Partial argentation resin chromatography (PARC): III. the effects of sodium ion incorporation and solvent on the separation of mixtures of fatty acids,of fatty esters,and of triglycerides

Partial argentation resin chromatography (PARC) was used to separate mixtures of fatty acids, of fatty esters and of triglycerides. Various eluting solvents (methanol, acetone, ether, benzene) were investigated and acetone was the preferred solvent. To prevent acid-catalyzed condensation of the acetone, the sulfonic acid protons of the XN1010 resin were replaced with sodium ions before silver ion incorporation. Atomic absorbtion spectrophotometry was used to determine the number of silver ions incorporated per volume of resin. A mixture containing stearic, elaidic and oleic acids was separated on a saturated silver resin column. A commercially available sample of linoleic acid was purified on a 53% PARC column (meaning 53% of the available sulfonic acid protons in the resin were replaced by silver ions). A 39% PARC column was used to separate a mixture of linseed acids. Methyl linoleate, linolenate and arachi-donate also were isolated on a 39% PARC column. A mixture containing triolein, trilinolein and trilinolenin was separated on a 32% PARC column.     

Use of argentation column chromatography in the identification of fish oil fatty acids by GLC: Application to cod liver oil

The fatty acid methyl esters from cod liver oil have been fractionated into seven fractions, depending on degree of unsaturation, by a silver nitratetreated silicic acid column. The fractions have been analyzed by gas liquid chromatography and the components identified by usual techniques.     

The triglycerides of sable fish(Anaplopoma fimbria). I. Quantitative fractionation by column chromatography on silica gel impregnated with silver nitrate

The triglycerides of sable fish muscle oil were eluted from a silver-nitrate-impregnated silica gel column with petroleum ether containing increasing amounts of ethyl ether as solvents. Eighteen peak fractions were analyzed for fatty acid composition. Calculated iodine values progressed regularly from 38 to 115. The detailed data show that the fractionation did not depend upon the presence of individual fatty acids but on total unsaturation. Not all of the triglycerides containing polyunsaturated fatty acids were eluted.     

Fractionation of commercial frying oil samples using sep-pak cartridges

Commercial frying oil samples were fractionated by column chromatography on hydrated silicic acid according to the standardized DGF-IUPAC-AOAC method. The non-polar fraction was isolated using a mixture of petroleum ether:diethyl ether (87:13), while the polar fraction was eluted by diethyl ether. These used frying oil samples were also fractionated using Sep-Pak cartridges. The non-polar fraction was eluted with 20 ml of a mixture of petroleum ether:diethyl ether (92:8), while the polar fraction was eluted with methanol. The purity of each fraction was studied by thin layer chromatography (TLC) and by the Iatroscan TLC/FID system using a mixture of hexane:tetrahydrofuran:acetic acid (97:3:1) as solvent system. The Sep-Pak and the standardized methods gave similar results. This indicates that the state of degradation of a frying oil (detection of polar components) could be studied using Sep-Pak cartridges, which is less time- and solvent-consuming than column chromatography.     

Säulen-chromatographische Bewertung der Oxydationsstufe von Fetten und Ölen

Column Chromatographic Estimation of the Extent of Oxidation of Fats and Oils Oxidized fats and oils can be separated into oxidized and unoxidized fractions by column chromatography with aluminium oxide as adsorbent and benzene and ethanol-diethyl ether mixture as eluting solvents. The method is suitable for the evaluation of advanced oxidation where chemical methods can lead to false results.     

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.     

Lipids in sea water

Acidified and filtered sea water samples which were extracted with petroleum ether and ethyl acetate have been shown to contain a variety of lipid compounds in trace amounts. Concentrations of these solvent-soluble substances ranged from 0.5 to 6.0 mg/liter, the lower concentrations being found in offshore waters. The solvent extracts of the sea water were separated into eight lipid classes by column chromatography on silicic acid. The fractions eluted with solvents of increasing polarity were characterized by thin-layer chromatography, infrared and ultraviolet absorption and gas chromatography. These techniques revealed a complex mixture of alkanes, alkenes, fatty acids, steroids, phospholipids and many as yet unidentified components. Twenty to thirty alkanes were present as indicated by gas chromatography. No aromatic hydrocarbons were detected. Chromatography of the methyl esters of the fatty acids indicated the presence of acids with chain lengths varying from 14 to 22 carbons, both saturated and unsaturated. In many samples the unsaturated fatty acids containing 18 to 22 carbons predominated. The lipid components varied somewhat in composition as well as concentration from location to location and with season and depth.     

Ultramicro fatty acid analysis of polar lipids: Gas-liquid chromatography after column and thin-layer chromatographic separation

A procedure is described for the analysis of the fatty acid composition of polar lipid classes in the nanogram range. The lipids are first fractionated by column chromatography followed by further separation into pure lipid classes by thin-layer chromatography. Lipid spots scraped from the thin-layer plates are esterified directly (i.e., without prior elution) with 6% sulfuric acid in methanol. The methyl esters are then analyzed by gasliquid chromatography with a hydrogen flame ionization detector. Samples of 200 nanograms or less give accurate results with helium as carrier gas, oxygen rather than air to support combustion, careful adjustment of the recorder and general attention to optimum electrical connections, dissociation of the column oven from the recorder and electrometer, and careful preconditioning of columns. Under proper conditions the base line is stable and a 10% of full scale deflection of the recorder can be obtained from 1 nanogram of a methyl ester, allowing highly precise analyses of fatty acid composition from the amount of lipid obtainable from one spot on a thin-layer chromatogram. Control studies demonstrated that extraneous peaks did not arise from the procedure or from the sphingosine and dihydrosphingosine of sphingolipids. The thin-layer chromatographic procedure did not influence the fatty acid composition of a pure sample of glucocerebroside isolated by column chromatography and the method was applied to lecithin and sphingomyelin or normal and pathological human brain specimens.     

Application of Methoxy-Bromomercuric-Adduct Fractionation to the Study of Cyclic Fatty Acid Monomers from a Heated Linseed Oil

A linseed oil was heated at 275° C for 12 hr under nitrogen. The triglycerides were isolated from the polymers by gel permeation chromatography on 2μ-styragel columns using tetrahydrofuran (THF) as solvent. One part of the isolated triglyceride fraction was submitted to HPLC on a C₁₈ reverse phase column. The resulting 8 fractions were transformed into methyl esters and hydrogenated on PtO₂ The content of cyclic fatty acid monomers (CFAM) in the different hydrogenated fractions was determined by gasliquid chromatography (GLC) on a Silar-10 C column. The other part of the isolated triglyceride fraction was submitted to the action of BF₃/MeOH. The resulting methyl esters were fractionated by thin-layer chromatography of the corresponding methoxybromomercuric adducts. Five bands were obtained using a mixture of hexane: dioxane (60:40) as the solvent system. A band between the diene and the triene bands contained 40.6% of the total cyclic fatty acid monomers (CFAM) while the diene and the monoene band contained respectively 38.9 and 11.2% of these cyclic components. The diene band and the band between the diene and the triene bands were hydrogenated on PtO₂ The structures of the resulting hydrogenated cyclic fatty acid monomers were studied using a gas-liquid chromatograph coupled with a mass spectrometer (GC-MS).     

Determination of the country of origin of pistachio nuts by DSC and HPLC

The laboratories of the U.S. Customs Service frequently encounter the major analytical problem of determining the country of origin of imported commodities. This is particularly challenging with natural products. This paper will describe the process for one such commodity —pistachio nuts. Two approaches were taken, high performance liquid chromatography (HPLC) and Differential Scanning Calorimetry (DSC). It was found that the results of these two techniques were complimentary and confirmatory. The HPLC data was obtained on the petroleum ether soluble fraction of the pistachio nuts while the DSC runs evaluated both this fraction and the whole nut. The HPLC results were further investigated by collecting fractions from the eluting peaks, derivatizing them to form the methyl ester of the fatty acids, and profiling by gas chromatography to confirm the triglycerides originally present. The U.S. Customs Service is currently using DSC area ratios to screen imported pistachio nuts for country of origin.     

Studies on lipid and fatty acid composition of human hepatoma tissue

Studies are reported on the composition of the lipids of human liver and hepatoma tissues from male adults. Liver tissues were obtained from individuals who died from causes other than liver disease or cancer. The hepatoma tissues were obtained from individuals shortly after they succumbed to cancer. The total lipid of each tissue was fractionated quantitatively by silicic acid column chromatography into neutral lipid, glycolipid, and phospholipid fractions. These fractions were analyzed by thin layer chromatography and converted to methyl esters for analysis of their constituent fatty acids by gas liquid chromatography. In comparison to liver tissue, the total amount of lipid in the hepatoma tissues was generally higher and more variable; the lipid of one hepatoma was ca. 92% of the dry wt of the tissue. The greater lipid content of the hepatoma tissues was due to the high percentage of neutral lipid. Except for one specimen, there was ca. the same amount of glycolipid in the hepatoma as in the liver tissues, but the composition of the glycolipid fraction of the hepatoma lipid differed considerably, particularly in the ganglioside fraction. The phospholipid fraction of hepatoma lipid was much lower than that of liver but exhibited only quantitative differences in composition. No glyceryl ether diesters and only traces of plasmalogens of phosphatidyl choline or phosphatidyl ethanolamine were detected in the liver and hepatoma lipids. The levels of monoenoic acids were higher and those of linoleic and polyunsaturated fatty acids lower in the hepatoma lipids. Positional isomers of trienoic acids not normally present in liver tissue were detected in hepatoma lipids. The abnormalities observed in lipid composition indicated interferences in the regulatory processes of lipid metabolism in human hepatoma similar to those observed in animals.     

Liquid Chromatographic fractionation of oil-sand and crude oil asphaltenes

Asphaltenes extracted from Alberta oil sands (Athabasca, Cold Lake, and Peace River) and crude oils (Taber South and Fenn-Big Valley) were fractionated by sequential elution solvent chromatography (SESC) involving 10 organic solvents on a silica column. Athabasca asphaltenes and SESC fractions were further studied by elemental analysis, i.r., u.v., and n.m.r. spectroscopy. Incomplete extraction of maltenes from the oil-sand bitumens increased the yields of the first two SESC fractions, the saturates and aromatics, of oil-sand asphaltenes relative to the crude oil asphaltenes. About 55 wt% of the asphaltenes elute in fractions 3–5. Two distinct molecular types are present in the asphaltenes; namely, lower functionality species with lower heteroatom content and the higher functionality species with higher heteroatom content. Compounds eluting in fractions 3–10 are predominantly polynuclear aromatics with alkyl substituants and probably bridged by cycloalkanes. The extent of bridging as well as the location, number and type of heteroatoms determines the fraction in which each compound appears. Complexity of compounds eluting increases with time: earlier fractions are composed of smaller-size polynuclear aromatic centers and contain heteroatoms in predominantly ring locations, whereas later fractions contain a larger proportion of complex species and more functional heteroatom groups.     

Fatty acids of cows' milk. B. Composition by gas-liquid chromatography aided by other methods of fractionation

A fresh sample of cows' milk was converted directly to the methyl esters by methanolysis and the esters were fractionated into eleven distilled fractions and an undistilled portion. The latter, which contained the bulk of the polyunsaturated and long chain esters, was further fractionated by adsorption chromatography on a silicic acid column. Each fraction from the distillation and adsorption chromatography was analyzed by gas-liquid chromatography on columns containing polyester stationary phases of different polarity. Twenty-seven minor components, including some not previously reported, were present each in less than 0.1%. The fatty acid distribution of the major components fell within the range generally reported. Presented at the 53rd Meeting of the American Oil Chemists' Society, October 17–19, 1960, New York. Eastern Utilization Research and Development Division, Agricultural Research Service, United States Department of Agriculture.     

High density lipoproteins from bovine plasma and follicular fluid do not possess a high affinity for glycosaminoglycans

Possible interactions between glycosaminoglycans and high density lipoproteins (HDL) in plasma and follicular fluid were examined. Total lipoproteins (d<1.21 g/ml) were obtained from plasma of five Holstein cows by ultracentrifugation and fractionated by gel filtration. Every other fraction from the HDL peak or fractions corresponding to the base and ascending portion of the HDL peak were composited and applied to a heparin-Sepharose affinity chromatography column. Elution profiles from both composites showed a peak that did not bind to the column that contained HDL devoid of apolipoprotein-E as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining and immunoblot analysis. Elution of lipoproteins from the ascending portion of the HDL peak resulted in a second minor peak eluting at 0.35 M NaCl, which was low density lipoprotein (LDL) contamination. Lipoproteins (d<1.21g/ml) isolated from follicular fluid obtained from small, medium or large follicles also were subjected to heparin-Sepharose affinity chromatography. Two peaks were observed, one corresponding to the lipoprotein that did not bind to the column, the other eluted at 0.5M NaCl and accounted for less than 2% of the protein applied. The second peak did not contain apolipoprotein-E or LDL. Bovine follicular fluid glycosaminoglycans (GAG) were isolated and subjected to HDL-Sepharose affinity chromatography. Less than 2% of the total GAG bound to the HDL column. Therefore, HDL in bovine specimens did not interact appreciably with heparin or GAG isolated from follicular fluid.     

The fatty acid composition of purified fractions of cold-pressed peanut oil

Cold-pressed peanut oil was separated into chromatographically homogeneous fractions by column chromatography. The fatty acid composition of the major fractions was determined by gas chromatography. The phosphatides, and especially the cephalins, had a higher palmitate content than did the triglycerides. Palmitate was the dominant fatty acid in the phosphatidyl-serines.     

Chromatographie analysis of seed oils. Fatty acid composition of castor oil

The fatty acid composition of a number of domestic and foreign castor oils was determined by consecutive column and gas-liquid Chromatographic analysis. After saponification of the oils and removal of the unsaponifiables, the nonhydroxy, monohydroxy, and dihydroxy acids were fractionated by partition chromatography on silicic acid. The amount of acid in each fraction was determined by titration or weighing. Gravimetric data were in good agreement with the titrimetric data. The acids obtained by saponification were converted to methyl esters with diazomethane and similarly subjected to partition chromatography. The methyl esters from various fractions were analyzed by gas-liquid chromatography. Components were tentatively identified by their comparative retention times and confirmed Presented at the AOCS meeting in Chicago, 1961. A laboratory of the Western. Utilization Research and Development Division, Agricultural Research Service, U.S.D.A.     

Thin layer and anion exchange chromatography of soybean saponins

Twenty-two solvent systems were evaluated for TLC of soybean saponins on silica gel. A maximum of four fractions separated by single development with the different solvents. Six successive developments with chloroformmethanol-water (65:25:4) separated soybean saponins into 10 or more fractions. Column chromatography of soybean saponins on an anion exchange resin with a linear gradient of acetic acid yielded seven fractions. Multiple development TLC of the saponins separated by column chromatography showed that a definite fractionation occurred. Several column chromatographic fractions contained components with identical TLC R_f values, but the components were clearly different on the basis of colors detected with sulfuric acid and on the basis of their elution positions from the ion exchange resin. Soybean saponins are more complex mixtures than previously recognized but can be fractionated by chromatography on thin layer plates and anion exchange resins. No. Utiliz. Res. Dev. Div., ARS, USDA.     

Distribution of cholesteryl esters and other lipid in subcellular fractions of the adrenal gland of the pig

Total lipids from whole pig adrenal glands as well as from their mitochondria, microsomes, liposomes, and cell sap were extracted and fractionated first into neutral lipids and phospholipids. The highest percentage of neutral lipids was found in the cell sap, and the lowest in the microsomal fraction. Neutral lipids were subfractionated into cholesteryl esters, free cholesterol, triglycerides, and free fatty acids. Cholesteryl esters were distributed throughout the liposomes. Free fatty acids represented a substantial part of cell sap lipids, but were present also in the mitochondria, microsomes, and liposomes. Fatty acids of all fractions were analyzed by gas liquid chromatography. Free fatty acids and cholesteryl ester fatty acids from all cellular fractions were similar in composition and were characterized by considerable quantities of linoleic and arachidonic acid. Triglycerides were characterized by an increased percentage of palmitic and a low content of arachidonic acid. Phosphatidyl choline, phosphatidyl ethanolamine, diphosphatidyl glycerol, and sphingomyelin plus phosphatidyl inositol were isolated from the lipids by preparative thin layer chromatography, and their fatty acids analyzed by gas liquid chromatography. Phosphatidyl choline and phosphatidyl ethanolamine from mitochondria, microsomes, and cell sap were very similar in respect of their fatty acid composition. Sphingomyelin plus phosphatidyl inositol was characterized by a high content of C22:2omega6. Diphosphatidyl glycerol was present in mitochondria and in the cell sap.     

Comparative evaluation of solvent extraction methods for the determination of neutral and polar lipids in beef

Samples of lean (< 5% fat), medium (13–15%) and high-fat (> 20%) ground beef were extracted for total lipid by 4 methods of wet extraction employing chloroform/methanol (CM), n-hexane/iso-propanol (HIP) and ethyl alcohol/ethyl ether (AE), and by 3 methods of soxhlet extraction of freeze-dried material by petroleum ether (PE) or eithyl ether (EE), CM and methylene chloride/ methanol (MM). The purified lipid was fractionated into neutral and polar lipid fractions by silicic acid chromatography and the frac-tions were analyzed for fatty acid distribution by gas liquid chroma-tography (GLC). The soxhlet procedure employing either PE or EE extracted less than 75% of total lipid, 89% of triglycérides and 15% of polar lipids from lean beef as compared to other methods, and as the fat content increased from 3 to 20%, extracted amounts of polar lipid which increased to 40% of that extracted by other methods. The fatty acid distribution of the fractionated triglycerides and polar lipids was generally within experimental error for each frac-tion, irrespective of the method of extraction. The percentages of 16:0 and 18:1 were significantly less in polar lipids than in trigly-cerides. In addition to significantly higher percentage of 18:2, the polar lipids contained up to 20% of long-chain fatty acids not detected in triglycerides. The soxhlet procedures with CM or MM were as effective as wet extraction procedures in extracting neutral and polar lipids. Presented at the 73rd AOCS annual meeting, Toronto, 1982. Contribution No. 512, Food Research Institute, Agriculture Canada.     

Chemical reactions involved in the deep-fat frying of foods: VIII. Characterization of nonvolatile decomposition products of triolein

Triolein was heated under simulated deep-fat frying conditions at 185 C for 74 hr. The thermally oxidized triolein was converted into methyl esters and then fractionated by urea exclusion. The urea adduct-forming ester (89.2%) was found to be methyl oleate unchanged by the frying treatment. The nonurea adduct-forming esters (10.8%) were further fractionated by silicic acid column chromatography into nine fractions with molecular weights ranging from 304 to 742. Physical and chemical analyses of the fractions indicated that some of them contained oxygen atoms which could not be accounted for by ordinary functional group analyses. The polymers isolated were formed by both carbon-to-carbon and carbon-to-oxygen linkages. The nonpolar dimers were further purified by thin layer and gas chromatography. Structure elucidation revealed that they consisted of a noncyclic dimer and a noncyclic dimer containing a carbonyl group, each of which amounted to 1.36% of the triolein originally used. The polar polymers were studied by depolymerization and the analysis of the depolymerized products. It was estimated that the triolein used for simulated deep fat frying contained 1.1% trimers formed through carbon-to-carbon linkages, 1.9% dimers and trimers joined through carbon-to-carbon linkages, and 3.1% dimers and trimers joined through carbon-to-oxygen or carbon-to-carbon linkages in the same molecule and also dimers and trimers in which all the monomeric units were joined through carbon-to-oxygen linkages. The precise form of the oxygen linkages are not known. However, the fact that it is not cleavaged by HC1 and HI suggests ether linkages.