Analysis of lipid classes by solid-phase extraction and high-performance size-exclusion chromatography

An improved method to analyze lipid classes of edible oils and fats by solid-phase extraction (SPE) and high-performance size-exclusion chromatography (HPSEC) is presented. A mixture of lipid standards was fractionated by the solid-phase extraction procedure (NH₂ phase) into polar and nonpolar fractions; these were then submitted to analysis by HPSEC. The size-exclusion chromatographic columns were three styrene/divinylbenzene columns with pore sizes of 100 Å and 50 Å. Light-scattering was used for the detection system, and the parameters of the detector were optimized to minimize the difference between the responses of the compounds studied. With this procedure it was possible to separate the following lipid classes: triacylglycerols, diacylglycerols, monoacylglycerols and free fatty acids, sterols, sterol esters, tocopherols and carotenoids. Quantitative analysis was studied for a light-scattering detector with several lipid standards of different molecular weights and unsaturation levels.     

Trace constituents in milk fat: Isolation and identification of oxofatty acids

Ca. 1% of the glycerides of milk fat contain oxofatty acids. The isolation, fractionation, and characterization of oxofatty acids were accomplished using the following sequence of steps: (A) transmethylation, (B) conversion into 2,4-dinitrophenylhydrazones, (C) adsorption of the 2,4-dinitrophenylhydrazones on magnesium oxide to eliminate the colorless lipid, (D) fractionation of the 2,4-dinitrophenylhydrazones into non-oxofatty acid and oxofatty acid fractions on alumina, (E) separation of the oxofatty acid 2,4-dinitrophenylhydrazones into saturated and unsaturated classes by argentation column chromatography, (F) separation of these classes by chain length using liquid-liquid column and thin layer partition chromatography, (G) resolution of positional isomers by thin layer chromatography, (H) regeneration of the positional isomer 2,4-dinitrophenylhydrazones, and (I) analysis of the parent oxofatty acids by gas liquid chromatographymass spectrometry. In this manner, 36 saturated and 11 unsaturated oxofatty acids were identified tentatively or positively. The saturated oxofatty acids ranged in chain length from C₁₀–C₂₄, predominantly C₁₈ and C₁₆, and generally contained an even number of carbon atoms. The unsaturated oxofatty acids ranged from C₁₄–C₁₈, with C₁₈ predominating.     

Phospholipids of the pulmonate land snailCepaea nemoralis (L.)

The phospholipids of the snailCepaea nemoralis, comprising the major lipid fraction (65%) in this terrestrial pulmonate, were investigated by thin-layer and column chromatography. Detailed gas chromatographic analyses of liberated fatty acid fractions and amino acid analyses of the water soluble moieties of isolated phospholipid classes were carried out. Phosphatidyl choline (47%) and phosphatidyl ethanolamine (21%) were found to be the predominant phospholipid classes, while phosphatidyl serine (8%), phosphatidyl inositol (6%), diphosphatidyl glycerol (3%), ceramide amino-ethylphosphonate (7%), lysophosphatidyl choline (1%), and phosphatidic acid (1%) were present in lesser amounts. In the phosphatidyl ethanolamine and phosphatidyl serine fractions, minor quantities of plasmalogen analogues were detected. Fatty acid profiles of the various phospholipid classes appeared to be strikingly diverse, e.g. a characteristic component, such as linoleic (18∶2ω6) acid, ranging from 3–54%. In vivo radioisotope studies using 1-¹⁴C-acetate demonstrated the high biosynthetic rate of all phospholipid classes and their respective fatty acid fractions. Results are discussed in relation to data on the phospholipids from other invertebrate species.     

Analysis of lipid classes and lipofuscin substances by high performance liquid chromatography

The fractionation and analysis of the lipid classes and fluorescent substances of animal tissues by high performance liquid chromatography (HPLC) using a combination of fluorescence and flame ionization detectors is described. The lipid classes and fluorescent substances are extracted from rat kidney and liver tissue by a new method that involves preextraction of nonlipid and aqueous-soluble fluorescent substances with hot dilute (0.05 N) acetic acid. The lipid classes and organic-soluble fluroscent substances are extracted from the residual tissue in three extractions: the first with chloroform/methanol, 1:1, v/v; the second with chloroform/methanol, 1:2, v/v; and the third with methanol. The fractionation of these compounds by HPLC is carried out with a column 0.2×45 cm, packed with a special adsorbent prepared by reacting silicic acid with ammonium hydroxide. The eluent is passed through a fluorescence detector, which provides a profile of the fluorescent compounds, and then to a flame ionization detector for analysis of the lipid classes. The method is demonstrated on rat blood serum, liver and kidney tissue.     

Fractionation of menhaden oil and partially hydrogenated menhaden oil: Characterization of triacylglycerol fractions

Menhaden oil (MO) and partially hydrogenated menhaden oil (PHMO) were dry-fractionated and solvent-fractionated from acetone. After conversion to fatty acid methyl esters, the compositional distribution of saturated, monounsaturated, trans, and n−3 polyunsaturated fatty acids (PUFA) in the isolated fractions was determined by gas chromatography. Acetone fractionation of MO at −38°C significantly increased the n−3 PUFA content in the liquid fractions over that of starting MO (P<0.05). For PHMO, liquid fractions obtained by low-temperature crystallization (−38, −18, and 0°C) from acetone showed significant increases (P<0.05) in monounsaturated fatty acid (MUFA) content over that of the starting PHMO. For selected MUFA-enriched fractions, reversed-phase high-performance liquid chromatography (HPLC) was used to separate, isolate, and characterize the major triacylglycerol (TAG) molecular species present. Thermal crystallization patterns for these fractions also were determined by differential scanning calorimetry (DSC). The results demonstrated that under the appropriate conditions it is possible to dry-fractionate or solvent-fractionate MO and PHMO into various solid and liquid fractions that are enriched in either saturated, monounsaturated, polyunsaturated, or the n−3 classes of fatty acids. Moreover, characterization of these TAG fractions by reversed-phase HPLC gives insight into the compositional nature of the TAG that are concentrated into the various fractions produced by these fractionation processes. Finally, the DSC crystallization patterns for the fractions in conjunction with their fatty acid compositional data allow for the optimization of the fractionation schemes developed in this study. This information allows for the production of specific TAG fractions from MO and PHMO that are potentially useful as functional lipid products.     

Transfer of Lauric and Myristic Acid from Black Soldier Fly Larval Lipids to Egg Yolk Lipids of Hens Is Low

Implementing insects, such as the black soldier fly larvae (BSFL), as animal feed commonly includes the previous removal of substantial amounts of fat. This fat may represent an as yet underutilized energy source for livestock. However, transfer of lauric and myristic acid, prevalent in BSFL fat and undesired in human nutrition, into animal-source foods like eggs may limit its implementation. To quantify this, a laying hen experiment was performed comprising five different diets (10 hens/diet). These were a control diet with soybean oil and meal and a second diet with soybean oil but with partially defatted BSFL meal as protein source. The other three diets were based on different combinations of partially defatted BSFL meal and fat obtained by two different production methods. Lauric acid made up half of the BSFL fat from both origins. Both BSFL fats also contained substantial amounts of myristic and palmitic acid. However, in the insect-based diets, the net transfer from diet to egg yolk was less than 1% for lauric acid, whereas the net transfer for myristic and palmitic acid was about 30% and 100%, respectively. The net transfer did not vary between BSFL originating from production on different larval feeding substrates. The results illustrate that hens are able to metabolize or elongate very large proportions of ingested lauric acid and myristic acid, which are predominant in the BSFL lipids (together accounting for as much as 37 mol%), such that they collectively account for less than 3.5 mol% of egg yolk fatty acids.     

Beeswax Fractions Used as Potential Oil Gelling Agents

The objective of this work was the fractionation of beeswax to investigate the phase behavior of binary blends of the fractions and their potential use as gelling agents in edible oleogels. We have extracted seven distinctive fractions, using preparative flash chromatography with eluents permitted to be used in the processing of food raw materials. The odor profile of the fractions was characterized. The high purity of collected fractions was shown through thin-layer chromatography, Fourier-transform infrared spectroscopy, and high-performance liquid chromatography with an evaporative light scattering detector methods for hydrocarbons (94.8%), monoesters (97.6%), and mixed mono-, di-, and triesters fraction (98.7%). Free fatty acids and fatty alcohol fractions had lower purity which equals 82.6% and 39%, respectively. The analysis of the binary pseudo phase diagram revealed that all fractions combined with hydrocarbons express eutectic behavior. Combinations of all other fractions resulted in the formation of solid solutions. This study shows that the texture of oleogels can be improved by using a combination of various fractions of beeswax instead of native wax.     

Seasonal Variations of Lipid Content and Composition in <Emphasis Type="Italic">Perna viridis</Emphasis>

The total lipid content, composition of main lipid classes, composition of sterols and composition of fatty acids in the main glycerolipids of Perna viridis were analyzed through four seasons using TLC-FID and GLC. Mussel samples were collected during different seasons between 2003 and 2004 from Shengsi Island, Zhejiang Province, China and stored frozen prior to freeze-drying and lipid extraction. Ten grams of dried mussel powder of each season were analyzed. Total lipid content ranged from 14.5 g/100 g in spring month to 7.8 g/100 g dried mussel powder in autumn month. The predominant lipid in spring month was triacylglycerol (TAG), however, in the other three seasons the phospholipids (PL) was the main lipid class. The most abundant fatty acid in TAG, PL and phosphatidylcholine (PC) was 16:0, with the summer samples having the highest proportion (24-30% of total fatty acid) and winter the lowest (14-22%). In phosphatidylethanolamine (PE), the spring samples had the highest proportions of 16:0. The predominant polyunsaturated fatty acids (PUFA) were 22:6n-3 and 20:5n-3 in TAG, PL, PE and PC (25-40%). The proportions of 22:6n-3 and 20:5n-3 were higher in spring than in other seasons in PL and PE. There were nine sterols identified, with cholesterol being the predominant sterol, and other main ones were desmostersol/brassicasterol and 24-methylenecholesterol. Proportions of other fatty acids in different lipid fractions and the sterol compositions as well also varied seasonally. There were subject to the seasonal variations. Differences in lipid content and composition, fatty acid composition in different lipid fractions may be caused by multiple factors such as lifecycle, sex, variation of plankton in different seasons and temperature, which could influence physiological activities and metabolism.     

Präparative Schichtchromatographie phosphatidhaltiger Lipide

Preparative Layer Chromatography of Lipids Containing Phosphatides According to the method described by H. Halpaap, the separations of microgram amounts of lipids containing phosphatides on precoated silicagel plates have been scaled up to milligram level using 2 mm precoated layers. Following this procedure the composition of lipid mixtures containing phosphatides, such as those from soybean and some commercial preparations, was determined by gravimetry. In order to check the fractionation by preparative layer chromatography, the isolated fractions were re-chromatographed on thin-layer plates, following the dry transfer technique. This simple technique combined with gas chromatography was employed for the determination of fatty acid composition of bile lipid fractions.     

Nonpolar-volatile lipids from soy protein isolates and hexane-defatted flakes

Lipid extracts from two samples of commercial soy protein isolates (SPI) and two samples of commercial hexane-defatted flakes were fractionated by silici acid-column chromatography. The material eluted with 100% chloroform was collected, further fractionated by silica solid-phase extractions, and analyzed by gas chromatography-mass spectroscopy by using mass spectra, retention times of authentic standards, and Kovats indices for identification. Thirty-eight compounds were identified and quantitated in the lipid fractions from soy protein isolates (SPI); 23 of these are reported for the first time as components of SPI. An additional 13 compounds are reported for the first time as components of hexane-defatted soybean flakes. The major classes of compounds reported for the first time associated with SPI include: butyl, methyl, and ethyl esters of fatty acids; phenols, diphenyls and phenyl esters; and abietic acid derivatives. Dehydroabietinal at 0.180 to 0.191 ppm of the protein isolates was the most abundant aldehyde in the SPI lipid extracts. The third most abundant aldehyde found in SPI after dehydroabietinal and hexanal was 2-butyl-2-octenal (0.065 to 0.086 ppm). Dehydroabietic acid methyl ester was present in SPI (0.309 to 0.459 ppm). Dehydroabietene (0.628 ppm) and abietatriene (0.396 ppm) were tentatively identified in one sample of hexane-defatted flakes.     

从雨生红球藻藻泥中选择性提取虾青素

以雨生红球藻湿藻泥为原料,研究了不同有机溶剂对胞内油脂和虾青素选择性提取分离的影响,通过酸解破壁提高虾青素和油脂的提取效率。结果表明,连续乙醇提取可对胞内色素和油脂有效分级提取,先提取出极性组分(叶绿素和极性脂),再提取中性组分(类胡萝卜素和中性脂)。中等极性溶剂或溶剂体系对类胡萝卜素的选择性和提取率较好;乙醇/乙酸乙酯混合溶剂提取类胡萝卜素的总得率(干重)达25.31 mg/g,提取率为69.35%。对雨生红球藻湿细胞进行酸解破壁处理有助于提高虾青素和油脂的提取率。在最优酸解破壁条件(盐酸浓度1 mol/L,温度60℃,时间60 min)下,含水80%的雨生红球藻藻泥的油脂总得率(干重)达418 mg/g,总脂提取率达97%。     

Lipids of cultured hepatoma cells: V. Distribution of isomeric monoene fatty acids in individual lipid classes

Monoenoic acid fractions were isolated from phosphatidylcholines, phosphatidylethanolamines, triglycerides, and cholesterol esters derived from minimal deviation hepatoma 7288C cells cultured on 11 media containing varying levels of serums and lipids. Hexadecenoate (16∶1), octadecenoate (18∶1), and eicosenoate (20∶1) fractions were subjected to ozonolysis and the isomeric composition of the monoene fractions determined quantitatively by gas liquid chromatography. The 16∶1 fractions consisted of palmitoleic acid, the Δ9 isomer (85–90%), and the Δ11 isomer (10–15%) in most of the cases; growth media and lipid class origin had little effect upon composition. The predominate acids of the 20∶1 fraction were the Δ13 and Δ11 isomers. Generally, the Δ13 isomer was present in the highest concentration, and this isomer was higher in phosphatidylcholines than the other classes. Vaccenic acid represented 33–66% of the 18∶1 fraction, and the balance was oleic acid. Oleic acid concentrations decreased, and vaccenic acid levels increased as the growth medium serum and lipid levels decreased. Lipid classes did not exhibit any distinct preference for either isomer. These data represent the first quantitative isomeric analysis of monoenoic acids derived from individual lipid classes and are the first to show the occurrence of high levels of vaccenic acid in neoplastic cells. This study suggests that the elevated levels of oleic acid, one of the most frequently observed changes in tumor lipids, may, in fact, represent elevated levels of vaccenic acid.     

A high hydroxyl group content found in high molecular weight fractions of poly(oxyethylene glycol)

The results of a fractionation of high molecular weight poly(oxyethylene glycol) with the mixture benzene—isooctane are presented. The fractions are characterized by gel permeation chromatography (GPC), infrared spectroscopy, viscometry, and dialysis. A high hydroxyl content was found for the higher fractions, which is not compatible with a linear polyoxyethylene glycol molecule with hydroxyl endgroups. The presence of hydroxyl groups on the chain is improbable. The dialysis of the higher fractions in CCl₄ and toluene shows that a surprising amount passes through the dialysis bag. The possibility of degradation of the polymer is considered. However, GPC analysis of the products of the dialysis suggest that the high molecular weight is made up of aggregates of middle-sized molecules and low molecular weight ones, held together by hydrogen bonding between hydroxyl and ether groups. Some results of a fractionation in water with the lower critical solubility temperature at 99°C. are given.     

Analysis of complex lipid mixtures by thin-layer chromatography and complementary methods

Conclusion Further work has been done on the application of thin-layer adsorption chromatography to the fractionation of complex lipid mixtures into classes. New methods, the use of siliconized silicic acid plates and the application of thin-layer adsorption chromatography combined with the complementary techniques of gas-liquid chromatography and paper chromatography, are presented for the resolution of classes of lipids into their constituents. In contrast to such elaborate conventional techniques as column chromatography, analyses using the methods reported in this paper can be performed rapidly in large numbers on a routine basis. Supported in part by the U. S. Public Health Service National Institutes of Health Research Grant 5817, and the National Science Foundation.     

In vitro incorporation of elongated fatty acyl products into lipid classes in the housefly,Musca domestica L. and the American cockroach,Periplaneta americana (L.)

Thein vitro incorporation of elongated fatty acyl products into various lipid classes was studied in the American cockroach,Periplaneta americana (L.) and the houseflyMusca domestica L. Stearoyl-CoA (18∶0-CoA) and linoleoyl-CoA (18∶2-CoA) were each elongated in microsomal preparations from abdominal epidermal tissue of the adult cockroach. Incorporation of radioactive tracer into different lipid classes was determined by thin-layer chromatography (TLC). In the American cockroach, 40–45% of the total radioactive label was incorporated into the free fatty acid fraction, with smaller amounts in the triglyceride (12–31%) and phospholipid (12–19%) fractions. Of the elongated products analyzed by radio-high performance liquid chromatography (HPLC), 53–60% was found in the free fatty acid fraction. In the housefly, the substrates 18∶0-CoA and 18∶1-CoA were used to determine into which lipids the elongated products would become incorporated. The saturated fatty acyl elongated products were found mainly in the free fatty acid (41%), triglyceride (23%), and acyl-CoA (17%) fractions. The monounsaturated fatty acyl elongated products were found in the triglyceride (44%), free fatty acid (11%), acyl-CoA (35%) and phospholipid (10%) fractions in three-day-old males. In three-day-old females, the elongated products were found in the triglyceride (45%), free fatty acid (28%), acyl-CoA (11%) and phospholipid (15%) fractions. From these data, it is not possible to determine the identity of the substrate for the conversion of the elongated fatty acyl products to the corresponding hydrocarbon (Hy). In the cockroach, incubations with 18∶0-CoA and with 18∶2-CoA resulted in small incorporations into 25∶0 Hy and into 27∶2 Hy, respectively. In the housefly, incubations with 18∶1-CoA resulted in a very small production of 27∶1 Hy in mature males and 23∶1 Hy in mature female houseflies. These data support the idea that the preparation of subcellular fractions results in an uncoupling of fatty acid chain elongation from the conversion of the fatty acid to the corresponding hydrocarbon in both insects.     

New TLC and OPTLC Methods for the Separation of Serum Lipids

Rapid methods for the separation of major lipid classes varying in polarity from cholesterol esters to lysophosphatidylcholine are presented, which were used for the analysis of extracts obtained from human sera. Solvent systems for lipid separation by overpressured thin-layer chromatography and thin-layer chromatography are described. These techniques are suitable to separate unsaturated and saturated cholesterol esters according to the number of carbon atoms, double bond numbers of their fatty acid contents. Fourteen lipid fractions may be separated using two successive developments in the same direction.     

Lipid composition of beef brain,beef liver,and the sea anemone: Two approaches to quantitative fractionation of complex lipid mixtures

Two new schemes for fractionation of complex lipid mixtures are presented. Their use for the study of lipids of beef brain, beef liver, and the sea anemone are described. Apparatus and techniques for working in an inert atmosphere, evaporation of solutions in the cold under nitrogen, use of infrared spectroscopy for examination of lipids and their hydrolysis products, preparation and clution of diethylaminoethyl (DEAE) cellulose and silicic acid-silicate columns and general column combinations that can be used to fractionate complex lipid mixtures are considered in detail. The first scheme, employing DEAE cellulose columns followed by thin layer and paper chromatographic examination of the fractions, was applied to liver lipids. The many components, some of them new lipids not previously detected, are clearly seen with this technique but are not seen when paper or thin layer chromatography alone or silicic acid chromatography are used. The second scheme employing DEAE for initial fractionation, followed by complete separation on silicic acid and silicic acid-silicate columns, was applied to lipids of the sea anemone and beef brain. Typical lecithin and phosphatidyl ethanolamine were isolated, but sphingomyelin was not found. A new sphingolipid, ceramide aminoethylphosphonate, with a free amino group and a direct carbon to phosphorus bond was isolated and characterized. The methods used for quantitative isolation, the infrared spectra, and the amounts of cholesterol, ceramide, cerebroside, galactosylglyceride, sulfatide, sphingomyelin, lecithin, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, triphosphoinositide, phosphatidic acid, cardiolipin, and ganglioside of beef brain are presented. Finally, the types of lipid-nonlipid interactions disclosed by column chromatography and their potential application to biological problems are discussed.     

Nachweis gesättigter cycloaliphatischer und aromatischer Fettsäuremethylester durch Dünnschicht- und Gas-Chromatographie

Detection of the Methyl Esters of Saturated Cycloaliphatic and Aromatic Fatty Acids with Thin-layer and Gas Chromatography Homologous series of methyl esters of saturated cycloaliphatic (CFA) and aromatic fatty acids (AFA), which were prepared by the alkali isomerisation of fish oil followed by hydrogenation and fractionation of the cyclised mixture as well as with the help of cyclising hydrogenation, were analysed by thin-layer and gas chromatography. Even 0.4% CFA and AFA, which do not form urea-adducts, can be detected in the fractions as a substance class in thin-layer chromatography. The equivalent carbon numbers (stearic acid = 18) of the principal isomers of CFA and AFA homologues (C₁₈ to C₂₂), which were obtained as a class in the thin-layer chromatographic separation, were determined by the gas chromatography.     

A critical evaluation of thermal fractionation of butter oil

Butter was fractionated on the basis of temperature (17–29°C) without agitation using slow cooling of melted anhydrous fat in conjunction with gentle vaccum filtration to produce four solid and four liquid fractions. Each of the fractions was analyzed for fatty acid composition, triglyceride profile, and characterized by gel permeation high performance liquid chromatography and differential scanning calorimetry thermograms. Fatty acid analysis indicated that the solid fractions had slightly higher amounts of palmitic and stearic acid and lower levels of oleic acid, while the remaining analyses did not indicate any substantial compositional differences between the fractions. Although the 29°C solid fraction (∼10%) could be said to be somewhat unique, the natural variation in the normal seasonal composition of butterfat was almost equal to that obtained by fractionation. The experimental physicochemical data obtained for the fractions in this study extend and verify previous work on butteroil fractionation, and indicate that thermal fractionation has marginal merit. On the other hand, literature describing more positive thermal butteroil fractionation results obtained by the properietary Tirtiaux process (Fleurus, Belgium), indictes that it may be a more expedient avenue to explore and let market forces determine whether fractionation has a future in Canada and North America.     

Analysis of soybean lecithin by thin layer and analytical liquid chromatography

The application of thin layer and analytical liquid chromatography to the analysis of two samples of commercial soybean lecithins is described. A combination of column chromatography and quantitative thin layer chromatography showed that these products consisted of ca. 82% mixture of the major phospholipids of soybeans, phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl inositol. The remainder of these products contained essentially the entire spectrum of lipid classes found in soybean oil-some 24 known and unknown glycolipids and phospholipids, in addition to the neutral lipids. Applications of analytical liquid chromatography to these lecithins gave a composition profile of the lipid classes comparable to two-dimensional thin layer chromatography. The potential of this method for the complete analysis of complex lipids, such as soybean lecithins, is indicated.