Analysis of triglycerides by consecutive chromatographic techniques. II. Ucuhuba kernel fat

The triglycerides from ucuhuba kernel fat (Virola surinamensis) were analyzed using thinlayer adsorption chromatography (TLC) followed by gas-liquid chromatography (GLC). The triglycerides were first separated into three fractions containing 0, 1, and 2 or more double bonds per molecule on silica gel TLC plates impregnated with AgNO₃. The total triglycerides and each individual TLC fraction were then analyzed by GLC for the molecular weights of their component triglycerides and for their fatty acid composition. Quantitation of the TLC fractions was achieved by GLC analysis of their fatty acids using an added internal standard and confirmed by solving simultaneous equations derived from GLC analysis of their triglycerides and fatty acids. Application of these combined chromatographic techniques separated the ucuhuba kernel fat into 23 triglyceride components. Trimyristin and laurodimyristin comprised over half the total triglycerides, which was expected since the fat contained 20.0 mole % laurie and 71.3% myristic acids. Presented at the AOCS Meeting in Houston, Texas, 1965. Supported in part by grants from the National Institutes of Health (AM-06011) and the Corn Products Institute of Nutrition.     

Some practical aspects of thin-layer chromatography of lipids

This report describes methods for the thin-layer chromatography (TLC) of lipids and some practical aspects of the methods. In order to present some basis for choosing the correct powder for particular separations, some properties of several widely used silica gel powders are compared. The effect of binder material such as calcium sulfate in silica gel is studied. The three systems, silica gel as a polar phase, silver nitrate-impregnated silica gel, and reversed phase systems are described with application to neutral lipids. Also included are the applications of TLC to the polar lipids, such as phospholipids, cerebrosides, sulfatides, sphingomyelin and other glycolipids from various sources. The pitfalls and precautions involved in these separations are discussed in detail.     

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.     

Quantitative determination of alk-1-enyl- and alkyl-glyceryl ethers in neutral lipids and phospholipids

A quantitative method for the simultaneous determination of alk-l-enyl- and alkyl-glyceryl ethers is described. Complete hydrogenolysis of carboxylate and phosphate esters of neutral lipids and phospholipids was achieved with lithium aluminum hydride. The hydrogenolysis products of the glyceryl ether containing lipids, alk-l-enyl- and alkyl-glyceryl ethers and alcohols, were identified by thin-layer chromatography (TLC), gas-liquid chromatography (GLC), and infrared spectroscopy. The alk-l-enyl- and alkyl-glyceryl ethers were quantitated by TLC photodensitometry. The specificity of this method can also be extended when used in conjunction with GLC, ion-complexing TLC, zonal scanning, and autoradiography to study composition, isomeric form, and the biosynthesis of glyceryl ethers in neutral and phospholipids. The percentage of alk-l-enyl- and alkyl-glyceryl ethers in bothtthe neutral lipids and phospholipids of various rat tissues was determined by the described method. Glyceryl ether glycerides represent 0.3–1.2% of the total neutral lipids whereas the glyceryl ether phosphatides of brain, heart, marrow, muscle, and spleen represent 4.5–12.0% of their total phospholipids. Higher concentrations of glyceryl alkyl than of alk-l-enyl ethers were found in the neutral lipids whereas the glyceryl alk-l-enyl ethers were found to predominate in the phospholipids. Presented at the AOCS Meeting, Chicago, October 1967. Under contract with the U. S. Atomic Energy Commission.     

Über die Zusammensetzung des Samenöles aus Traubenholunder (Sambucus racemosa L.)

Composition of the Seed Oil of Clustered Elder (Sambucus racemosa L.) The seeds of clustered elder (Sambucus racemosa L.) contain 28% of an oil. Its separation to groups of components was carried out by adsorption chromatography. Single groups of substances were further analysed by TLC and GLC in combination with physico-chemical methods (IR and MS). Thus the following substances were found and identified: n-alkanes (C₁₇–C₃₃), 2-methyl- and 3-methylalkanes, squalene, a mixture of esters, triglycerides, β-sitosterol, campesterol and a mixture of diglycerides. The conditions for the separation of esters by silica gel column chromatography were also found. The mixture of esters was thus separated into a group of aliphatic wax esters (C₃₄–C₄₄) and four groups of esters of steroidal alcohols (mainly β-sitosterol and campesterol) with aliphatic unsaturated acids (predominantly C_18:1, C_18:2 and C_18:3). In the triglycerides representing the main fraction of the oil (93%), the acids C_16:0, C_18:1, C_18:2 and C_18:3 are present. The same acids were also found in diglycerides.     

The composition of beef heart cardiolipins isolated by solvent and chromatographic fractionation

A method is described for the isolation of cardiolipin from beef heart lipids by a single pass through a silica gel column. The isolated cardiolipin was free of neutral lipids and phospholipids and accounted for 10% of total phospholipid phosphorus. It was compared to commercial cardiolipins prepared by the Pang-born method of selective precipitation. Analysis for fatty acids, glycerol and phosphorus revealed a molar ratio of 2∶1.5∶1 for both preparations, provided the fatty acid esters were assayed colorimetrically. If the fatty acids were determined by titration, the commercial cardiolipin had a molar ratio of fatty acid to phosphorus of 1.6, while ours remained at 2.0. Upon hydrolysis with acetic acid, the former yielded 76% water-soluble phosphorus, the latter only 2%. Both cardiolipins contained over 90% C-18 fatty acids, with our preparation containing 76% linoleate, the commercial preparation, 90%. After alkaline hydrolyses a component was isolated from the commercial cardiolipin which represented 15.5% of the total weight. It developed an interfering pigment in the fatty acid ester determination but has not been identified. After its removal analysis demonstrated three fatty acids per molecule of the commercial cardiolipin.     

Identification and composition of turnip root lipids

Two varieties of turnip, Laurentian and Wye, were examined for their lipid and fatty acid composition. Lipids extracted with 80% ethanol contained variable quantities of phosphatidic acid, which was considered to be an artifact. Crude lipids were fractionated by TLC, and fatty acids and sterols were analyzed by GLC. Among the common phospholipids, cardiolipid and phosphatidyl glycerol were abundant components. Linolenic acid comprised 60% of the total fatty acids. β-Sitosterol was the principal sterol, and about half of the carotenoids was lycopene. No great differences between the two varieties studied were observed however. Contribution No. 53 of the Food Research Institute, Canada Department of Agriculture, Ottawa.     

Analytik plasmalogenhaltiger Phosphatide aus Herzgewebe mittels extracellulärer Phospholipase A2

The Analysis of Phospholipids from Cardiac Membranes by Phospholipase A₂. Phospholipase A₂ from snake venom has been employed to analyse the positional distribution of fatty acids in glycerolipids from guinea pig and pig cardiac membranes. It is known that phospholipase A₂ hydrolyses the fatty acids in sn-2 position of 1,2 diacylglycerophospholipids. In cardiac membranes phosphatidylcholine and phosphatidylethanolamine contain along with diacyl esters the corresponding alkenylether analogues of phospholipids. In the present experiments the alkenylether moieties were slowly hydrolysed by phospholipase A₂ from snake venom. We therefore separated by TLC lysophosphatides from fatty acids and unattacked phospholipids. The latter were the plasmalogens. The separated lipids were characterized by GLC. In some experiments the phospholipids were labelled with ¹⁴C-fatty acids before they were hydrolysed by phospholipase A₂. The alkenyl ether chain of plasmalogens seems to negativly influence the hydrolysis of fatty acids in sn-2 position of those phospholipids.     

FA determination in cold water marine samples

The determination of FA in cold water marine samples is challenging because of the presence of large proportions of a variety of labile PUFA. This study was undertaken to establish optimal methods for FA analysis in various sample types present in the marine environment. Several techniques used in FA analysis, including lipid fractionation, FAME formation, and picolinyl ester synthesis, were examined. Neutral lipids, acetone-mobile polar lipids, and phospholipids (PL) were readily separated from each other on columns of activated silica gel, but recoveries of PL were reduced. Deactivation of the silica gel with 20% w/w water produced variable recoveries of PL (66±22%). FAME formation with BF₃ gave optimal recoveries, and a method to remove hydrocarbon contamination from these samples before GC analysis using column chromatography was optimized. Picolinyl derivatives of FA are useful in structural determinations with MS, and a new base-catalyzed transesterification method of their synthesis from FAME was developed. Finally, a series of calculations, combining FA proportions with acyl lipid class concentrations, was designed to estimate FA concentrations. In algae and animal samples, these estimates were in good agreement with actual FA concentrations determined by internal standards.     

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.     

Fatty acids in phospholipids isolated from human red cells

Total lipids of packed erythrocytes from healthy men 22 to 25 years old were extracted with chloroform-methanol mixture. Phospholipid classes were separated from neutral lipids and pigments on a silicic acid column. Phosphatidyl inositol (PI) was freed of its contaminants phosphatidyl ethanolamine (PE) and phosphatidyl serine (PS) on an aluminum oxide column. Additional silicic acid columns with modified solvent systems were needed for complete separation of other overlapped phospholipid classes. The identification of phospholipids in each eluted fraction was accomplished by TLC, using the appropriate spray tests and reference compounds, and confirmed on each of the isolated phospholipids by IR spectrophotometry. The total content of phospholipids as determined by phosphorus analysis was found to be 2.63 mg/ml of packed cells. These phospholipids were found to have the following composition (in per cent of total phospholipid): PI, 2.3; PE, 13.4; ethanolamine plasmalogen (EP), 14.5; PS, 3.9; lecithin (L), 34.2; choline plasmalogen (CP), 1.4; sphingomyelin (Sph), 28.4 and lysolecithin (LL), 1.7. The fatty acid composition of each phospholipid was determined by GLC. The average number of double bonds per fatty acid in the isolated phospholipids was found to be as follows: PI, 1.5; PE, 1.9; EP, 3.6; PS, 2.1; L, 1.0; CP, 2.0; Sph, 0.2 and LL, 0.5. The positional distribution of fatty acids in both L and PE was ascertained by selective enzymatic hydrolysis with phospholipase A. Saturated fatty acids of L were esterified predominantly in the α′-position, whereas in PE only 63.9 mole per cent of the saturated fatty acids were found in this position. Presented in part at the AOCS Meeting in Los Angeles, April 1966. Dept. of Health, Education and Welfare, USPHS.     

A complete separation of lipids by three-directional thin layer chromatography

A novel three-directional thin layer chromatography (TLC) method is reported by which all the polar and neutral lipids are isolated on a single TLC plate. Following resolution of the phospholipids by two-directional TLC, lipids are visualized by ultraviolet light after spraying with 2′,7′-dichloro-fluorescein. A line is drawn across the plate, parallel to the second direction of development, separating the resolved phospholipids and the neutral lipids concentrated along the solvent front. The TLC plate is then chromatographed in the reverse direction of the second development to resolve the neutral lipids. By exposing the lipids to HCl fumes after the first development, the plasmalogen content of the lipids may also be determined. This new technique is rapid and lends itself to qualitative and quantitative analyses of total lipids. Contribution no. 1163 from the Animal Research Centre.     

Chromatographic analysis of polyglycerols and their fatty acid esters

Polyglycerols and their fatty acid esters have been analyzed by gas-liquid chromatography (GLC) as trimethylsilyl ether derivatives. Linear diglycerols and triglycerols were isolated from commercial polyglycerols by vacuum distillation. Mono- and di-fatty acid esters were synthesized in the laboratory. Two isomers of diglycerol have been separated and identified. GLC analysis was carried out on columns packed with 3% JXR on Gas Chrom Q. Response factors for diglycerol and triglycerol relative to glycerol have been established. Commercial polyglycerol esters are shown to be mixtures of glycerol, free fatty acids, mono- and diglycerides, and mono- fatty acid esters of diglycerol and triglycerol. Separation of free polyglycerols and their esters is also demonstrated by thin-layer chromatography on Silica Gel-G containing 4.0% boric acid.     

Gas chromatography in lipid investigations

Biological problems involving long chain compounds and steroids can now be studied more effectively than ever before through use of GLC techniques. When combined with TLC methods for the separation of classes of compounds, these procedures are the most valuable analytical methods now known for lipid investigations. The fact that both qualitative and quantitative data may be obtained at the same time, and that the methods may be used to study complex mixtures at the microgram and sub-microgram level, suggests that many new applications will be found in the fields of chemistry, biology and medicine. The procedures described in this paper are chiefly those developed over the last few years in the laboratories of the authors. Thin-film columns prepared with diatomaceous earth supports have been used since 1960 in many studies. A new procedure, utilizing a thin-film column, was developed for the separation and estimation of long chain fatty acid methyl esters. The method has been used in conjuction with TLC separations using silica gel and silica gel-silver nitrate plates. The relationship between retention time behavior and the structure of steroids has been studied, and the “steroid number” concept has been used to described GLC properties of steroids. Procedures have been found for the inactivation of supports, for the modification of liquid phase properties, for the preparation of a variety of special derivatives useful in GLC work and for the study of several groups of steroids important in human metabolism. A number of quantitative analytical separations were also developed.     

Stereospecific analysis of fatty acid esters of chloropropanediol isolated from fresh goat milk

The fatty acid esters of chloropropanediol isolated from goat milk fat in small quantities were subjected to a stereospecific analysis via phospholipase C and phosphocholine esters as intermediates. Synthetic rac-1-chloro-2,3-dioleoyl-propanediol was prepared by standard methods and was used as a control. The stereospecific analyses were performed following a release of the fatty acids from the primary positions of each chloropropanediol diester with pancreatic lipase. The resulting X-1-chloro-2-acylpropanediols were then converted into the corresponding phosphocholine derivatives by a stepwise reaction with phosphorus oxychloride and choline chloride. The X-1-chloro-2-acyl-3-phosphocholinepropanediols were subjected to hydrolysis with phospholipase C (C. perfringens), which hydrolyzed 50% of the phosphatide within two min and the rest of it in two hr. From previous experience with glycerol esters, it was assumed that the more rapidly hydrolyzed molecules were the sn-1-chloro-2-acyl-propanediol derivatives and the more slowly hydrolyzed ones the sn-2-acyl-3-chloropropanediol derivatives. A hydrolysis with phospholipase A₂ (Crotalus adamanteus) released 50% of the total fatty acid along with the corresponding lyso compound within 10 min, after which there was no further reaction. The hydrolysis products were assayed directly by gas liquid chromatography (GLC) or were isolated by thin layer chromatography (TLC) prior to quantitation by GLC. Both naturally occurring and synthetic chloropropanediol diesters behaved similarly on stereospecific analysis and were therefore concluded to be racemic.     

The distribution of labeled palmitic acid into the diglycerides and triglycerides of rat adipose tissue

After in vitro incubation of rat epididymal fat pads with radioactive palmitic acid, the distribution of the label in the different lipid classes and in different triglycerides was determined by silica gel and silver nitrate-silica gel thin-layer chromatography (TLC). The radioactivity of the diglycerides was approximately half of the triglycerides. This ratio did not change with alteration in the time of incubation. It remained unaltered even after a subsequent 10-min incubation in a nonradioactive medium. When the fat pads were incubated, first with¹⁴C-, then with³H-labeled palmitic acid, the³H/¹⁴C ratio was slightly lower in diglycerides than in triglycerides. The fully saturated molecules contained 38% of the radioactivity of triglycerides. Addition of oleic acid or norepinephrine to the labeled palmitic acid-containing medium decreased this value. Subsequent incubation with these compounds did not alter the distribution of radioactivity.     

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.     

Synthesis of monoglycerides by glycerolysis of rapeseed oil using immobilized lipase

Reaction conditions for the synthesis of monoglycerides (MG) by enzyme-catalyzed glycerolysis of rapeseed oil using Lipozyme® IM have been studied. Silica gel was used to adsorb the glycerol to overcome the problems of low glycerol solubility in the organic phase. An experimental design was used where temperature, time, the ratio of silica gel to glycerol (w/w), the water activity (a) _w , the isooctane concentration, and the ratio of glycerol to triglycerides (mol/mol) were varied. Response surface methodology was used to evaluate initial reaction rate and yield for the different products. The best yield of MG achieved under the studied conditions was 17.4% (mol fatty acid in substance/total mol fatty acid in mixture) (75°C, 20 h, silica gel/glycerol 2:1, a _w =0.17, 48% isooctane, glycerol/triglycerides 6:1). The same conditions yielded 36.8% diglycerides, 13.6% free fatty acids (FFA), and 36.9% triglycerides. This is at the same level as the equilibrium yield. The yield of MG is low compared to the final yield achieved with solid-phase glycerolysis. However, in solid-phase glycerolysis the reaction mixture becomes solid, and therefore the solid-phase process is not suitable for industrial application. The formation of FFA was very fast compared to the synthesis of MG. Equilibrium for FFA was reached within 2 h, and the yield was strongly affected by the a _w . Increasing a _w greatly increased the formation of FFA. In the a _w ratio 0.06–0.3, the yield of FFA increased from 4 to 19% while the yield of MG was nearly unaffected. As FFA is an undesired product, it is important to keep the a _w as low as possible.     

Partition coefficient of diglycerides in crystallization of palm oil

Crystallization of palm oil with and without solvent was carried out over a temperature range of 10–25°C. The yields of olein, the diglyceride contents, and compositions of the stearin and olein phases were determined by thin-layer and gas-liquid chromatography. The three major diglycerides, analyzed as C₃₂, C₃₄, and C₃₆, are mainly dipalmitoyl glycerol, palmitoyloleoyl glycerol, and dioleoyl glycerol. In crystallization without solvent, C₃₂ (PP) had a strong affinity for the stearin fraction and C₃₆ diglycerides concentrated in the olein phase. The partition coefficient of diglycerides between the olein and stearin phases was temperature-dependent and was influenced by the type of solvent used. Although solvent enhances the diglyceride partition into the olein phase, partitioning is more effective at low temperatures and with acetone as the solvent for fractionation.