The isolation of omega-3 polyunsaturated fatty acids and methyl esters of fish oils by silver resin chromatography

Multigram quantities of the highly unsaturated ω3 component from samples of fish oil fatty acids and esters were isolated by silver resin chromatography. An XN1010 resin column saturated with silver ions was utilized. Polyunsaturated fatty acid (PUFA) esters from fish oil concentrate (FOC) were fractionated based on the number of double bonds by using solvent programming (acetonitrile in methanol). Larger samples (4–9 g) of FOC acids and esters and menhaden acids and esters were enriched in ω3 polyunsaturates to 82–99% (95–99% total PUFA) by use of a large 100% silver resin column and isocratic elution with 30, 35 or 45% acetonitrile in acetone.     

Comparative autoxidative susceptibility of fatty esters with 0–6 methylene-interrupted double bonds

Disappearance of unsaturated fatty esters due to autoxidation was estimated by gas liquid chromatography. The stability of a mixture containing from 0–6 methylene-interrupted double bonds was significantly increased by chromatography over silver nitrate-silicic acid. The relative stability of the esters is a linear function of the number of double bonds.     

A rapid method for concentrating highly unsaturated fatty acid methyl esters in marine lipids as an aid in their identification by GLC

The selective solubility of unsaturated fatty acid methyl esters in nitromethane at temperatures down to −20C can be used to concentrate highly unsaturated methyl esters. With a typical sample of marine oils methyl esters having an iodine value of 110–190, a concentrate can be ready for GLC analysis in an hour or less and the nitromethane layer can be injected directly for analysis in GLC apparatus with ionization detectors. Examples of the use of the method in the identification of component fatty acids in herring oil are given.     

Gas chromatographic separation of cholesteryl esters of fatty acids of different degrees of unsaturation

Cholesteryl esters prepared from the fatty acid methyl esters of linseed oil, pig liver lipids, and Japanese anchovy oil have been separated on the basis of their chain lengths and number of double bonds by gas lipid chromatography on a cyanosiloxane SILAR 10C column. The equivalent chain lengths of cholesteryl esters having acyl groups with 14–22 carbons and 0–6 double bonds are presented. A significant influence of the column temperature on the equivalent chain lengths of the polyenoic fatty acid cholesteryl esters has been found. Separation of the cholestanyl and epicholestanyl esters of linseed oil fatty acids, respectively, under the same conditions is also described.     

Preparation of fatty 3,5-disubstituted isoxazole compounds from FA esters

Long-chain fatty compounds containing an isoxazole heterocyclic ring system substituted at the 3- and 5-ring positions were prepared in moderate to good yields (40–64%) in one pot by condensing FA esters such as methyl palmitate, stearate, oleate, or linoleate with the lithiated anion of N-(isopropylidene)isopropylamine followed by dehydrative cyclization. This approach allows readily available FA esters to be utilized and incorporated into the construction of the isoxazole ring system. These novel products were isolated then characterized by NMR, IR spectroscopy, GC-MS, and m.p. Mass spectra of the fatty isoxazole compounds, derived utilizing EI ionization, showed distinctive cleavage patterns that occurred uniformly along the fatty alkyl chain allowing the position of double bonds to be readily located. Two prominent ions at m/z 97 and 110 were common to all the fatty isoxazole compounds examined and were presumably from a McLafferty rearrangement and a cyclization displacement reaction, respectively.     

Ultrasound-assisted oxidative cleavage of acetylenic and ethylenic bonds in unsaturated fatty esters with potassium permanganate

The effect of ultrasound on the oxidation of unsaturated fatty esters was investigated. Unsaturated fatty esters containing acetylenic or olefinic bonds or both were oxidatively cleaved with KMnO₄ in aqueous acetic acid (AcOH/H₂O, 1:1, vol/vol for olefinic fatty esters and AcOH/H₂O, 9:1, vol/vol for acetylenic fatty esters) to yield the corresponding carboxylic acid moieties in quantitative yields, when the reactions were conducted under ultrasound (20 kHz, 53 W/cm², 8–15 min). The methyl ester derivatives of the carboxylic acid moieties formed were identified by gas-liquid chromatographic analysis. The procedure constitutes a facile method for the determination of the position of the unsaturated center (olefinic or acetylenic) in an unsaturated fatty ester substrate, especially where the unsaturated system consists of an acetylenic bond.     

Distribution and identification of the fatty acids from the coho salmon,Oncorhynchus kisutch (Walbaum)

To study the fatty acids of the coho salmon, entire fish were homogenized and the total lipids extracted with methanol-chloroform. The fish ranged in size from 75 to 85 mm total length and contained from 2.1%–6.9% lipid in the tissues. Methyl esters of the fatty acids were produced with anhydrous methanol and HCl. Qualitative identification of the fatty acid methyl esters was accomplished by gas-liquid chromatography. Thin layer silver nitrate-silicic acid plates were used to separate the component methyl esters according to the number of double bonds. Location of the ethylenic groups of the unsaturated fatty acid methyl esters was established by reductive ozonolysis and identification of the aldehydes and aldehyde-esters produced. The number of carbons in the unsaturated fatty acid methyl esters was determined by hydrogenation of each of the fractions. Fatty acids found in the highest concentrations were: 16∶0, 16∶1, 18∶0, 18∶1, 18∶4, and 22∶6. Fatty acids 16∶0, 18∶1, 18∶2, 20∶5, and 22∶6, differed markedly from concentrations found in tubificid worms, the exclusive diet of the fish during the experiment. Technical Paper No. 2059, Oregon Agricultural Experiment Station Work supported in part by research Grant No. EF105, U.S.P.H.S.     

Displacement of the double bonds during hydrogenation of unsaturated fatty acid methyl esters

Summary The displacement of the double bond of several unsaturated fatty acid methyl esters during hydrogenation with a nickel-kieselguhr catalyst at 180°C. was investigated. The analysis of the dicarboxylic acids (obtained by oxidation of the reaction products with KMnO₄ in acetic acid solution) by means of partition chromatography enabled a reliable semiquantitative determination of the position isomers formed. During hydrogenation of methyl esters of oleic, elaidic, petroselinic, and linoleic acid formation of large amounts of position isomers was proved to occur. Migration of the double bonds in both directions took place but was in all cases strongly pronounced in a direction opposite the ester group. The place and configuration (cis or trans) of the double bonds in the starting material apparently were of little importance in this respect. It follows that hydrogenation of fatty acid esters leads to products which are far more complicated, as is generally known. This is especially of importance with respect to the application of hydrogenated fatty oils in the food industries.     

Analysis of north atlantic and baltic fish oil triacylglycerols by high-performance liquid chromatography with a silver ion column

Triacylglycerols from Atlantic herring (Clupea harengus), sandeel (Ammodytes sp.) and Baltic herring (Clupea harengus membras) have been fractionated by silver ion high-performance liquid chromatography. An ion exchange column loaded with silver ions was the stationary phase, and a gradient in the mobile phase from 1,2-dichloroethane/dichloromethane (1∶1, v/v) to acetone and then to acetone/acetonitrile (2∶1, v/v) was used to effect the separation with light-scattering (i.e., mass) detection. Fractions were collected via a streamsplitter, and fatty acid methyl esters were prepared by transesterification in the presence of an internal standard for identification and quantification by gas liquid chromatography. Triacylglycerols were separated according to the number of double bonds in the fatty acyl residues. Resolution was excellent at first, when the least unsaturated molecules eluted (trisaturated to dimonoene-monodiene fractions). Base-line resolution could no longer be achieved when molecules containing trienoic or more highly-unsaturated fatty acids began to elute because of overlapping components. Nonetheless, some valuable separations of species containing two saturated and/or monoenoic fatty acids and one polyenoic fatty acid were achieved. Double bond indices (average number of double bonds in each triacylglycerol molecule) were calculated to estimate the separations possible. Fractions containing at least 11–14 double bonds per molecule were obtained.     

Evidence of thermal decomposition of fatty acid methyl esters during the synthesis of biodiesel with supercritical methanol

New evidence on the thermal decomposition of fatty acid methyl esters during biodiesel synthesis in supercritical conditions is presented. Thermal decomposition products were detected chromatographically, by applying the UNE-EN 14105:2003 standard, as a broad single peak during the determination of glycerides in the reaction samples. These degradation products could be quantified chromatographically by the above standard because the area of the peak was proportional to the disappearance of the polyunsaturated fatty acid methyl esters, which contain two or more double bonds (methyl linoleate and linolenate), generated during biodiesel synthesis from soybean oil. In the experimental conditions tested, thermal decomposition reactions of these unsaturated fatty acid methyl esters began to appear at 300 °C/26 MPa, and were more intense as the temperature rose. For its part, the main saturated fatty acid methyl ester (methyl palmitate) generated during the reaction was hardly decomposed at all in the experimental conditions tested and only began to disappear at 350 °C/43 MPa.     

Heats of combustion of fatty esters and triglycerides

Gross heats of combustion (HG) have been measured for three classes of fatty esters and two classes of triglycerides (TGs). The esters included saturated methyl esters, Me 6:0–22:0; saturated ethyl esters, Et 8:0–22:0; and unsaturated methyl esters, Me 12:1–22:1, Me 18:2 and Me 18:3. The TGs included the saturated TGs, C 8:0–22:0, and unsaturated TGs, C 11:1, C 16:1, C 18:1, C 18:2, C 18:3, C 20:1 and C 22:1. HG were measured in a Parr adiabatic calorimeter according to a modification of ASTM D240 and D2015. Linear regression analysis (LINREG) yielded equations that related HG to carbon number (CN) or chain length, electron number (EN) or number of valence electrons and molecular weight (MW). Calculated HG values from CN, EN, or MW were nearly identical. Thus, any one of these three variables can be used to predict HG satisfactorily. R squared values for all equations were 0.99. Equations for correlating HG of saturated or unsaturated TGs with molecular characteristics of these molecules have not been reported. With LINREG, we developed equations that permitted predictions of HG from structures of the saturated and unsaturated TGs. Equations for predicting HG of methyl and ethyl esters were compared to those in the literature and were found to be more accurate and precise. Presented in part at the AOCS meeting in New Orleans, LA, in May 1987.     

Analysis of autoxidized fats by gas chromatography-mass spectrometry: V. Photosensitized oxidation

The role of singlet oxygen in oxidation was studied by analyzing hydroperoxide isomers in unsaturated fats and esters by gas chromatography-mass spectrometry (GC-MS). On oxidation photosensitized with methylene blue at 0 C, methyl oleate produced a 50–50% mixture of 9- and 10-hydroperoxides, linoleate a mixture of 66% conjugated (9+13) and 34% unconjugated (10+12) hydroperoxides, and linolenate a mixture of 75% conjugated (9+12+13+16) and 25% unconjugated (10+15) hydroperoxides. Cottonseed, safflower, and corn oil esters showed, as in soybean esters, the presence of varying amounts of 12-hydroxy esters derived from the corresponding hydroperoxide at low peroxide values. Since these oils do not contain linolenic acid, a likely source of the 12-hydroperoxide is linoleic acid by photosensitized oxidation. Several lines of evidence support the conclusion that singlet oxygen may contribute to the unique hydroperoxide composition of vegetable oil esters at low levels of oxidation. In the presence of photosensitizers such as methylene blue and chlorophyll, the unique hydroperoxide composition (high levels of 10- and 12-hydroperoxides) obtained in soybean esters was similar to that produced by oxidation at low peroxide values. In contrast, a normal hydroperoxide composition was produced, as expected from the fatty acid composition of soybean oil esters, when singlet oxygen quenchers such as β-carotene and α-tocopherol were used and when the esters were treated with carbon black to remove natural photosensitizers. GC-MS analyses of the derived unsaturated alcohols provided indirect evidence for 12-hydroperoxy-9,13-diene in soybean esters as expected by photosensitized oxidation of linoleate. Presented at the AOCS Meeting, San Francisco, California, April 29–May 3, 1979. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Analysis of the dark-colored impurities in sulfonated fatty acid methyl ester

A fractionally distilled C₁₄−C₁₆ fatty acid methyl ester, derived from palm oil, was sulfonated with gaseous SO₃ in a falling film reactor to form an α-sulfo fatty acid methyl ester (α-SF; unbleached and unneutralized form). The included dark-colored impurities were then separated from α-SF as a diethyl ether-insoluble matter. After purification by thin-layer chromatography, the colored species were analyzed by ion-exchange chromatography, gel-permeation chromatography, and nuclear magnetic resonance spectrometry. These data suggested that the colored species were polysulfonated compounds with conjugated double bonds. Minor components in the raw fatty acid methyl ester, found by gas chromatography/mass spectrometry, were spiked into the purified methyl palmitate and then sulfonated. The unsaturated methyl ester and hydroxy ester showed the worst color results. The methyl oleate and methyl 12-hydroxystearate were then sulfonated and analyzed. Deep black products were obtained, which showed the same properties as the colored species in α-SF. It was concluded that low levels of unsaturated fatty acid methyl esters and hydroxy esters in the fatty acid methyl ester are the main causes of the coloring.     

Lipid composition of selected vegetable oils

This paper gives analytical data on the composition of 14 selected consumer-available liquid vegetable oils, including soybean, soybean-cottonseed blends, corn, safflower, peanut, olive and apricot kernel oils. Label information identified six samples as specially processed or refined and three samples as cold pressed with no preservative added; the labels of the remaining five samples did not mention processing. Data are given for fatty acid composition,trans content, location of the double bonds in the unsaturated fatty acids, percent conjugation, tocopherol content, fatty acid composition of the 2-poisition of the triglycerides, polyunsaturated to saturated fatty acid (P/S) ratio, and the ratio of α-tocopherol to polyunsaturated fatty acids (α-T/P). The ranges of values found were: conjugated unsaturation, 0.18–1.09%; α-tocopherol, 0.01–0.60 mg/gm; total tocopherol 0.14–1.52 mg/gm; P/S, 0.5–8.7; and α-TP, 0.03–2.26. The compositions of the fatty acids on the 2-position and on the 1,3-position of the triglycerides were compared with those calculated by the Evans’ hypothesis and found to agree well for all but olive and apricot kernel oils. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable. Deceased     

Hydrogenation of unsaturated fatty esters with copper-chromite catalyst: Kinetics,mechanism and isomerization

Hydrogenation of linolenate with copper chromite produced a large amount of conjugated diene and minor amounts of nonconjugatable dienes. The double bonds in conjugated dienes and monoenes were scrambled all along the chain. This product distribution can be explained if it is assumed that conjugation of the double bonds is followed by hydrogenation. In competitive hydrogenation, fatty esters with conjugated double bonds were reduced preferentially over fatty esters with methylene-interrupted double bonds. Isomerization of conjugated double bonds (geometric and positional) occurred more rapidly than reduction. Reduction of conjugated double bonds in the presence of deuterium resulted in a majority of the products containing no deuterium. Most of the added deuterium was incorporated into the unreacted material. Mechanisms are proposed to account for the products formed during the hydrogenation of linolenate, linoleate and their isomers. One of 10 papers to be published from the Symposium “Hydrogenation,” presented at the AOCS Meeting, New Orleans, April 1970. No. Utiliz. Res. Dev. Div., ARS, USDA.     

Identification of C18 Intermediates Formed During Stearidonic Acid Biohydrogenation by Rumen Microorganisms In Vitro

In vitro batch incubations were used to study the rumen biohydrogenation of unsaturated fatty acids. An earlier study using increasing supplementation levels of stearidonic acid (18:4n-3), revealed that the rumen microbial population extensively biohydrogenates 18:4n-3 after 72 h of in vitro incubation, though several intermediates formed were not completely characterized. Therefore, in the present study, samples were reanalyzed in order to identify the 18:2, 18:3 and 18:4 biohydrogenation intermediates of 18:4n-3. Gas–liquid chromatography coupled to mass spectrometry was used to characterize these intermediates. The acetonitrile chemical ionization mass spectrometry of the fatty acid methyl esters derivatives enabled the discrimination of fatty acids as non-conjugated or conjugated biohydrogenation intermediates. In addition, the acetonitrile covalent adduct chemical ionization tandem mass spectrometry yielded prominent ions indicative of the double bond position of the major 18:3 isomers, i.e. Δ5,11,15 18:3. Furthermore, the 4,4-dimethyloxazoline derivatives prepared from the fatty acid methyl esters enabled the structure of novel 18:2, 18:3 and 18:4 biohydrogenation intermediates to be elucidated. The intermediates accumulated in the fermentation media after 72 h of incubation of 18:4n-3 suggest that similar to the biohydrogenation pathways of linoleic (18:2n-6) and α-linolenic (18:3n-3) acids, the pathway of the 18:4n-3 also proceeds with the formation of conjugated fatty acids followed by hydrogenation, although no conjugated dienes were found. The formation of the novel biohydrogenation intermediates of 18:4n-3 seems to follow an uncommon isomerization pattern with distinct double bond migrations.     

The fatty acids ofEntomophthora coronata

The total lipids and fatty acid composition ofEntomophthora coronata were determined. The fungus was grown on a chemically defined medium and a chemically nondefined medium (Sabouraud dextrose yeast extract) for a period of 26 days. The organism contained from 16.2% to 44.6% total lipids depending upon the days of growth. The major fatty acids were 12∶0 (5.5–9.0%), 13∶0 (1.2–8.2%), 14∶0 (33.5–43.5%), 16∶0 (9.7–13.9%), 18∶1₉ (20.4–22.4%), and 18∶2_9,12 (3.5–10.5%). Lesser amounts of 15∶0, 16∶1, 16∶2, 17∶0, 18∶0, two other 18∶2 (both having conjugated double bonds), 18∶3_6,9,12, another 18∶3 (conjugated double bonds), 20∶3_8,11,14, 20∶4_5,8,11,14, another 20∶4 (conjugated double bonds), and 24∶1 acids were found. Trace amounts of 20∶0, 20∶1, 20∶2, 22∶0 and 24∶0 were also present. The relative percentage of most of the fatty acids did not vary appreciably with growth. However, 18∶2_9,12 and 20∶4_5,8,11,14 increased with age of the chemically defined culture. Peak E (18∶2, conjugated double bonds) increased and 13∶0 and 18∶3_6,9,12 decreased with age of the chemically nondefined culture. The fatty acids were predominately saturated (56.9–69.1%) and contained a high percentage of shorter chain fatty acids (C 12 to C 15). The fatty acids of the chemically defined culture were more unsaturated than the Sabouraud culture and the unsaturation increased with age of the culture.     

cis-5-Monoenoic fatty acids in some chenopodiaceae seed oils

Methyl esters from seed oils of four Chenopodiaceae species are unusual in that they contain methylcis-5-hexadecenoate (4.6–12%) and methyl 5-octadecenoate (1.1–1.2%). There are indications of small amounts of 18∶2^5,9 and 18∶3^{5, 9, 12} along with unsaturated acids commonly found in seed oils-oleic (14–21%), linoleic (53–57%) and linolenic (3.5–7.8%). Fatty acid composition of the oils was determined by gas chromatography, and positions of the double bonds were established by application of gas chromatography-mass spectrometry to the methoxylated methyl esters. N. Market. Nutr. Res. Div., ARS, USDA.     

Formation of triacylglycerol core aldehydes during rapid oxidation of corn and sunflower oils with tert-butyl hydroperoxide/Fe2+

The lipid ester core aldehydes formed during a rapid oxidation (7.8 M tert-butyl hydroperoxide, 90 min at 37°C) of the triacylglycerols of purified corn and sunflower oils were isolated as dinitrophenylhydrazones by preparative thin-layer chromatography and identified by reversed-phase high-performance liquid chromatography with on-line electrospray ionization mass spectrometry and by reference to standards. A total of 113 species of triacylglycerol core aldehydes were specifically identified, accounting for 32–53% of the 2,4-dinitrophenylhydrazine (DNPH)-reactive material of high molecular weight representing 25–33% of the total oxidation products. The major core aldehyde species (50–60% of total triacylglycerol core aldehydes) were the mono(9-oxo)nonanoyl- and mono(12-oxo)-9,10-epoxy dodecenoyl- or (12-oxo)-9-hydroxy-10,11-dodecenoyl-diacylglycerols. A significant proportion of the total (9-oxo)nonanoyl and epoxidized (12-oxo)-9,10-dodecenoyl core aldehydes was found in complex combinations with hydroperoxy or hydroxy fatty acyl groups (6–10% of total triacylglycerol core aldehydes). Identified were also di(9-oxo)nonanoylmonoacylglycerols (0.5% of total) and tri(9-oxo)nonanoylglycerols (trace). The identification of the oxoacylglycerols was consistent with the products anticipated from tert-butyl hydroperoxide oxidation of the major species of corn and sunflower oil triacylglycerols (mainly linoleoyl esters). However, the anticipated (13-oxo)-9,11-tridecadienoyl aldehyde-containing acylglycerols were absent because of further oxidation of the dienoic core aldehyde. A significant proportion of the unsaturated triacylglycerol core aldehydes contained tert-butyl groups linked to the unsaturated fatty chains via peroxide bridges (2–9%). The study demonstrates that rapid peroxidation with tert-butyl hydroperoxide consitutes an effective method for enriching natural oils and fats in triacylglycerol core aldehydes for biochemical and metabolic testing.     

Determination of Polyunsaturated Fatty Esters (PUFA) in Biodiesel by GC/GC–MS and <Superscript>1</Superscript>H-NMR Techniques

In the present work, methods based on Gas Chromatography (GC), Gas Chromatography–Mass Spectrometry (GC–MS) and ¹H-NMR techniques was developed for the characterization and estimation of Polyunsaturated Fatty Esters (PUFA) in biodiesel. The GC method enables the separation, identification and estimation of these polyunsaturated fatty acid methyl esters to be carried out using a highly polar capillary column (100% cyanopropyl silicon). GC–MS was utilized for unambiguous identification and estimation of esters and isomers present in biodiesel. The estimation of PUFA content is important because PUFA content is a part of the EN 14214 specifications of biodiesel. There is no existing standard method for the estimation of total PUFA content in biodiesel. The developed GC method also quantifies EPA and DHA (C20:5 and C22:6) fatty acid methyl esters which can be used as markers for the estimation of fish oil biodiesel. The presence of EPA and DHA indicates the contamination of fish oil in biodiesel. Further, the ¹H-NMR technique has also been employed to identify and estimate PUFA containing ≥3 double bonds. The PUFA content was estimated from the integral intensities of the chemical shift region corresponding to all type of double bonds in the ¹H-NMR spectra. Based on the developed method, GC fingerprinting of various biodiesel samples was carried out to estimate a PUFA content as low as 1,000 ppm.