Supercritical Fluid Science and Technology

Chapter 27

Supercritical Fluid Carbon Dioxide Extraction in the Synthesis of Trieicosapentaenoylglycerol from Fish Oil

Downloaded by UNIV OF ARIZONA on November 12, 2012 | http://pubs.acs.org Publication Date: August 29, 1989 | doi: 10.1021/bk-1989-0406.ch027

W. B. Nilsson, V. F. Stout, E. J. Gauglitz, Jr., F. M. Teeny, and J. K. Hudson U.S. Department of Commerce, National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Northwest Fishery Center, Utilization Research Division, 2725 Montlake Boulevard East, Seattle, WA 98112 Supercritical fluid carbon dioxide (SC-CO ) fractionation of fish oil ethyl esters (EE) was employed to prepare EE of two omega-3 fatty acids, all cis-5,8,11,14, 17-eicosapentaenoic acid (EPA) and all cis-4,7,10,13, 16,19-docosahexaenoic acid (DHA) in 90% purity and to separate the synthetic triacylglycerols (TG), trieicosapentaenoylglycerol (tri-EPA), and tridocosahexaenoylglycerol (tri-DHA) in≥92%purity from other reaction mixture components. In the synthesis, glycerine reacted with EE and sodium glyceroxide catalyst to form TG. The desired TG (50-60 wt%) was accompanied by fatty acids (2-5%), mono- and diglycerides (10-15%), unidentified byproducts (5-10%), and unreacted EE (15-25%). The TG products were isolated using supercritical fluid fractionation employing pressure programming at 60 °C with pure CO and CO plus 4 wt% ethanol. Analyses of the isolated EE and TG indicated that reactant EE are incorporated into the TG with little or no selectivity regardless of chain length or degree of unsaturation. 0mega-3 (w3) fatty acids have been the subject of many recent reports due to their reputed medicinal properties in the treatment of cardiovascular and autoimmune/inflammatory diseases (1-4). Most of these studies have suggested that consumption of fish oils, the main source of w3 fatty acids in the human diet, has significant implications for long-term health and the prevention of numerous diseases. However, before w3-contalning substances can be recommended in nutritional supplements or as pharmaceutical agents, the physiological properties of individualw3fatty acids must be defined. Twow3fatty acids that have drawn widespread attention are all cis-5,8,ll,14,17-eicosapentaenoic acid (EPA or 20:5w3) and all cis-4,7,10,13 16,19-docoeahexaenoic acid (DHA or 22:6w3). The designation 20:5w3 connotes a straight-chain, 20-carbon fatty acid with 5 methylene-interrupted double bonds beginning at the third carbon counting from the terminal methyl group. This chapter not subject to U.S. copyright Published 1989 American Chemical Society 2

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In Supercritical Fluid Science and Technology; Johnston, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


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NILSSON E T A L .

Synthesis ofTrieicosapentaenoylglycerolfivmFish Oil

The o v e r a l l goal of the f i s h o i l research program i n our laboratory, which dates back to the mid-1950's, has been to develop various methodologies to produce materials f o r e l u c i d a t i n g the n u t r i t i o n a l and medicinal properties of ui3-containing substances not only i n humans, but also i n t e r r e s t r i a l (5) and aquatic animals (6). The d i v e r s i t y of f a t t y acids present i n f i s h o i l s , and the inherent i n s t a b i l i t y of highly unsaturated f i s h o i l s have hampered these e f f o r t s . In some early work, molecular d i s t i l l a t i o n combined with forced urea complexing of the hydrolyzed native t r i a c y l g l y c e r o l s , here referred to as t r i g l y c e r i d e s , effected i s o l a t i o n of nearly 90% DHA (7). The fate of other ui3 f a t t y acids was not determined, but the method may well be less e f f e c t i v e f o r i s o l a t i n g shorter chain ui3 f a t t y acids. At best, the process required two molecular d i s t i l l a tions at elevated temperatures (up to 180 °C) and one multi-stage urea f r a c t i o n a t i o n , r e s u l t i n g i n some degradation of the highly polyunsaturated products of i n t e r e s t . Recently, s u p e r c r i t i c a l f l u i d carbon dioxide extraction (SFE) has shown promise i n applications requiring the separation of complex mixtures of high b o i l i n g unstable substances at s i g n i f i c a n t l y lower temperatures than used i n processes such as f r a c t i o n a l d i s t i l l a t i o n . One of many examples i s the p u r i f i c a t i o n of the monomer diacetone acrylamide (8). S u p e r c r i t i c a l f l u i d CO2 (SC-CO2) has the further advantage of avoiding the use of flammable organic solvents. Carbon dioxide i s r e a d i l y a v a i l a b l e , and does not leave toxic residues, making its use a t t r a c t i v e i n food and pharmaceutical a p p l i c a t i o n s . Augmenting the work of Eisenbach (9), two reports from t h i s laboratory have shown that EPA and DHA i n p u r i t i e s above 90% can be obtained from f i s h o i l ethyl esters using SFE at temperatures as low as 80 °C (10-11). In addition, a recent report describes a countercurrent continuous process f o r producing large quantities of both f a t t y acid esters of s i m i l a r p u r i t y (12). Up to now, most p h y s i o l o g i c a l studies on i n d i v i d u a l ui3 f a t t y acids have used methyl or e t h y l esters d i r e c t l y . Concerns have been raised over the s u i t a b i l i t y of this form of l i p i d f o r several reasons. Hydrolysis leads to highly toxic methanol or less toxic ethanol. Beyond the question of the l i b e r a t e d alcohol, the monoesters might themselves be t o x i c because of d i f f e r e n t i n t r i n s i c properties vis-à-vis the t r i g l y c e r i d e s . The monoester, because i t i s not the usual form or i s much l e s s viscous, may e i t h e r be r e s i s t a n t to absorption or may i n t e r f e r e with other processes i n the gastrointest i n a l t r a c t . In f a c t , a recent report claims that when EPA-containing materials were fed to f a s t i n g human subjects, free f a t t y acids, arginine s a l t s , and t r i g l y c e r i d e s were metabolized more r a p i d l y and completely than ethyl esters (13). The obvious a l t e r n a t i v e to monoesters, excluding the i r r i t a t i n g free f a t t y a c i d , i s the t r i g l y c e r i d e composed of a s i n g l e f a t t y acid, f o r example, EPA. Such t r i g l y c e r i d e s have'reportedly been Isolated i n minor amounts from some f i s h (14), but synthesis from the pure ester or f a t t y acid i s the only p r a c t i c a l source. Although saturated f a t t y acids or t h e i r esters react r e a d i l y to form t r i g l y c e r i d e s , highly unsaturated f a t t y acids are much more r e s i s t a n t . The method of Lehman and Gauglitz (15) requires free f a t t y acids and elevated temperatures. From our SFE work, substantial quantities of p u r i f i e d (ca. 90%) esters of EPA and DHA were a v a i l a b l e . We wished to


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TECHNOLOGY

incorporate them d i r e c t l y i n t o t r i g l y c e r i d e s without the extra step of hydrolysis of the esters to the acids, e s p e c i a l l y since the acids are even more readily autoxidized than the highly reactive u>3 esters (16). The generation of ester reactants and the p u r i f i c a t i o n of the synthetic t r i g l y c e r i d e s are the main subjects of t h i s paper.


Experimental SFE Apparatus. Concentrates of the ethyl esters of EPA and DHA were obtained by f r a c t i o n a t i o n of menhaden o i l esters which previously had been urea fractionated (17). The apparatus i s shown schematically i n Figure 1. Unless otherwise noted, I d e n t i f i c a t i o n of vendors has been made elsewhere (10). The heart of the system consists of a pair of 10,000 p s i double-ended, diaphragm-type compressors i n s t a l l e d i n p a r a l l e l . Process pressure was controlled using a back pressure regulator. Compressed CO2 was pumped through 1/4" OD high pressure 304 s t a i n l e s s s t e e l (SS) tubing into a one-foot 1/4" ID SS pipe which sometimes was used as a preheater. The extraction vessel was a s i x foot long, 3.5" OD χ 1.25" ID 304 SS pipe (Temco, Inc., Tulsa, OK). (The use of trade-names i n t h i s p u b l i c a t i o n does not imply endorse ment by the National Marine F i s h e r i e s Service.) The column was packed with 0.16" Propak which i s a protruded 316 SS d i s t i l l a t i o n packing material ( S c i e n t i f i c Development Co., State College, PA). The ester feedstock was loaded into the column through a port located 1 foot from the bottom. In addition to an i n t e r n a l thermocouple probe at the top of the column, several thermocouple ports were d r i l l e d at 7" i n t e r v a l s along the side of the column. The thermocouple probes, the t i p s of which l i e at the center of the column, provided simul taneous measurement and control of the process temperature at several positions along the length of the column. Each probe was wired to an on/off temperature c o n t r o l l e r (Syscon Int. Inc., South Bend, IN) which controlled the process temperature at the probe position by supplying power to a s i l i c o n rubber heater wrapped around the column. As shown i n Figure 1, there were 7 i n d i v i d u a l l y controlled heaters. Thus a thermal gradient could be introduced along the length of the column. In a l l ester f r a c t i o n a t i o n s , a gradient was used i n which the temperature increased from the bottom to the top of the column. SC-CO? Extraction Methodology. In a t y p i c a l batch cycle, the column was loaded with ca. 100 g of esters. The system was then purged with CO2 and the column heaters allowed to e s t a b l i s h the desired tempera ture gradient. Once the column was pressurized, CO2 passing through the esters at the bottom of the column dissolved a portion of the charge into the f l u i d phase. The ester-laden f l u i d was forced upwards through the increasing temperature gradient. Process temper atures and pressures were selected such that ester s o l u b i l i t y decreased with increasing temperature, that i s , underwent retrograde condensation. Therefore, i n each temperature "zone" of the gradient, l o c a l heating caused p r e f e r e n t i a l condensation of less soluble ester components thereby enriching the f l u i d phase with respect to more soluble components present at a given point i n the f r a c t i o n a t i o n . Ester-laden f l u i d emerging from the top of the column was expanded through a heated expansion valve to i s o l a t e the extract.


Synthesis of Trieicosajmdaenoylgtycerolfrom Fish Oil


NDLSSON ET AL.


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TECHNOLOGY

T r i g l y c e r i d e Synthesis. The synthetic procedure was a modification of that discussed previously (18). T r i g l y c e r i d e s were synthesized from glycerine and ethyl esters v i a the glyceroxide formed i n s i t u from c a t a l y t i c amounts (4 16:4ω1 18:4tu3 20:4u>6 20:5u>3 21:5u>3 22:6u>3

90% EPA

2.0 2.6 90.1 1.0 3.3

PUFA 5.0 5.6 7.5 1.5 47.7 1.4 22.5


90% DHA

2.7 1.5 89.5

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NILSSONETAL.

Synthesis of Trieicosapentaerwyîgîycerolfrom Fish Oil


SC-CO? P u r i f i c a t i o n of Reaction Mixture. For p u r i f i c a t i o n of smaller batches (20 g) of the crude reaction mixture, a scaled down version of the large v e s s e l , a 6-foot length of 1.0" OD χ 11/16" ID SS pipe, was used. In addition, several p u r i f i c a t i o n s were performed using 4 + 0.5 wt% ethanol (EtOH) as a cosolvent. The EtOH was injected at a constant volumetric flow rate into the preheater where indicated i n Figure 1 using a Shimadzu LC-6A HPLC pump. A n a l y t i c a l Procédures» Glyceride f r a c t i o n s from p u r i f i c a t i o n of reaction mixtures were analyzed by thin layer s i l i c a gel chromatography (TLC) and HPLC. In addition, several preparative TLC's were performed on the i s o l a t e d t r i g l y c e r i d e s . The p u r i f i e d t r i g l y c e r i d e s were e s t e r i f l e d d i r e c t l y to methyl esters by a modification of the method of Morrison and Smith (19), and the f a t t y acid p r o f i l e determined by gas chromatography using conventional techniques described elsewhere (10). Results and Discussion I s o l a t i o n of Ester Concentrates. A d e t a i l e d discussion of the method used to obtain concentrates of EPA and DHA has been presented i n two previous reports (10-11). Those data were c o l l e c t e d from 20-g charges i n the smaller 6' long, 11/16" ID column. Figure 2 shows f r a c t i o n a t i o n curves f o r selected components of the feedstock mixture with a 100-g charge i n the larger 6' long, 1.25" ID column. The maximum temperature of the gradient was 80 °C, the SC-CO2 flow rate was ca. 50 standard l i t e r s / m i n , and the run was pressure programmed as indicated by the step-curve scaled on the right-hand ordinate. Of the EPA present i n the feed, ca. 65% was recovered i n the 90% concentrate; s i m i l a r l y ca. 75% of the DHA was recovered i n the 90% DHA concentrate. I s o l a t i o n of trl-EPA Using SC-CO?. EPA ester of 90% p u r i t y obtained i n the process described above was used as a reactant to synthesize t r i g l y c e r i d e s . We w i l l r e f e r to the product as tri-EPA i n subsequent discussion although i t should be emphasized that t r i g l y c e r i d e s synthesized from 90% pure EPA are not expected to contain t r i e i c o s a pentaenoylglycerol i n 90% p u r i t y . In f a c t , i f a l l components present i n the o r i g i n a l ester s t a r t i n g material have equal p r o b a b i l i t y of incorporation i n t o a t r i g l y c e r i d e , random s t a t i s t i c s predict that the f i n a l product would contain (0.9)3 100 or 73% tri-EPA. The remainder would mainly be comprised of mixed t r i g l y c e r i d e s containing two EPA moieties. In a l l syntheses of tri-EPA, the crude product mixture contained mono- and d i g l y c e r i d e intermediates and a s i g n i f i c a n t quantity of unreacted esters. In addition, f r e e f a t t y acids and other uncharacterlzed byproducts were present. This finding i s e s s e n t i a l l y i n l i n e with that observed by Lehman and Gauglitz (15), who p u r i f i e d t h e i r crude reaction mixture by molecular d i s t i l l a t i o n at temperatures above 250 °C. F r a c t i o n a t i o n of highly unsaturated glyceride mixtures using s u p e r c r i t i c a l f l u i d CO2 i s an a t t r a c t i v e a l t e r n a t i v e to molecu l a r d i s t i l l a t i o n because of the p o s s i b i l i t y of I s o l a t i n g the product at much lower temperatures. Brunner and Peter (20) used a bench scale countercurrent continuous apparatus with an unspecified X


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Figure 2. Fractionation curves generated i n a pressure pro grammed f r a c t i o n a t i o n of 100 g of urea-fractionated ethyl esters (see 'PUFA, Table 1)· Column temperatures from top to bottom (see Figure 1) were 80, 71, 63, 56, 50, 45, 40 °C. Pressures used are represented by the step-curve which i s scaled on the right-hand ordinate. The left-hand f r a c t i o n a t i o n curve, designated Σ16, i s the sum of 16:3m4 and 16:4uil.


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NILSSONETAL

Synthesis of Trkicosapentaenoylglycerolfrom Fish Oil


s u p e r c r i t i c a l f l u i d to obtain monoglycerides of ca. 99% purity from a mixture containing 40% diglycerides by weight. Panzer et al. (21) stated that l i t t l e separation of glyceride mixtures could be achieved using s u p e r c r i t i c a l f l u i d carbon dioxide, although no experimental evidence was given to support t h i s claim. While i t may be true that SC-CO2 can e f f e c t l i t t l e separation of mono- and d i g l y c e r i d e s , we have found i t to be useful i n i s o l a t i n g unreacted esters and t r i g l y c e r i d e s from our crude mixtures. Table I I provides a summary of experimental conditions and results for the f r a c t i o n a t i o n of a crude tri-EPA reaction mixture.

Table I I .

Data from the f r a c t i o n a t i o n of the crude tri-EPA product mixture with SC-CO2. A l l heated zones at 60 °C

F r a c t i o n number Feed 1 2 3 4 5 6 7

Fraction wt (g) 20.00 3.92 1.34 1.56 1.58 4.16 4.11 0.31

Pressure bar 138 186 207 241 310 310 310

Figure 3 i s a photograph of a TLC of the crude product mixture and each of the seven f r a c t i o n s (an ester and mixed glyceride standard of 18:3u>3 was spotted on right-hand side of the p l a t e ) . As i s apparent by v i s u a l inspection of the TLC of f r a c t i o n 1, unreacted ethyl esters, accounting f o r about 20% of the mixture by weight, are cleanly extracted early i n the f r a c t i o n a t i o n . By gas chromatography f r a c t i o n 1 was found to have a composition e s s e n t i a l l y i d e n t i c a l to that of the o r i g i n a l ester reactants (see '90% EPA Table 1), thus providing good evidence that f o r t h i s mixture the composition of the synthetic t r i g l y c e r i d e s i s the same as that of the s t a r t i n g material. It i s also useful to point out that the recovered unreacted esters, which i n themselves have s i g n i f i c a n t value, can be recycled i n a subsequent synthesis. The T L C s of f r a c t i o n s 2-3 show them to cont a i n the bulk of the mono- and diglyceride intermediates as well as the free f a t t y acids and other u n i d e n t i f i e d byproducts. Fractions 4-6 accounting for nearly 50% of the feed by weight are seen to contain most of the synthetic t r i g l y c e r i d e product. HPLC analyses indicate that taken together, these three f r a c t i o n s contain t r i g l y c erides of at least 93% purity. Since the HPLC analyses were performed a few weeks after the f r a c t i o n a t i o n and some autoxidation i s known to have occurred, 93% should be considered a conservative figure. 1

I s o l a t i o n of Tri-EPA Using SC-CO? and 4 wt% Ethanol as a Cosolvent. Although the purity of tri-EPA obtained i n the above f r a c t i o n a t i o n i s s a t i s f a c t o r y the solvent-to-feed r a t i o , defined as the weight of CO2 necessary to fractionate a unit weight of feedstock, was 490. Reduct i o n of the S/F would be desirable. One approach to increase the


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Figure 3. Thin layer s i l i c a gel chromatogram of the feed and fractions obtained i n the f r a c t i o n a t i o n of the tri-EPA reaction mixture using SC-C02« MG « monoglyceride, DG - d i g l y c e r i d e , FFA - free f a t t y acid, TG - t r i g l y c e r i d e , EE - ethyl ester. Other spots are unidentified byproducts.



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NILSSONETAL.

Synthesis of Trkiœsapentaenoylglycerolfrom Fish Oil

s o l u b i l i t y of solutes i n SC-CO2 and thus reduce the S/F i s to add a small quantity (ca. 10% or less by weight) of a miscible compound to CO2 as a "coeolvent". In t h e i r work on the f r a c t i o n a t i o n of g l y c erlde mixtures with SC-CO2, Brunner and Peter (20) investigated a number of cosolvents including acetone, benzene, methylene chloride, and ethanol (EtOH). Panzer (21) selected hexane as a coeolvent i n s i m i l a r work. A l l of these solvents are toxic to some degree and therefore must be removed from the product. Since i t i s d i f f i c u l t to guarantee the complete removal of solvent residues, EtOH i s an a t t r a c t i v e choice due to its r e l a t i v e l y low t o x i c i t y . Preliminary data on the s o l u b i l i t y of soybean t r i g l y c e r i d e s i n CO2 at 60 °C without coeolvent and with 2 and 4 wt% EtOH between 207 and 310 bar are shown i n Figure 4. Values obtained with pure CO2 are i n good agreement with those reported by F r i e d r i c h (22). The data i n Figure 4 suggested that small quantities of added EtOH could be used to increase the s o l u b i l i t y of t r i g y c e r i d e s (and presumably mono- and d i g l y c e r i d e s ) i n CO2 at pressures s i m i l a r to those used i n the f r a c t i o n a t i o n with pure CO2, thereby reducing the S/F. However, i n order that a comparably successful f r a c t i o n a t i o n be r e a l i z e d , the increased loading of the solute i n the f l u i d phase must not be accompanied by a large decrease i n the s e l e c t i v i t y of the f l u i d f o r one component over another. Brunner and Peter (20) present evidence that the s e l e c t i v i t y of SC-CO2 f o r free f a t t y acids i n palm o i l i s actually enhanced by use of c e r t a i n cosolvents including EtOH. To confirm t h i s r e s u l t , a crude tri-EPA reaction mixture containing 90% tri-EPA was fractionated using SC-CO2 with a 4 + 0 . 5 wt% EtOH coeolvent. The rate at which EtOH wae introduced by the HPLC pump wae predetermined baeed upon a CO2 flow rate of ca. 10 etandard l i t e r e per minute. Table I I I provides a eummary of the results of the test.

Table I I I .

Data from the f r a c t i o n a t i o n of the crude tri-EPA product mixture with SC-CO2 containing 4 wt% ethanol. A l l heated zones at 60 °C

Fraction number Feed 1 2 3 4 5

Pressure (bar) 138 186 207 310 431

F r a c t i o n wt 23.53 4.68 3.58 1.84 10.17 2.38

(g)

The pressures were e s s e n t i a l l y the same as those i n the f r a c t i o n a t i o n without coeolvent (see Table I I ) . As anticipated from the data of Figure 4, the observed S/F of 200 for the test with 4 wt% EtOH i s s i g n i f i c a n t l y less than that found using CO2 alone. V i s u a l inspect i o n of the TLC's shown i n Figure 5 indicates that f r a c t i o n 4, accounting f o r about 45% (w/w) of the feed i s t r i g l y c e r i d e of high p u r i t y . HPLC of f r a c t i o n 4 indicated that i t contains t r i g l y c e r i d e s of at least 92% purity. Again, because of delays i n performing the HPLC analyses, t h i s i s considered a conservative estimate.


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Synthesis of Trieicompentaenoyiglycerolfrom Fish Oil

Figure 5 . Thin layer s i l i c a gel chromatogram of the feed and fractions obtained i n the f r a c t i o n a t i o n of the tri-EPA reaction mixture using SC-CO2 and a 4 wt% EtOH cosolvent. MG « monoglyeeride, DG - d i g l y c e r i d e , FFA - free f a t t y a c i d , TG - t r i glyceride, EE · ethyl ester. Other spots are u n i d e n t i f i e d byproducts.


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I n c i d e n t a l l y , the mono- and diglycerides present i n the reaction mix ture were miscible with the EtOH recovered i n the receiver while the highly unsaturated t r i g l y c e r i d e was not. This observation provided a useful v i s u a l indicator of where to cut f r a c t i o n s . Another advantage of using an EtOH cosolvent suggested by Brunner and Peter (20) was that increased solute s o l u b i l i t y allows reduction of processing pressures. Table IV summarizes the r e s u l t s of the f i n a l f r a c t i o n a t i o n of the crude tri-EPA mixture. The pres sures used i n t h i s test are generally ca. 35-70 bar lower than those i n the previous two f r a c t i o n a t i o n s . Fractions 6-7 comprise ca. 60 wt% of the feed. HPLC analyses estimated the purity of both to be better than 98%. As expected, the S/F was considerably higher (ca. 450) than i n the previous test at elevated pressures. On the other hand, the y i e l d and p u r i t y of the product were apparently improved.

Table IV· Data from the f r a c t i o n a t i o n of the crude tri-EPA product mixture with SC-CO2 containing 4 wt% ethanol at reduced pressures. A l l heated zones at 60 °C Fraction number Feed 1 2 3 4 5 6 7 8

Pressure (bar) 138 159 172 172 172 241 241 241

Fraction wt 20.00 2.76 1.26 1.14 0.95 0.48 5.95 6.18 0.62

(g)

Fractionation of Other Synthetic T r i g l y c e r i d e Product Mixtures. To obtain trl-DHA, a mixture derived from the 90% DHA esters was f r a c tionated with 4 wt% EtOH cosolvent. A tri-DHA f r a c t i o n accounting for 44% of the feed by weight was i s o l a t e d . The p u r i t y of t h i s f r a c t i o n with respect to t r i g l y c e r i d e s was estimated by HPLC to be 93% or better. In a l l of the fractionations discussed to t h i s point, the compo s i t i o n of the unreacted esters recovered early i n the test was found to be very s i m i l a r to that of the ester reactants. From t h i s e v i dence, i t can be concluded that a l l components i n the 90% EPA and DHA reactants are incorporated into the t r i g l y c e r i d e product with equal p r o b a b i l i t y . This i s probably not s u r p r i s i n g , as the components i n both concentrates d i f f e r l i t t l e i n chain length or degree of unsatur a t l o n . To provide a more rigorous t e s t , the product mixture from a synthesis s t a r t i n g with mixed chain-length polyunsaturated f a t t y acid esters ( PUFA i n Table I) was fractionated using CO2 with 4 wt% EtOH. As observed In previous t e s t s , the f i r s t f r a c t i o n contained e s s e n t i a l l y a l l of the unreacted ethyl esters. Gas chromatographic analysis of t h i s f r a c t i o n gave the following composition i n GC peak area %: 16:3ω4, 3.9%; 16:4uil, 4.2%; 18:4ω3, 7.3%; 20:4ui6, 1.5%; 20:5UJ3, 49.3%; 21:5cu3, 1.4%; 22:6ui3, 21.3%. Although some d i f f e r ences i n composition vis-à-vis the s t a r t i n g material do e x i s t , they f

f


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NILSSON E T A L .

Synthesis of Trieicosapentaenoylglycerolfrom Fish Oil

are c e r t a i n l y not large and may well f a l l within experimental error. Lehman and Gauglitz (15) reported s i m i l a r observations f o r the pro cedure they developed using free f a t t y acids. F r a c t i o n a t i o n of t h i s mixture proved to be somewhat more d i f f i c u l t than f r a c t i o n a t i o n of the tri-EPA. The wider d i s t r i b u t i o n of chain lengths i n the g l y c erides synthesized from the ΡUFA mixture required the more d i f f i c u l t separation of, f o r example, C22-containing d i g l y c e r i d e s from C16-containing t r i g l y c e r i d e s .


Conclusions I s o l a t i o n of t r i g l y c e r i d e s from mixed glycerldes can be accomplished using pure s u p e r c r i t i c a l f l u i d CO2 at moderate temperatures by pres sure programming. A few wt% of added ethanol can s i g n i f i c a n t l y reduce the solvent-to-feed r a t i o i f comparable pressures are used or a l t e r n a t i v e l y permit reduction of pressures during the f r a c t i o n a t i o n . Regardless of the composition of esters used as reactants, there appears to be l i t t l e or no s e l e c t i v e incorporation of i n d i v i d u a l components i n t o the t r i g l y c e r i d e product. Acknowledgement We wish to express appreciation to Jeanne Joseph, Bob Ernst, and coworkers of the National Marine F i s h e r i e s Service, Charleston, SC, f o r supplying urea-fractionated menhaden o i l esters.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Lands, W. Ε. M. Fish and Human Health; Academic Press, Inc.: Orlando, FL, 1986. Kinsella, J. E. Seafoods and Fish Oils in Human Health and Disease; Marcel Dekker, Inc.: New York, 1987. Leaf, A. New Eng. J. Med. 1988, 318, 549. Dehmer, G. J.; Popma, J. J.; van den Berg, E. K.; Eichhorn, E. J.; Prewitt, J. B.; Campbell, K. D.; Jennings, L. J.; Willerson J. T.; Schmitz, J. M. New Eng. J. Med. 1988, 319, 733. Miller, D.; Leong, K. C.; Knobl, G. M.; Gruger, Ε., Jr. Proc. Soc. Exp. Biol. Med. 1964, 116, 1147. Boggio, S. M.; Hardy, R. W.; Babbitt, J. K.; Brannon, E. L. Aquaculture. 1985, 51, 13. Stout, V. F. J. Am. Oil Chem. Soc. 1963, 40, 40. McHugh, Μ. Α.; Krukonis, V. J. Supercritical Fluid Extraction Principles and Practice; Butterworks: Boston, 1986, p. 178. Eisenbach, W. Ber. Bunsenges. Phys. Chem. 1984, 88, 882. Nilsson, W. B.; Gauglitz, E. J., Jr.; Hudson, J. K.; Stout, V. F.; Spinelli, J. J. Am. Oil Chem. Soc. 1988, 65, 109. Nilsson, W. B.; Gauglitz, E. J., J r . ; Hudson, J. K.; Teeny, F. M. Paper presented at the 194th ACS National Meeting, New Orleans, LA, Paper AGFD 0051. Krukonis, V. J. In Supercritical Fluid Extraction and Chroma tography; Charpentier, Β. Α., and Sevenants, M. R., Eds.; ACS Symposium Series 366; American Chemical Society: Washington, DC, 1988, pp. 34-36.


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