Fatty acids of long chain length in baltic herring lipids

The component fatty acids with carbon numbers exceeding 22 in flesh lipids of Baltic herring caught in May and September 1967 in the Turku archipelago have been studied. The total lipid content of the flesh of the herring was 3.5% on average in May and 7.2% on average in September. The fatty acids in the lipids were converted to methyl esters which were resolved and analyzed by urea adduct fractionation, thin layer chromatography and programmed temperature gas liquid chromatography (GLC). The lipids of the herring caught in May were found to contain 15 fatty acids with 24–32 carbon atoms, whereas the lipids of the herring caught in September were found to contain only nine fatty acids with 24–28 carbon atoms. The differences are probably due to nutritional factors. The long chain fatty acids in the lipids of the herring caught in September were isolated by preparative GLC and their structures were studied by UV spectroscopy before and after alkali isomerization, by IR spectroscopy and by GLC of their ozonization products. The identified acids were tetracosanoic, 15-tetracosenoic, 12,15,18,21-tetracosatetraenoic, 9,12,15,18,21-tetracosapentaenoic, 6,9,12,15,18,21-tetracosahexaenoic, 17-hexacosenoic, 11,14,17,20,23-hexacosapentaenoic, 8,11,14,17,20,23-hexacosahexaenoic and 4,7,10,13,16,19,22-octacosaheptaenoic acids. The proportion of the fatty acids containing over 22 carbon atoms in the lipids of fall herring is much higher than has been found earlier in the lipids of marine teleost fish; the reason may be due at least partly to differences in analytical methods.     

A rapid low temperature method for preparation of methyl esters of fatty acids

A rapid method for preparation of methyl esters of fatty acids in lipids has been accomplished by forming the sulfuric acid complex of the lipid in ethyl ether at the temp of a dry ice-acetone bath. Decomposition of the complex with methanol results in direct formation of methyl esters of the fatty acids. A comparison was made of gas liquid chromatography (GLC) analysis of fatty acid composition of several fats using methyl esters prepared by this and by two other methods. Results of this comparison reveal that the method is not only rapid but provides complete reaction with no apparent changes in the fatty acids. Mich. Agr. Exp. Sta. Pub. No. 3503. Presented at the AOCS meeting in Chicago, 1964.     

Cyclopropenoid Fatty Acids in Malagasy baobab: Adansonia grandidieri (Bombacaceae) Seed Oil

Adansonia grandidieri (bombacaceae family) seed oil gives a positive Halphen test. Composition analysis of derivatized fatty acid methyl esters, in presence of silver nitrate in anhydrous methanol, after chromatography fractionation on silicagel column, were made by gas-liquid chromatography (GLC) using a packed DEGS column. Presence of malvalic and sterculic acids were detected. GLC analysis using glass capillary columns coated with Carbowax 20 M and BDS shows that A. grandidieri seed oil contains mainly palmitic (41%), oleic (22%) and linoleic (12%) acids. Cyclopropenic fatty acid concentration was 14% with 6% for malvalic and 8% for sterculic acids. A slight proportion of dihydrosterculic acid (1.5%) was observed. GLC fatty acid methyl esters analysis, without derivatization, on the two glass capillary columns coated with Carbowax 20 M and BDS gave the same results for cyclopropenic acids content.     

Dihydrosterculic acid,a major fatty acid component ofEuphoria longana seed oil

The seed oil ofEuphoria longana, Sapindaceae, contains 17.4% of 9,10-methyleneoctadecanoic (dihydrosterculic) acid. This identification is based on information from thin layer chromatography, infrared analysis, gas liquid chromatography, nuclear magnetic resonance and mass spectroscopy. Since GLC of the oil showed components that emerged between the usual triglycerides, the cyclopropanoid acid is apparently a triglyceride constituent. The presence of smaller amounts, less than 1%, of cyclopropanoid fatty acids of different chain lengths is indicated by GLC and TLC analyses of the methyl esters. The other major fatty acids in this oil are: 16∶0 (19%), 18∶0 (7%), 18∶1 (36%), 18∶2 (6%), 18∶3 (5%) and 20∶0 (4%).Euphoria oil contains considerably larger amounts of cyclopropanoid fatty acids than previously reported in other seed oils. Presented at the AOCS-AACC Joint Meeting, Washington, D.C., April 1968. No. Utiliz. Res. Dev. Div.; ARS, USDA.     

GLC and TLC analysis of isopropylidene derivatives of isomeric polyhydroxy acids derived from positional and geometrical isomers of unsaturated fatty acids

Gas-liquid chromatography (GLC) and thin-layer chromatography (TLC) were used to investigate the isomeric positional geometrical isopropylidene derivatives of nine isomeric dihydroxy esters, four isomeric methyl 9,10-12-trihydroxystearates, and eight isomeric methyl 9,10-12,13-tetrahydroxystearates prepared from unsaturated fatty acids. The isopropylidenes derived fromcis andtrans monounsaturated fatty acids were easily separated on both polar and nonpolar columns. Positional isopropylidenes derived from positional isomers of monounsaturated fatty acids were not separated on either liquid phase but were resolved by TLC. Four of the eight isomeric isopropylidenes derived from the four geometrical isomers of linoleic acid were resolved on the polar column; the other four isomers eluted as a single peak. The four isomeric isopropylidene-trifluoroacetate derivatives derived from ricinoleic and ricinelaidic acids were also resolved on the polar column. GLC analyses were carried out with liquid phases of ethylene glycol succinate methyl silicone polymer (EGSS-X) and methyl silicone polymer (SE-30) packed columns. Isopropylidenes, in addition to their applicability for the resolution of polyhydroxy acid mixtures, are particularly useful for the determination of double bond positions and geometrical configurations of fatty acids without cleavage. Under contract with the U. S. Atomic Energy Commission.     

Separation of petroselinic (cis-6 18:1) and oleic (cis-9 18:1) acids by gas-liquid chromatography of their isopropyl esters

Petroselinic (cis-6 18:1) and oleic (cis-9 18:1) acids that occur together in Umbelliferae seeds can be resolved by gasliquid chromatography (GLC) of their methyl or isopropyl esters on a 50 m × 0.25 mm fused-silica capillary column coated with a 100% cyanopropyl polysiloxane stationary phase (CP Sil 88). The use of isopropyl esters instead of methyl esters increases the difference between equivalent chainlengths from 0.06 carbon unit up to 0.08. This is sufficient to obtain an almost base-line resolution between the two components.cis-Vaccenic acid is completely separated from oleic acid in both derivative forms. GLC of fatty acid isopropyl esters on an appropriate capillary column thus appears to be the simplest means to simultaneously and accurately quantitate petroselinic, oleic andcis-vaccenic acids.     

Cyclopropane fatty acid metabolism: Physical and chemical identification of propane ring metabolic products in the adipose tissue

Twelve male weanling rats were distributed equally into 3 groups and placed on fat-free diets. The diets of groups 1 and 2 were supplemented with 0.54% of recemic methylcis-9,10-methylene octadecanoate (CMO) and racemic methyltrans-9,10-methylene octadecanoate (TMO), respectively. Group 3 served as a control. Gas liquid chromatography (GLC) analyses of the adipose tissue methyl esters indicated at the level fed, that cyclopropane fatty acids do not affect normal fatty acid metabolism as has been shown for cyclopropene fatty acids. GLC analyses of groups 1 and 2 revealed the presence of a different unidentified fatty acid for each of the acids fed in addition to the CMO and TMO acids themselves. Each of the unidentified acids and the CMO and TMO acids were isolated and purified by preparative GLC. The absolute identity of the CMO and TMO acids fed and isolated from body fat was established by IR, NMR, and mass spectra. The biodegradation products of the CMO and TMO esters were shown to becis- andtrans-3,4-methylene dodecanoic acid, respectively. Unequivocal proof of structure was established through synthesis followed by comparison of IR, NMR, and mass spectra and melting points, GLC retention times, and elemental analyses with those obtained for the degradation products. Neither member of the racemic mixtures of either thecis or thetrans cyclopropane acids was preferentially utilized by the rat as shown by the lack of octical activity in the degradation products and the CMO and TMO acids isolated from the body fat. The accumulations of the 3,4-methylene dodecanoic acids in the adipose tissue of the rats fed CMO and TMO cyclopropane fatty acids suggest the inability of the beta oxidation enzyme system to proceed past the cyclopropane ring in a fatty acid chain. The synthesis ofcis- andtrans-3-dodecenoic acids, intermediates in the synthesis of the 3,4-methylene dodecanoic acids, and the geometrical cyclopropane isomers are discussed. This work to be submitted in partial fulfillment of the requirement for Ph.D.     

Column types for the chromatographic analysis of oleochemicals

Chromatography has developed into one of the principle methods of analysis of oleochemicals. Gas chromatography has been used extensively for the analysis of long-chain fatty acids as well as for the analysis of triglycerides and plant sterols. In recent years, high pressure liquid chromatography (HPLC) has been used for the analysis of triglycerides as well as for other related materials. Specialized gas chromatography columns have been developed for the separation of long-chain fatty acids such as the methyl esters. These columns have generally used high polarity stationary phases which separate fatty acids by degree of unsaturation. A specialized use of these high polarity stationary phases is separation ofcis-trans isomers as well ascis-cis andtrans-trans isomers. In this paper, packed and capillary columns are compared for the separation of thecis-trans isomers of fatty acid methyl esters prepared from a hydrogenated vegetable oil. For HPLC separations, the presence of a double bond is approximately equivalent chromatographically to shortening the alkyl chain by two carbons. The long-chain polyenic acids or ethyl esters thus elute near but are resolved from the short-chain saturated fatty acids or esters. HPLC is the method of choice for relatively complex, high molecular weight, or labile esters, such as those of retinyl or cholesterol. Glyceryl esters are particularly well resolved by HPLC in terms of both total chain length and degree of unsaturation. This technique is also useful for lipid class separations and for the analysis of modified fatty acid products, such as prostaglandins and related materials. In general, these analyses are conducted with octadecyl bonded phase column packings.     

Analysis of hydroxylated fatty acids from plant oils

A novel process has been described recently for the preparation of hydroxylated fatty acids (HOFA) and HOFA methyl esters from plant oils. HOFA methyl esters prepared from conventional and alternative plant oils were characterized by various chromatographic methods (thin-layer chromatography, high-performance liquid chromatography, and gas chromatography) and gas chromatography-mass spectrometry as well as¹H and¹³C nuclear magnetic resonance spectroscopy. HOFA methyl esters obtained fromEuphorbia lathyris seed oil, low-erucic acid rapeseed oil, and sunflower oil contain as major constituents methylthreo-9,10-dihydroxy octadecanoate (derived from oleic acid) and methyl dihydroxy tetrahydrofuran octadecanoates, e.g., methyl 9,12-dihydroxy-10,13-epoxy octadecanoates and methyl 10,13-dihydroxy-9,12-epoxy octadecanoates (derived from linoleic acid). Other constituents detected in the products include methyl esters of saturated fatty acids (not epoxidized/derivatized) and traces of methyl esters of epoxy fatty acids (not hydrolyzed). The products that contain high levels of monomeric HOFA may find wide application in a variety of technical products.     

Hydrierung von Lipiden als Hilfsmittel zur Identifizierung und Quantifizierung von Phosphatiden aus Membransystemen des Herzmuskels

Hydrogenation of Lipids for Identification and Quantification of Phosphatides from Pellicle Systems of Cardiac Muscle. It was the aim of our research to show that hydrogenation of lipids is an auxiliary technique in phospholipid analysis of cardiac membranes. This is of interest if a preliminary overview on lipid fractions containing highly unsaturated fatty acids is needed. The fatty acids and the diglycerides from phospholipids were hydrogenated according to the procedure described by Appelqvist (A simple and convenient procedure for the hydrogenation of lipids on the micro- and nanomole scale, J. Lipid Res. 13 (1972), 146) with platinum oxide as a catalyst. The lipids (fatty acid methyl esters or acetylated diglycerides) were taken to dryness in a test-tube under nitrogen and flushed with hydrogen. The catalyst, suspended in methanol was injected through a septum. For identification purposes thin-layer chromatography on silica gel and on silica gel impregnated with silver nitrate was combined with gas chromatography before and after hydrogenation. After hydrogenation the fatty acid profile is much simpler and the fatty acid methyl esters can easily be differentiated from dimethyl acetals, as the latter are more volatile. Diacylglycerides and alkenylacylglycerides were also separated by thin-layer chromatography in individual subclasses before they were analysed by gaschromatography. Hydrogenating the lipids makes it possible to circumvent in part difficulties which arise often with polyunsaturated fatty acids. As the chain length of C20 and C22 are mainly represented by C20:4 , the arachidonic acid and C22:6 the docosahexaenoic acid, both fatty acids can be assessed after hydrogenation. The fatty acid profile of phosphatidylcholine and phophatidylethanolamine of cardiac muscle from rat, guinea pig and pig was determined. Each sample was analysed before and after hydrogenation. The fatty acids with the same chain length were summed up and compared to the corresponding chain length after hydrogenation.     

Lipid,sterol and fatty acid composition of antarctic krill (Euphausia superba Dana)

The lipid classes, fatty acids of total and individual lipids and sterols of Antarctic krill (Euphausia superba Dana) from two areas of the Antarctic Ocean were analyzed by thin layer chromatography (TLC), gas liquid chromatography (GLC) and gas liquid chromatography/mass spectrometry (GLC/MS). Basic differences in the lipid composition of krill from the Scotia Sea (caught in Dec. 1977) and krill from the Gerlache Strait (caught in Mar. 1981) were not observed. The main lipid classes found were: phosphatidylcholine (PC) (33–36%), phosphatidylethanolamine (PE) (5–6%), triacylglycerol (TG) (33–40%), free fatty acids (FFA) (8–16%) and sterols (1.4–1.7%). Wax esters and sterol esters were present only in traces. More than 50 fatty acids could be identified using GLC/MS, the major ones being 14∶0, 16∶0, 16∶1(n−7), 18∶1(n−9), 18∶1(n−7), 20∶5(n−3) and 22∶6(n−3). Phytanic acid was found in a concentration of 3% of total fatty acids. Short, medium-chain and hydroxy fatty acids (C≤10) were not detectable. The sterol fraction consisted of cholesterol, desmosterol and 22-dehydrocholesterol.     

The fatty acids of degras

Degras contains a complex mixture of lipids comprised of branched and normal chain fatty acids, hydroxy acids, sterols, sterol esters and long chain wax esters. There are no glycerides in degras. This paper is a report on the composition of the branched and normal chain fatty acids. Preparative techniques of thin-layer chromatography were used to isolate the fatty acids from the other lipid classes. Gas chromatography was used on three different stationary phase separations of the fatty acid methyl esters. Identifications of the composition were based on a combination of techniques and known standards. Authorized for publication on April 20, 1965, as paper No. 3003 in the journal series of The Pennsylvania Agricultural Experiment Station.     

Cyclic fatty acid monomer formation in frying fats. I. Determination and structural study

The formation of monomeric cyclic fatty acids was studied in a model system in which partially hydrogenated soybean oil (PHSO) was heated intermittently for 80 hr of simulated deep fat frying. Oil samples (fresh and heated) and their methyl esters were fractionated according to their molecular size using gel permeation chromatography (GPC). Oils and GPC fractions were analyzed for cyclic monomers by the following steps: (i) preparation of fatty acid methyl esters (FAME); (ii) microhydrogenation of FAME; (iii) urea fractionation of hydrogenated FAME; (iv) analysis by capillary gas liquid chromatography (GLC), and (v) structural characterization of cyclic monomer peaks by mass spectrometry (GC-MS). Under simulated frying conditions the concentration of cyclic monomers increased from 736 ppm (0.07%) in fresh oil to 1803 ppm (0.18%) in heated oil. GC-MS with capillary columns allowed the identification of several C₁₈ α-disubstituted cyclohexane and cyclopentane isomers as hydrogenated methyl esters. Other noncyclic and contaminant compounds eluting within the expected GLC retention range of cyclic monomers also were identified in all the samples and GPC fractions.     

Composition of the fat extracted from the seeds of theobroma bicolor

The fat fromTheobroma bicolor was analyzed for glyceride content by thin layer chromatography (TLC), and for fatty acid composition and triglyceride (carbon number) composition by gas liquid chromatography (GLC). The fat was then separated into glycerides of different degrees of unsaturation by means of silver nitrate TLC. Then, the bands were examined by GLC before and after conversion to methyl esters. From the results obtained, the distribution of the fatty acids on the individual glycerides was calculated. The fat consisted of 96.5% triglyceride with only 2.5% diglyceride and 1.7% free fatty acid. The major fatty acids present were 42.3% C18:0, 45,2% C18:1, and 6.0% C16:1. Most of the triglycerides were of carbon number 52 (18.0%) and 54 (77.6%). The major triglycerides were 38.6% 1-stearyl-2,3 diolein (SOO), 25.4% 2-oleyl-1,3 distearin (SOS) and 13.8% 1-palmito-2-oleyl-stearin (POS). Only 44.3% of the fat consisted of monounsaturated triglycerides.     

Fatty acid desaturase specificity in tetrahymena

The ciliate,Tetrahymena, was provided a supplement of the fatty acid [1-¹⁴C] 18∶2Δ6,9. After a period of growth the cells were claimed, the lipids extracted, the polar lipids recovered and the mild alkali-labile fatty acid methyl esters generated. The fatty acids were resolved by high pressure liquid chromatography (HPLC), the 18∶3Δ6,9,12 (γ-linolenic acid) was recovered and its identity verified by high pressure liquid chromatography (HPLC), gas liquid chromatography (GLC), hydrogenation and oxidation. Fifty-three percent of the cell-associated label was found in γ-linolenic acid; thus, a Δ12 fatty acid desaturase converts the 6,9 octadecadienoic acid to the 6,9,12 derivative. No carboxyl or methyl terminus restriction appears on Δ9 monoenoic or dienoic fatty acid desaturation in this cell as is found in higher plants and animals.     

Application of methoxy-bromomercuri-adduct fractionation to the analysis of fatty acids of partially hydrogenated marine oils

The fatty acids of a refined and of a partially hydrogenated menhaden oil, iodine value (IV) 84.5, were separated into different classes (e.g., monoene, diene, including pentaene and hexaene) by thin layer chromatography (TLC) of their methoxy-bromomercuri-adducts (MBM). In the solvent system hexane: dioxane, the separation of fatty acids occurred according to the degree of unsaturation. No influence was exerted by either the geometry or the position of the ethylenic bonds. The effect of the various chain lengths (C₁₄−C₂₂) was to broaden the bands, but no overlap occurred among the chain lengths. A wide range of C₂₀ unsaturated fatty acids were prepared by the hydrazine reduction of 20∶5-Δ5,8,11,14,17. These were separated into groups as MBM adducts and identified by comparison of their experimental and calculated equivalent chain lengths (ECL) in gas liquid chromatography (GLC) on SILAR-5CP and SILAR-7CP columns. This confirmed that GLC did not totally separate all groups of isomers of different degrees of unsaturation. The quantitative analysis of both refined and partially hydrogenated (IV-84.5) menhaden oils by GLC was effected by the recovery of the fatty acid methyl esters from the MBM adduct TLC bands with the addition of methyl heptadecanoate (17∶0) as an internal standard, followed by analysis of the different fractions on open-tubular columns coated with SILAR-5CP. For methylene- and nonmethylene-interrupted unsaturated acids, 100% recovery from the MBM adducts was achieved, but in the case of the conjugated dienes the maximal recovery was 70%.     

Regioselective analysis of triacylglycerols by lipase hydrolysis

A modified procedure for the regiospecific analysis of triacylglycerols (TAG) with a 1,3-specific lipase is described. After partial lipase hydrolysis of the triacylglycerol, the released free fatty acids (FFA) and 1,2(2,3)-diacylglycerols (DAG) were isolated by thin-layer chromatography (TLC) and converted to fatty acid methyl esters (FAME). The FAME were analyzed by gas-liquid chromatography (GLC). The 1,3-specific lipases used in this study included supported preparations from strains ofMucor miehei andRhizopus oryzae. The method also was applied to the regiospecific analyses of tung nut and Chinese melon seed oil triacyglycerols, both of which contain high proportions of α-elaeostearic acid. The TAG composition of the oils was substantiated in parallel analysis of the oils by highperformance liquid chromatography with chemical ionization mass spectrometric detection of intact TAG.     

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.     

An analysis of separation factors applicable in the gas-liquid chromatography of unsaturated fatty acid methyl esters on a polyester substrate

The employment of gas-liquid chromatography (GLC) separation factors between methyl esters of unsaturated fatty acids is feasible as a means of tentative identification, either between acids of one chain length and differing numbers of double bonds, or between acids of one chain length and the same number of double bonds in differing positions, provided the acid structures are appropriately grouped by end carbon chain. The modification of separation factors by temperature, chain length, number of double bonds, or position of double bonds is apparent from examination of a larger number of examples than was hitherto available. Examples of the usefulness of separation factors in identifying unknowns or predicting retention times are given.     

Jojoba oil wax esters and derived fatty acids and alcohols: Gas chromatographic analyses

HCl-catalyzed ethanolysis followed by saponification readily surmounts the resistance of long chain wax esters to direct hydrolysis by alkali. Additionally, choosing ethyl instead of methyl esters allows baseline separations between long-chain alcohols and corresponding esters in gas liquid chromatographic (GLC) analysis of total alcohol and acid components before saponification. Liquid wax esters were analyzed on a temperature-programmed 3% OV-1 silicone column. Geographical and genetic effects on the variability of jojoba oil composition were investigated with five different seed samples. Major constituents in jojoba seed oil from shrubs in the Arizona deserts, as indicated by GLC analyses of oil, ethanolysis product, isolated fatty alcohols and methyl esters of isolated fatty acids, were C₄₀ wax ester 30%, C₄₂ wax ester 50% and C₄₄ wax ester 10%; octadecenoic acid 6%; eicosenoic acid 35%, docosenoic acid 7%, eicosenol 22%, docosenol 21% and tetracosenol 4%. Oil from smaller leaved prostrate plants growing along California’s oceanside showed a slight tendency toward higher molecular size than oils from the California desert and Arizona specimens. The wax esters are made up of a dispro-portionately large amount of docosenyl eicosenoate and are not a random combination of constituent acids and alcohols.Lunaria annua synthetic wax ester oil was used as a model for evaluating the analytical procedures. Presented at the AOCS Meeting, Chicago, September 1970 No. Utiliz, Res. Dev. Div., ARS, USDA.