36 MPa AND 55 °C. E x t r a c t i o n Number (Refer to F i g u r e 4-19) R e s i d u a l o i l l e f t F a t t y A c i d 1 2 3 i n v e s s e l C8:0 % 3.91 3.27 2.01 1.19 C10:0 % 3.76 3.02 1 .74 1 .06 C1 2: 0 % 45.9 44.0 43.2 41.5 C14:0 % 17.5 17.8 16.2 16.1 C16:0 % 9.22 9.91 11.1 12.1 C18:0 % 4.72 5.89 7.17 7.90 C18: 1 % 14.0 14.8 16.1 17.0 C18:2 % 0.92 1.14 1 .56 1 .99 C18:3 % 0.09 0.10 0.59 0.66 C20:0 % 0.08 0.09 0.41 0.53 Table 4-4 and 4-5 l i s t the f a t y a c i d composition of each e x t r a c t . I t i s n o t i c e d that only small v a r i a t i o n s occur 91 F i g u r e 4-18: E x t r a c t i o n curve f o r pure cocoa b u t t e r . E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C. 92 F i g u r e 4-19: E x t r a c t i o n curve f o r pure palm k e r n e l o i l . E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C. 93 i n the f a t t y a c i d composition of each e x t r a c t d u r i n g each stage of the e x t r a c t i o n p r o c e s s . In both cases, the o i l e x t r a c t s c o l l e c t e d at the i n i t i a l stage were s l i g h t l y r i c h e r i n l i g h t e r f a t t y a c i d s (e.g. C12:0 i n palm k e r n e l o i l and C16:0 i n cocoa b u t t e r ) , while those obtained from the l a t t e r stages show a s l i g h t i n c r e a s e in h e a v i e r f a t t y a c i d s (e.g. C18:1 i n palm k e r n e l o i l and C18:2 i n cocoa b u t t e r ) . The small v a r i a t i o n of the f a t t y a c i d composition c o u l d be due to the type of f a t t y a c i d d i s t r i b u t i o n i n the t r i g l y c e r i d e s of the o i l s . The t r i g l y c e r i d e s present i n cocoa b u t t e r (Table 4-6) having 55 and 75 carbon atoms c o n s t i t u t e a l a r g e p o r t i o n of the o i l , being 61% and 31% by weight r e s p e c t i v e l y . TABLE 4-6 : TRIGLYCERIDE COMPOSITION OF COCOA BUTTER. ( H i l d i t c h and W i l l i a m , 1964) T r i g l y c e r i d e Carbon Weight Number Percent P a l m i t o - d i s t e a r i n 55 2 O l e o - d i p a l m i t i n 53 6 O l e o - p a l m i t o - s t e a r i n 55 52 O l e o - d i s t e a r i n 57 19 P a l m i t o - d i o l e i n 55 9 S t e a r o - d i o l e i n 57 1 2 These t r i g l y c e r i d e s , though d i f f e r e n t i n molecular s t r u c t u r e , have very s i m i l a r molecular weights. I t i s very l i k e l y t h a t they e x h i b i t s i m i l a r s o l u b i l i t i e s i n C0 2. As a r e s u l t , the m a j o r i t y of the t r i g l y c e r i d e s would be e x t r a c t e d 94 at the same r a t e and consequently the composition of the e x t r a c t remains constant f o r much of the e x t r a c t i o n . However, the behaviour e x h i b i t e d by palm ke r n e l o i l i s more d i f f i c u l t to r a t i o n a l i z e . The range of molecular weights of the t r i g l y c e r i d e s present i n t h i s o i l (Table 4-7) i s wider with carbon numbers ranging from 37 to 49. TABLE 4-7: TRIGLYCERIDE COMPOSITION OF PALM KERNEL OIL. ( H i l d i t c h and William,1964). T r i g l y c e r i d e Carbon Weight Number Percent C a p r o - d i l a u r i n 37 10 Capro-lauro-myr i s t in 39 5 T r i l a u r i n 39 1 C a p r o - l a u r o - p a l m i t i n 45 3 D i l a u r o - m y r i s t i n 41 27 D i l a u r o - p a l m i t i n 43 8 Lauro-myristo-pamitin 45 7 C a p r o - l a u r o - o l e i n 43 12 D i l a u r o - o l e i n 45 6 L a u r o - m y r i s t o - o l e i n 47 1 1 L a u r o - p a l m i t o - o l e i n 49 10 Quite unexpectedly, in t h i s study, the e x t r a c t i o n r a t e was found to be r e l a t i v e l y constant f o r much of the e x t r a c t i o n process ( F i g u r e 4-19). T h i s o b s e r v a t i o n c o u l d be the r e s u l t of an i n t e r a c t i o n between the three d i f f e r e n t f a t t y a c i d m o i t i e s w i t h i n each of the mixed t r i g l y c e r i d e s a f f e c t i n g the s o l u b i l i t y and there may a l s o be an i n t e r a c t i o n between the d i f f e r e n t mixed t r i g l y c e r i d e s i n the palm k e r n e l o i l r e s u l t i n g in changes i n the s o l u b i l i t i e s of the o i l s i n C0 2. Research on the e f f e c t of carbon c h a i n 95 l e n g t h and degree of f a t t y a c i d s a t u r a t i o n i n mixed t r i g l y c e r i d e s on o i l s o l u b i l i t y would help to separate the two p o s s i b l e e x p l a n a t i o n s . 4.3.3 CO, EXTRACTION OF OIL MIXTURES Mixtures of cocoa b u t t e r and palm k e r n e l o i l (weight r a t i o 75:25,50:50 and 25:75) were e x t r a c t e d with C0 2 at 55°C and 36 MPa. L i k e the r e s u l t s obtained with mixture of pure t r i g l y c e r i d e s , the e x t r a c t i o n curves ( F i g u r e s 4-20,4-21 and 4-22) i n d i c a t e that the amount of o i l removed per u n i t mass of C0 2 d i m i n i s h e s g r a d u a l l y with time, i n c o n t r a s t to the l i n e a r p a t t e r n obtained with pure o i l s . Again, the r e s u l t s i n d i c a t e that the s o l u b i l i t y of the mixture i s changing with time over the course of the e x t r a c t i o n . The e x t r a c t s from these experiments were a l s o c o l l e c t e d at d i f f e r e n t stages of the e x t r a c t i o n f o r a n a l y s i s of t h e i r f a t t y a c i d composition. 96 TOTAL C02 PASSED THROUGH BED (g) F i g u r e 4-20: E x t r a c t i o n curve f o r the mixture of cocoa b u t t e r and palm k e r n e l o i l i n the weight r a t i o of 75*25 E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C. 97 TOTAL C02 PASSED THROUGH BED (g) F i g u r e 4-21: E x t r a c t i o n curve f o r the mixture of cocoa b u t t e r and palm k e r n e l o i l i n the weight r a t i o of 50:50. E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C. F i g u r e 4-22: E x t r a c t i o n curve f o r the mixture of cocoa b u t t e r and palm k e r n e l o i l i n the weight r a t i o of 25:75. E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C. 99 TABLE 4-8 FATTY ACID COMPOSITION OF THE C0 2 EXTRACTS OF THE MIXTURE OF COCOA BUTTER AND PALM KERNEL OIL IN THE APPROXIMATE WEIGHT RATIO OF 75:25 (A),50:50 (B) AND 25:75 (C). EXTRACTION CONDITIONS: 36 MPa AND 55 °C. (A) E x t r a c t i o n Number (see F i g u r e 4-20) F a t t y I n i t i a l Weight Percent A c i d Mixture 1 2 3 4 C8:0 0.62 2.38 1 .53 0.29 0.18 C10:0 0.67 1 .98 1 .43 0.29 0.20 C12:0 12.2 25.2 20.0 5.55 3.92 CI 4:0 5.96 7.88 7.26 2.46 2.09 C16:0 20.3 17.7 19.8 24.5 22.8 C18:0 24.2 17.9 20.5 29.2 33. 1 C18: 1 30.5 23.2 25.6 33.4 33.7 C18:2 4.24 3.01 3.21 3.33 3.14 C18:3 0.31 0.22 0.21 0.25 0.25 C20:0 1.01 0.51 0.58 0.72 0.77 (B) E x t r a c t i o n Number (see F i g u r e 4-21) F a t t y I n i t i a l Weight Percent Ac i d Mixture 1 2 3 4 C8:0 1.71 3.74 2.46 0.51 0.07 C10:0 1 .55 3.14 2.42 0.60 0.09 C12:0 22.4 39.4 35.6 11.6 2.58 CI 4:0 8.28 11.9 12.4 6. 28 2.01 C16:0 17.8 12.0 13.7 22.3 23.8 C18:0 19.3 10.3 11.4 21.6 31.7 C18:1 25.4 16.5 19.2 31.9 35.2 C18:2 3.37 2.47 2.61 4. 34 3.88 C18:3 0.14 0.15 0.01 0.17 0.03 C20:0 0.67 0.40 0.24 0.55 0.77 100 (c) E x t r a c t i o n Number (see F i g u r e 4-22) Weight Percent F a t t y Ac i d I n i t i a l Mixture 2 3 4 C8:0 C1 0:0 C1 2:0 CI 4:0 CI 6:0 CI 8:0 C18:1 C18:2 C18:3 C20:0 2.43 2.24 32.6 12.2 13.1 11.1 21.6 3.31 0.24 0.46 4.07 3.45 44.8 14.2 9.82 6.52 14.5 2.33 0.16 0.23 3.44 3.12 42.8 14.5 10.6 6.80 15.8 2.54 0.18 0.24 I. 17 1 .33 24.0 I I . 3 17.1 13.7 26.5 4.21 0.19 0.49 0.57 0.66 12.7 5.67 17.3 27.3 31.2 3.22 0.27 1 .09 From the i n f o r m a t i o n d i s p l a y e d i n Table 4-8, i t i s evident that at the e a r l y e x t r a c t i o n stages the composition contained a high percentage of the l i g h t e r f a t t y a c i d s , such as C10:0, C12:0 and C14:0 which are the major c o n s t i t u e n t s of palm ke r n e l o i l . On the other hand, a l l the l a t e r e x t r a c t s seem to be e n r i c h e d with heavier f a t t y a c i d s (carbon number above 14) found i n cocoa b u t t e r , with the exception of C18:3 and C20:0. T h i s phenomenon i m p l i e s that some f r a c t i o n a t i o n has taken p l a c e d u r i n g e x t r a c t i o n of the cocoa b u t t e r and palm k e r n e l o i l mixtures which are composed of compounds with widely d i f f e r e n t s o l u b i l i t i e s . 4.4 EFFECT OF WATER CONTENT ON OIL SOLUBILITIES Biotechnology i s p r o v i d i n g o p p o r t u n i t i e s f o r the mass b i o l o g i c a l p r o d u c t i o n of many chemical compounds. Since most of these systems operate i n an aqueous medium, i t would be d e s i r a b l e to be able to e x t r a c t the products d i r e c t l y from 101 the l i q u i d medium. One of the o b j e c t i v e s of t h i s r esearch t h e r e f o r e was to examine the e f f e c t of sample water content on t r i g l y c e r i d e s o l u b i l i t y . Cocoa butter/water mixtures i n d i f f e r e n t p r o p o r t i o n s were e x t r a c t e d with C0 2 at 36 MPa, 55 °C and a C0 2 f l o w r a t e of 1.0 ml/min. In the f i r s t s e r i e s of e x t r a c t i o n s using a cocoa/water (90:10) mixture, the s o l u b i l i t y data obtained were q u i t e i n c o n s i s t e n t and not r e p r o d u c i b l e . Upon passin g through the mixture, C0 2 d i d not appear to be i n c o n t a c t with o i l d r o p l e t s long enough to a t t a i n a s t a t e of e q u i l i b r i u m . An a l t e r n a t i v e method was attempted. Two e x t r a c t i n g v e s s e l s were connected i n s e r i e s and used to e x t r a c t the o i l / w a t e r mixture. T h i s arrangement in e f f e c t lengthens the e x t r a c t i o n bed, and p r o v i d e s a longer o i l / C 0 2 contact time and a more e f f e c t i v e mass t r a n s f e r r a t e . The s o l u b i l i t y data generated f o r cocoa butter/water mixtures with up to 50 % water content i s shown in Table 4-9. 1 02 TABLE 4-9 : SOLUBILITY OF COCOA BUTTER IN C0 2 AT VARIOUS WATER CONTENTS. (EXTRACTION CONDITIONS : 36 MPa, 55 °C and 1.0 ml C0 2/min.) The water content from 0 to 50 % by weight had no a p p r e c i a b l e e f f e c t on the e q u i l i b r i u m s o l u b i l i t i e s of cocoa b u t t e r in s u p e r c r i t i c a l C0 2. The r e s u l t s from experiments using samples with water content higher than t h i s c o n c e n t r a t i o n were not r e p r o d u c i b l e ( i . e . a s a t u r a t i o n curve c o u l d not be obtained) probably due to the poor mass t r a n f e r r a t e between the o i l and C0 2. E x t r a c t i o n of sample at t h i s very high water content i s impossible with t h i s equipment without f u r t h e r m o d i f i c a t i o n s . An e x t r a c t i o n curve f o r 50:50 mass f r a c t i o n cocoa butter/water mixture at 36 MPa and 55 °C i s shown i n F i g u r e 4-23. E x t r a c t i o n was c a r r i e d out f o r 4.5 hours and about 92% of the o i l was recovered from the o i l / w a t e r mixture at the end of the p r o c e s s . As long as the o i l / C 0 2 c o n t a c t time i s s u f f i c i e n t , a water content up to 50 % had l i t t l e e f f e c t on the o i l Water Content (Weight %) S o l u b i l i t y (mg o i l / g C0 2) 0 1 0 20 30 40 50 10.3 10.0 9.78 10.3 10.1 9.91 103 1 I I I I I I I 1 I I . I I 7 OlO 36.0 72.0 108.0 144.0 180.0 216.0 252.0 TOTAL C02 PASSED THROUFGH BED (g) F i g u r e 4-23: E x t r a c t i o n curve for a mixture of 50:50 (wt %) of cocoa butter/water mixture. E x t r a c t i o n c o n d i t i o n s : 36 MPa and 55°C. 1 04 e x t r a c t a b i l i t y and the e q u i l i b r i u m s o l u b i l i t y of o i l in C0 2. The mass t r a n s f e r r a t e can be improved by using longer e x t r a c t i o n bed or smaller C0 2 f l o w r a t e . The optimum moisture content of the sample sub j e c t e d to e x t r a c t i o n should be determined from a f e a s i b i l i t y study of the d r y i n g process of the sample p r i o r to e x t r a c t i o n and the design of the e x t r a c t i o n equipment. Chapter 5 CONCLUSIONS The f i n d i n g s of t h i s study can be summarized as f o l l o w s : 1. The s o l u b i l i t y of t r i g l y c e r i d e s i n s u p e r c r i t i c a l C0 2 i s a d i r e c t f u n c t i o n of the C0 2 d e n s i t y and the e x t r a c t i o n temperature. R e s u l t s confirmed previous s t u d i e s that s o l u b i l i t y d i d inc r e a s e with i n c r e a s i n g temperature at constant d e n s i t y , and with i n c r e a s i n g C0 2 d e n s i t y at constant temperature. The e x t r a c t i o n of most t r i g l y c e r i d e s should be c a r r i e d out at pressure g r e a t e r than 15 MPa. 2. There e x i s t s a l i n e a r r e l a t i o n s h i p between the molecular weights of a group of s a t u r a t e d t r i g l y c e r i d e s and the lo g a r i t h m of t h e i r s o l u b i l i t i e s i n C0 2. The s o l u b i l i t y of any s a t u r a t e d t r i g l y c e r i d e i n the. same homologous s e r i e s may t h e r e f o r e be p r e d i c t e d from t h i s r e l a t i o n . 3. For the C18 t r i g l y c e r i d e s s t u d i e d , the presence of one double bond on each of the f a t t y a c i d chains caused a s i g n i f i c a n t i n c r e a s e i n the s o l u b i l i t y of the t r i g l y c e r i d e in C0 2. However, the a d d i t i o n of a second double bond to each of the f a t t y a c i d chains of the t r i g l y c e r i d e showed no f u r t h e r i n c r e a s e i n s o l u b i l i t y . 4. In the e x t r a c t i o n of the mixture of t r i m y r i s t i n and t r i o l e i n , s i g n i f i c a n t f r a c t i o n a t i o n o c c u r r e d d u r i n g the process; the i n i t i a l e x t r a c t s were mainly the l i g h t e r t r i m y r i s t i n while the e x t r a c t s at the l a t e r stage 105 106 c o n s i s t predominantly of the heavier t r i o l e i n . The mixture d i d not behave l i k e an i d e a l s o l u t i o n . I t i s b e l i e v e d that the i n t e r a c t i o n s between the s a t u r a t e d and unsaturated t r i g l y c e r i d e s are s i g n i f i c a n t . The e q u i l i b r i u m s o l u b i l i t y of palm ker n e l o i l in C0 2 was found to be higher than that of cocoa b u t t e r , as the palm k e r n e l o i l c o n s i s t s mainly of the l i g h t e r C12 and C14 t r i g l y c e r i d e s while cocoa b u t t e r i s r i c h i n the hea v i e r C18 compounds. No s i g n i f i c a n t change i n f a t t y a c i d composition of the e x t r a c t s was observed at v a r i o u s stages of e x t r a c t i o n when each o i l was e x t r a c t e d s e p a r a t e l y with SC-C0 2. However, when the mixture of these o i l s was e x t r a c t e d , s i g i f i c a n t f r a c t i o n a t i o n was observed. The water content of a sample from 0 to 50 % by weight had l i t t l e e f f e c t on the o i l e x t r a c t a b i 1 i t y and the e q u i l i b r i u m o i l s o l u b i l i t y i n the *SC-C02. Chapter 6 RECOMMENDATIONS The f o l l o w i n g recommendations are suggested f o r f u t u r e r e s e a r c h : 1. The e f f e c t of double bonds on the f a t t y a c i d c h a i n s on the e x t r a c t i o n process should be s t u d i e d f u r t h e r using other t r i g l y c e r i d e s with d i f f e r e n t carbon numbers and at other temperatures. 2. A study on the s u p e r c r i t i c a l C0 2 e x t r a c t i o n of mixed t r i g l y c e r i d e s w i l l p r o v i d e u s e f u l i n f o r m a t i o n on the i n t e r a c t i o n between the f a t t y a c i d c hains (with d i f f e r e n t carbon c h a i n l e n g t h and degree of un s a t u r a t i o n ) of the t r i g l y c e r i d e i n the s u p e r c r i t i c a l phase. 3. 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Swagelok f i t t i n g s , Nupro va l v e Hewlett-Packard Inc. HPLC, 2ym f r i t s Hyseco F l u i d Systems Ltd, Parker MV-200S val v e Malkin and Pinton I n d u s t r i a l S u p p l i e s s t a i n l e s s s t e e l b o l t s Medigas P a c i f i c L t d . carbon d i o x i d e , a i r , helium and hydrogen Rheodyne, Inc. Rheodyne v a l v e Sigma Chemicals Ltd, t r i g l y c e r i d e s , GC standards Spectrex L i m i t e d 0.5 mm g l a s s beads 1 1 2 Supelco, Inc. T e c h n i c a l Marketing Assoc. 1 1 3 GC columns, Hamilton s y r i n g e s , v i a l s SP4290 I n t e g r a t o r 

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