Quantitative isolation of lipids of partially defatted and whole peanuts by a dry column method

A dry column method of lipid extraction was found to be applicable to peanuts—partially defatted as well as whole. Total lipid was obtained from the peanut/sodium sulfate/Celite 545 columns by isocratic elution with dichloromethane/methanol 9:1. Moreover, neutral lipids were obtained by sequential elution that were completely free from polar lipids. First, dichloromethane eluted the neutral lipids, then the 9:1 solvent eluted the polar lipids—at times containing small quantities of neutral lipids. Total lipid values obtained by the column extraction method were slightly higher than those obtained by the standard Soxhlet extraction procedure. This was due in part to the more complete polar lipid isolation produced by the column method. In partially defatted peanuts produced by mechanical pressing, 99% of the polar lipids remained in the retained oil, and these were shown to be slightly less unsaturated than the neutral lipids.     

Two procedures to remove polar contaminants from a crude brain lipid extract by using prepacked reversed-phase columns

Two procedures were developed using prepacked, reversed-phase columns (Bond Elut) for the separation of lipids from water-soluble contaminants. A crude lipid extract from brain tissue, was diluted stepwise with a methanol/water (method 1) or a methanol/saline (method 2) mixture and, with each step, was passed through the column. As the polarity of the solvent was increased, all lipids became bound to the column, while the water-soluble compounds remained in the eluate. After three subsequent dilutions and column elutions, the eluate containing the more polar contaminants was discarded. The bound lipids were then eluted with a small volume of chloroform/methanol (1∶2, v/v). Alternatively two fractions were eluted, the first fraction eluted with methanol/water (12∶1, v/v), contained gangliosides, phosphatidyl-serine, phosphatidylinositol, phosphatidic acid and sulfatides. The second fraction, eluted with chloroform/methanol (1∶2, v/v), contained all remaining phospholipids, cerebrosides and cholesterol. For both methods a quantitative recovery of cholesterol and phospholipids was obtained. In method 2, when water was replaced by saline in the dilution solvent mixture, gangliosides were also bound and quantitatively recovered.     

Horse erythrocyte gangliosides: Preparation of the major hematoside NeuNG1-Lac-Cer

A simple method for the isolation of hematoside NeuNG1-Lac-Cer from horse erythrocytes is described. An aliquot of the crude ganglioside fraction was labeled by tritiated sodium borohydride after mild periodate oxidation. The compounds obtained were used as radioactive tracers in column chromatography. Gangliosides were applied onto a silicic acid column and eluted stepwise by solvents of steadily increasing polarity. The major ganglioside, NeuNG1-Lac-Cer, was eluted in a high yield by the solvent mixture chloroform/methanol/water (60∶35∶8, v/v/v).     

Quantitative analysis of lipid classes by liquid chromatography via a flame ionization detector

A method is described for the direct quantitative analysis of the lipid classes of mammalian tissue lipids using high performance liquid chromatography (HPLC) with a flame ionization detector (FID). The lipid is extracted from the tissue with chloroform/methanol after deactivation of hydrolytic enzymes and removal of nonlipid substances by extraction with hot dilute acetic acid (0.05N). Separation of the lipid classes is performed with a column (45 cm × 0.2 cm id) of 8 μm silica (Spherisorb, Phase Sep, Hauppague, NY) treated with concentrated ammonium hydroxide at a solvent flow rate of 0.5 ml/min, which requires a pressure of ca. 900 psi. Cholesteryl esters (CE) and triglycerides (TG) are eluted first with Skellysolve B/methylene chloride (1∶1, v/v); cholesterol (CH) is eluted with chloroform/methylene chloride (1∶2, v/v) and the phospholipids with methanol containing 6% ammonium hydroxide added to the latter solvent in a linear gradient. The neutral lipids are eluted in ca. 12 min and the phospholipids in an additional 30 min. The relative amount of each lipid class was determined from standard curves of the peak areas obtained according to response factors using erucyl alcohol as an internal standard. The method was applied to samples of kidney, liver and serum of rats. Duplicate analyses were generally within ca. 1.0% and good agreement was obtained in the analysis of the lipid classes of Azolectin and liver mitochondria lipid compared to thin layer chromatography (TLC) via photodensitometry of charred spots or phosphorus analysis of recovered phospholipids.     

Comparison of lipid composition ofCandida guilliermondii grown on glucose,ethanol and methanol as the sole carbon source

The carbon and energy source for aerobically grown cultures ofCandida guilliermondii profoundly influenced the neutral lipid content and the fatty acid composition of the individual lipid components. Methanol (0.80%, w/v) grown cells cultivated at 30 C in presence of 0.025% ammonium sulfate contained 12% total lipids, 67% of which was neutral lipids. Glucose (0.74%, w/v) or ethanol (0.53%, w/v) grown cells contained 21–22% total lipids, 80% of which was neutral lipids, under the same conditions. Methanol-grown cells contained a decreased 18∶1 acid (52–54% of total fatty acids) and an increased 18∶2 acid (23–25%), as compared with glucose- or ethanol-grown cells which contained 57–66% 18∶1 acid and 8–14% 18∶2 acid, in both neutral and polar lipid fractions. The relationship between methanol metabolism and desaturation of fatty acid in yeast was discussed.     

Fluorescent lipids of bovine brain white matter

Two fluorescent lipids were isolated from a chloroform-methanol (2∶1 v/v) extract of bovine brain white matter by two-step preparative thin layer chromatography on Silica Gel G. The first step was performed with chloroform-diethyl ether-acetic acid (70∶30∶1 v/v) as developing solvent and revealed a single fluorescent band below the solvent front. The band was scraped off, and the lipid was eluted in chloroform and reapplied to Silica Gel G plates. In the second step, benzene-methanol-ethyl acetate (85∶10∶5 v/v) as developing solvent revealed two fluoresent bands (A and B) with R_f values of 0.72 and 0.65. The lipids were eluted and the UV and visible absorption spectra were measured in heptane, as were the excitation and fluorescence spectra. Sulfuric acid absorption spectra (2 and 24 hr treatment at 22 C) as well as the resulting excitation and fluorescence spectra were also determined. The fluorescent lipids reacted positively in a number of nonspecific color tests for steroids, but the chemical nature of these minor components of the neutral lipid fraction remains to be established.     

Fatty acid composition of rat hearts as influenced by age and dietary fatty acids

Fatty acid composition of neutral and polar lipid fractions from rat hearts was determined in rats of different ages as their diet source changed. Piebald rats were weaned at 21 days and were fed standard lab chow. Lipids from rat hearts, mothers milk and lab chow were purified on a Sephadex G-25 fine column and separated into neutral and polar lipid fractions by silicic acid column chromatography. These lipid fractions were then hydrolyzed and methylated with BF₃ in methanol, prior to gas liquid chromatographic separation on a 1/8 in. × 10 ft aluminum column of 15% EGS on 80–100 mesh acid-washed Chromosorb W. Three major fatty acids in the neutral lipid fraction comprised 72% of total neutral lipid fatty acids from young hearts. At sexual maturity (at least 74 days old) C_18∶1 was the major fatty acid, followed by C_16∶0 and C_18∶0. The same three fatty acids comprised 83% of total polar lipid fatty acids, but C_18∶0 was the major fatty acid, followed by C_16∶0 and C_18∶1. The fatty acid composition of dietary lipids influenced the total neutral lipid fatty acid composition of the rat heart, but had little influence on the fatty acid composition of the polar lipid fraction. Presented in part at the AOCS Meeting, New Orleans, April 1970.     

Effect of extraction methods on lipid yield and fatty acid composition of lipid classes containing γ-linolenic acid extracted from fungi

The effect of extraction procedures on the lipid yield and fatty acid composition of total lipid and main lipid structures (phospholipids, diacylglycerols, triacylglycerols, free fatty acids, and sterol esters) of fungal biomass (Mucor mucedo CCF-1384) containing γ-linolenic acid (GLA) was investigated. Seventeen extraction methods, divided into three groups, were tested: six with chloroform/methanol, five with hexane/alcohols, and six with common solvents or mixtures. The chloroform/methanol procedure (2∶1) was selected as standard, where lipid yield (TL/DCW, total lipid per dry cell weight) was 17.8%, considered to be 100% of lipids present. All chloroform/methanol extractions yielded more than 83% recorvey of lipids. Use of hexane/isopropanol solvent systems led to a maximum of 75% recovery. The best lipid yield was achieved by a two-step extraction with ethanol and hexane (120%). Extraction efficiency of the other solvent systems reached a maximum of 73%. Triacylglycerols were the main structures of lipid isolated; only methanol-extracted lipid contained 58.5% phospholipids. The fatty acid content of total recovered lipid was variable and depended on both the lipid class composition and the solvent system. GLA concentrations in total lipids isolated by hexane/alcohol procedures (7.3–10.7%) are comparable with classical chloroform/methanol systems (6.5–10.0%). The maximal GLA yield was obtained with chloroform/methanol/n-butanol/water/0.1 M ethylenediaminetetraacetic acid (EDTA) (2∶1∶1∶1∶0.1, by vol) and after two-step extraction with ethanol and hexane (14.3 and 13.7 g GLA/kg DCW, respectively). The highest GLA content was analyzed in the phospholipid fraction (16.1%) after using chloroform/methanol/n-butanol/water/0.1 M EDTA (2∶1∶1∶1∶0.1, by vol). Remarkably low concentrations of polyunsaturated fatty acids were determined in the free fatty acid fraction.     

Lipid composition of rat superior cervical ganglion

Excised rat superior cervical ganglion were incubated in Krebs-Ringer solution, freeze-dried, and lipids extracted with chloroform: methanol (2∶1) v/v. Chromatography of lipid extracts in three separate thin layer chromatography solvents was accomplished on a single thin layer chromatography plate coated with acid-washed Silica Gel H. Individual lipids were eluted from the silica gel using a modified Swinny filter holder technique and transesterified with methanolic-BF₃. Fatty acid methyl esters were separated and quantitated by gas liquid chromatography, while phospholipids were determined with a Malachite green dye organic phosphorus assay. Quantitation of neutral lipids was accomplished with a micro Liebermann-Burchard technique and sphingolipids estimated colorimetrically as free sphingosine. The composition of lipids from freezedried rat ganglia resembles the lipid composition of rat brain. Phosphatidylcholine and phosphatidylethanolamine represent 76% of the glycerolphosphatides. The sphingolipids were comprised primarily of sphingomyelin with moderate levels of cerebroside and sulfatide. Cholesterol was the predominant neutral lipid. The major phospholipid fatty acids included palmitic, stearic, and oleic acids. Sumbitted in part as a partial requirement for the Ph.D. degree, State University of New York at Buffalo, Buffalo, N.Y.     

Analysis of lipids from cooked beef by thin-layer chromatography with flame-ionization detection

Lipids were extracted from cooked ground beef with methylene chloride/methanol (2:1). The lipids were separated on a silicic acid column, into neutral and polar fractions by elution with methylene chloride, followed by methanol. These fractions were analyzed by Iatroscan thin-layer chromatography with flame-ionization detection instrumentation on Chromarods S-III (silica gelcoated quartz rods). Comparison of cooked beef stored for 0, 4 and 7 d at 4°C indicated that storage caused a decrease in total lipids, an increase in neutral lipids and a decrease in polar lipids, specifically in phosphatidylcholine. These changes in the lipid fraction were associated with meat flavor deterioration and an increase in lipid oxidation.     

Analytical separation of nonlipid water soluble substances and gangliosides from other lipids by dextran gel column chromatography

A column chromatographic procedure is reported utilizing a dextran gel (Sephadex) for the complete separation of the major lipid classes from water-soluble nonlipids. Lipids other than gangliosides are eluted first with chloroform/methanol 19/1 saturated with water, gangliosides with chloroform/methanol/water containing acetic acid, and water-soluble nonlipids with methanol/water 1/1. Results for adult human whole brain, grey and white matter, and normal infant whole brain lipids are presented. With beef brain lipid as sample the ganglioside fraction is essentially pure, but with human brain lipid samples only about 70% of the second fraction is ganglioside. All ganglioside and water soluble nonlipid of a human spleen chloroform/methanol extract was separated from lipids with the procedure. Control studies with P³²O₄≡ and C₁₄ labelled glucose showed that all counts were present in fraction 3. Similar studies with C₁₄ labelled amino acids (glycine, serine, alanine, phenylalanine) showed that only phenylalanine counts were eluted in fraction 2 along with the gangliosides. The procedure was applied for removal of large amounts of ammonium acetate from DEAE cellulose column fractions and for complete removal of adsorbent and salts from lipids eluted from thin-layer chromatograms. After passage through the dextran gel columns, lipids eluted from thin-layer chromatograms were found to give infrared spectra identical to those of pure samples obtained by other procedures.     

Fungi pathogenic to insects: III. Neutral and polar lipids ofEntomophthora coronata

The neutral and polar lipid composition ofEntomophthora coronata was determined qualitatively. The fungus was grown on a chemically nondefined medium (Sabouraud dextrose yeast extract) and a chemically defined medium for a period up to 26 days. The lipids were characterized by thin-layer, column, gas chromatography, and selective sprays,³²P-labeling, and mass spectrometry. The neutral lipids consist of monoglycerides, diglycerides, cholesterol, free fatty acids, triglycerides, and cholesteryl esters. The polar lipids consist of phospholipids (phosphatidyl ethanolamine, phosphatidyl choline, lysophosphatidyl ethanolamine, lysophosphatidyl choline, and spingomyelin), a number of glycolipids including cerebrosides, and many unrecognizable lipids, most of which are present in trace amounts. The cerebrosides and spingomyelin are present in significant amounts, and their concentration increased with age of the culture. The major fatty acids (>10%) of the total, neutral, and polar lipids of the mycelia are 14∶0, 16∶0, 18∶1, 18∶3(γ), and 24∶1. The polar lipids of total culture (unsaturation index 0.88) and of the conidia (unsaturation index 1.48) are considerably more unsaturated than the corresponding neutral lipids (unsaturated index 0.50 and 0.49). The mycelial polar lipids, compared to the neutral lipids, possess less 14∶0 and 18∶1 but contain a greater percentage of 16∶0, 18∶2, 18∶3(γ), 24∶0, and 24∶1. The major fatty acid of the conidia (>10%) are 13∶0, 14∶0, 18∶1, 18∶2, 18∶3(γ), and 20∶4. Their polar lipids have a higher proportion of 18∶2, 18∶3(γ), and 20∶4. The cerebrosides possess 24.1 in high relative proportion (30.1%). Presented at the AOCS Meeting, Atlantic City, October 1971.     

Rapid separation of neutral lipids,free fatty acids and polar lipids using prepacked silica sep-Pak columns

A method is described for the separation of neutral lipid, free fatty acid and polar lipid classes using small (600 mg), prepacked silica Sep-Pak columns. Combinations of hexane and methyltertiarybutylether were used to progressively elute cholesteryl ester first then triglyceride from the column. After column acidification, fatty acids were eluted followed by cholesterol. Recoveries of these lipids were 96% or greater. Polar lipids were eluted from the column using combinations of methyltertiarybutylether, methanol and ammonium acetate. Phospholipid classes could not be separated completely from each other. Phosphatidylethanolamine and phosphatidylinositol eluted together, whereas the more polar phosphatidylcholine, sphingomyelin and lysophosphatidylcholine were eluted as a second fraction. Recoveries of each phospholipid was greater than 98%.     

Lipid composition of oats (Avena sativa L.)

Compositions of lipids extracted from a sample of Hinoat oat by seven solvent systems and that extracted with chloroform/methanol (2:1 v/v) from six selected cultivars representing high and low lipid contents are reported. Lipid components (steryl esters, triglycerides, partial glycerides, free fatty acids, glycolipids and phospholipids) were separated by silicic acid column chromatography and thin layer chromatography and quantitated by GLC analysis of fatty acids and phosphorus determinations. Twelve oat cultivars were examined for the fatty acid composition of lipid extracted with n-hexane. Lipids extracted from Hinoat by different solvent systems ranged from 5.6 to 8.8%. Quantitative distribution of lipid components extracted with chloroform/methanol from six cultivars containing 4.6 to 11.6% lipid showed a significant correlation (γ=0.99) between the total lipid and the neutral lipid content. Phospholipid content was similar in all cultivars, but glycolipids showed a two-fold increase in high lipid oats. Triglycerides contained less palmitic and more oleic acid than the glycolipids or phospholipids. Nine glycolipids and 11 phospholipids have been identified, and the polar lipid composition of Hinoat oat is presented.     

钝顶螺旋藻中γ-亚麻酸甲酯的提取研究

用１∶２∶０８（Ｖ／Ｖ）的氯仿 甲醇 水溶剂系统提取螺旋藻中总类脂,总类脂在硅胶柱上经氯仿、丙酮、甲醇依次洗脱,分离得到中性脂、总脂肪酸半乳糖脂、磷脂。总脂肪酸半乳糖脂与５∶９５（Ｖ／Ｖ）的氯乙酰 甲醇作用转变为总脂肪酸甲酯,再通过多次与尿素形成复合物,可从总脂肪酸甲酯中分离、纯化出 亚麻酸甲酯,其纯度达到了９６５％。     

Characterization of porcine omental lipids

Porcine omental lipid extracts were fractionated and the major lipid components characterized. Approximately 97% of the chloroform/methanol extract consisted of triglycerides containing primarily 16∶0, 18∶0, 18∶1, and 18∶2 fatty acids. Small quantities of free fatty acids, cholesterol, di- and monoglycerides were also detected. The phospholipid fraction, obtained by solvent partition and Unisil column chromatography and characterized by high performance liquid chromatography (HPLC) and HPLC-mass spectrometry, was found to consist primarily of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. The neutral glycolipids, isolated by solvent partition and Unisil column chromatography and identified by HPTLC and HPLC, were found to consist primarily of di-, tri-and tetraosylceramides. The complex glycolipid fraction, obtained from Folch upper phase solvent partition and characterized by HPTLC and immunoblotting, was found to consist primarily of ganglio-, globo-, and neolacto- neutral glycolipids and ganglio-, globo-, neolacto- and fucosylated gangliosides.     

Total and polar lipids in soybean protein meals

Soybean protein meals obtained by various oil extraction methods have different neutral oil content, and they may contain differnet amounts of polar lipids. Three soy protein meals obtained by different processing methods were extracted by two solvents consecutively, chloroform/methanol (2:1, vol/vol) and water-saturated butanol, for total lipid analysis. The organic flour (i.e., ground soybean) containted 15.52% total lipids; the high protein dispersibility index flour from extrusion-expelling processing and the white flour from conventional solvent extraction contained 11.20 and 1.84% total lipids, respectively. Organic flour contained more polar lipids than the other two protein meals on a dry-weight meal basis. Chloroform/methanol extracted most of the lipid from the meals, whereas water-saturated butanol resulted in an extract with more polar lipids than that from chloroform/methanol extraction.     

Simultaneous Quantification of Plant Glyceroglycolipids Including Sulfoquinovosyldiacylglycerol by HPLC–ELSD with Binary Gradient Elution

Membrane lipids of photosynthetic organisms consist of glycerophospholipids and glyceroglycolipids. We investigated a method for the simultaneous quantitative analysis of neutral and acidic lipids using HPLC–ELSD, and quantified monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG). Ten complex lipid classes were separated with a binary gradient system consisting of chloroform and methanol–acetone–water–acetic acid (30:60:9:1, v/v/v/v) with 0.3% triethylamine (pH 4), and were eluted within 16 min. The contents of SQDG in ten edible plants ranged from 3 to 101 mg/100 g, and were positively correlated to the neutral glyceroglycolipids contents.     

Efficiencies of Solvent Systems for Extraction of Legume Lipids

Ten solvent systems representing a wide range in polarity were highly variable in their abilities to extract the total lipids from field pea and lentil flours and starches. Propanol/water (3 : 1v/v) extracted the most lipid from lentil flour and both legume starches. Chloroform/methanol (2:1 v/v) and benzene/ethanol (4:1 v/v) gave consistently high recoveries of total lipids from both flours but appeared to be less effective with starches. Fractionation of the purified flour lipids demonstrated that propanol/water extracted phospholipids and glycolipids quite efficiently but chloroform/methanol and benzene/ethanol gave high recoveries of both nonpolar and polar lipids. These neutral, phospho- and glycolipids from the two legume flurs were highly polyunsaturated, containing 35–50% linoleic and 5–21% linolenic acid, and would be susceptible to oxidative rancidity. Purified lipids from legume starches were recovered as neutral lipids primarily and yields were very low.     

Accelerated Solvent Extraction Improves Efficiency of Lipid Removal from Dry Pet Food While Limiting Lipid Oxidation

Accelerated solvent extraction (ASE) was evaluated for extracting lipids from baked and extruded dry pet foods to determine factors controlling extraction efficiency and effects on lipid oxidation. Hydroperoxide decomposition and new lipid oxidation were minimal at 40 °C but increased at higher extraction temperatures without increasing yields. Maximum extraction required grinding samples to 250 µm particles, presence of polar solvents [chloroform, chloroform/methanol 2:1 (v/v) mixed, hexane/methanol 2:1 (v/v)], and a minimum of 20 min total static extraction time in repeat extraction cycles. Hexane and methanol injected into extraction cells simultaneously but separately was able to nearly duplicate extractions of chloroform/methanol, providing an option for replacing toxic chlorinated hydrocarbon solvents in ASE. However, lipid oxidation was higher in hexane. Yields were quantitative in baked biscuits but lower in extruded kibbles due to more dense, complex molecular structures. ASE extraction yields of 40 min or less were comparable to manual extraction yields of 24–48 h, with lower oxidation. Overall, one or two ASE extraction cycles with static times less than 20 min appeared to provide adequate lipid yields that accurately reflect lipid composition while inducing minimal modification when lipid oxidation products are the analytical endpoint.