Triglyceride separation by reverse phase high performance liquid chromatography

Rapid separations of triglycerides by chain length and degree of unsaturation were made by high performance liquid chromatography (HPLC) on a C-18 μ-Bondapak column with acetonitrile-acetone solvent mixtures. For saturated triglycerides, a linear relationship was observed between the carbon number and the log of the retention volume. Each double bond present in the triglyceride decreased the retention volume to approximately that of a saturated triglyceride with two carbon atoms less. Correlations of the fatty acid composition, as determined by gas liquid chromatography (GLC) with the HPLC data, provides much additional insight about triglyceride composition. To calculate triglyceride compositions, an internal standard tripentadecanoin was added to collected fractions before analysis by GLC.     

Die Zusammensetzung der Triglyceride von Kuhmilch und Humanmilch

Triglyceride Composition of Bovine and Human Milk The triglyceride composition in the fat portion of bovine milk (winter and summer) and mature human milk was calculated from the HPLC partition number and from the carbon number and fatty acid pattern in the HPLC fractions. The triglycerides identified (122 in bovine milk, 94 in human milk) and their contents in the HPLC fractions are tabulated.     

Structure of bovine milk fat triglycerides

The triglycerides of bovine milk fat globules were isolated and separated into short, medium and long chain lengths by thin-layer chromatography. The molecular weight distribution and the fatty acid composition of the component triglycerides was then separately determined by gas chromatography following argentation-thin-layer and preparative gas chromatography. Some 38 triglyceride types (28% of total), of which there could be up to 6 isomers, were specifically identified and quantitatively estimated. The quantitative estimates for the rest of the milk fat triglycerides were limited to much more complex glyceride groups. The results confirm the earlier claim that butyric and caproic acids occur in milk fat almost exclusively in combination with medium and long chain fatty acids. Presented in part at the AOCS Meeting, Philadelphia, October, 1966.     

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.     

Composition of the fat extracted from the seeds of theobroma bicolor

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

The determination of cocoa butter equivalents in chocolate

A method of determining cocoa butter equivalents in chocolate and cocoa butter is described. The method relies on a new approach for interpreting data obtained by triglyceride gas liquid chromatography (GLC). This technique provides information on the composition of a fat according to the carbon number of the triglycerides (C_n). Examination of the data for a wide range of cocoa butters shows that a straight line relationship between the C₅₀ and C₅₄ contents exists. This relationship has been used as the basis for a quantitative method determining the amount and type of cocoa butter equivalent added to chocolate. The application of the method to both plain and milk chocolate is described. The method is also used to determine the amount of milk fat in chocolate.     

Zur Analyse von Triglyceriden

Analysis of Triglycerides Only recently the analytic of triglycerides of natural fats and oils has found in HPLC an adaquate method. By means of HPLC it is possible to separate the glycerides of the fat with respect to partition number (PN) and to analyse the collected fractions by GLC with respect to carbon number (CN). This procedure yields a complete basis for calculation of the composition of triglyceride groups which are characterized by their carbon number and number of double bonds (e.g. C54:1). If the analytical data of each fraction are completed with the fatty acid composition, the fatty acid combination of the single triglycerides and their distribution can likely be characterized. This method was applied to triglyceride analysis of LOBRA oil. The obtained results are reported.     

Analysis of triglycerides by consecutive chromatographic techniques. i.cuphea llavia seed fat

The triglycerides ofCuphea llavia var.miniata seed fat were separated according to the number of double bonds/molecule using preparative thin layer chromatography (TLC) on silicic acid impregnated with silver ion. The recovered frac-tions were quantitated by the chromotropic acid technique. Each fraction was then separated ac-cording to mol wt using gas-liquid chromatog-raphy (GLC). This multiple chromatography procedure resolvedC. llavia triglycerides into 17 different components. The triglyceride composition ofC. llavia seed fat was calculated from the above results. Since the fat contains 91.2 mole % decanoic acid, it was expected that each triglyceride molecule would contain at least two molecules of decanoic acid. Results showed this to be generally true, but sev-eral minor component triglycerides not conform-ing to this pattern were found. Presented at the AOCS Meeting in Minneapolis, 1963.     

Determination of milk fat in chocolates by gas-liquid chromatography of triglycerides and fatty acids

The combination of two routine methods is proposed to determine the content of milk fat (MF) in chocolates, which is applicable even in the presence of lauric fats or others. The content of MF is obtained from the sum of C₄₀, C₄₂, and C₄₄ medium-chain triglycerides, determined by capillary gas-liquid chromatography (GLC). A new method, based on methyl esters of lauric acid and on minor acids situated between myristic and palmitic, is proposed. It enables detection and estimation of potential lauric fats, as well as the determination of the actual content of MF. The influence of other vegetable and animal fats is discussed. We analyzed 45 MF samples extracted from industrial milk powders and from pure or fractionated MF for chocolate manufacturing or pastry by GLC of triglycerides. We also analyzed by capillary GLC the methyl esters from 22 of those fats. Mixtures of these 22 MF samples with a cocoa butter also were used for chromatographic analyses of methyl esters and triglyceride. Results from the various analytical methods have been presented.     

Anwendungsmöglichkeiten der HPLC in der modernen Fettanalytik

The Use of HPLC in Modern Fat Analysis Although the general lipid detection problem has not yet found a satisfactory solution, HPLC is increasingly popular for the investigation of fats and their minor components. There are now numerous HPLC applications that may also prove useful in the identification of fats and the characterization of fat mixtures. Whereas fatty acid and sterol investigation is still a domain of GLC, triglyceride compositions and some minor components are usually analyzed by HPLC. Triglycerides are separated by RP-HPLC both according to chain length and number of double bonds. In this way characteristic differences are seen even in fats that do not differ much by their fatty acid composition, HPLC is widely used also in the investigation of tocopherols and other fat soluble vitamins, as well as for trace components such as chlorophyll degradation products, contaminants and additives. HPLC peak area ratios may permit conclusions as to the nature of a fat, its origin and history and possible adulteration.     

The simultaneous determination of both the quantity and the fatty acid composition of the triglycerides in three to ten microliters of plasma

An integrated system using chromatography on silica gel-impregnated glass paper, an internal standard and gas-liquid chromatography (GLC) makes possible the isolation of triglycerides, the determination of triglyceride, fatty acid composition, and the quantitative determination of triglyceride. Recoveries are good and values for total triglyceride obtained by this system compare favorably with values obtained by application of the hydroxamate colorimetric method to the triglyceride eluate from silicic acid columns. The fatty acid composition of the cholesteryl ester (CE) fraction is obtainable at the same time by GLC of methyl esters prepared from the CE fractions which also appears as a distinct spot on these chromatograms. Also, a densitometric method is described for triglyceride determination by photometry of the charred spots obtained after chromatography on the gel-paper. Presented at the AOCS Meeting, Houston, April, 1965     

Triglyceride analysis by gas chromatography in assessment of authenticity of goat milk fat

The triglyceride (TG) composition of 35 samples of milk collected at different times of year from five herds of goats was analyzed using short capillary column-gas chromatography. The distribution of TG in goat milk fat was unimodal, peaking at C₄₀ (12.6%); the sum of TG from C₃₈ to C₄₄ accounted for about 50%, whereas the three classes of TG from C₄₈ to C₅₂ did not exceed 6% each. These results were compared with the corresponding data for cow milk fat. Significant differences between herds were observed, mainly in long-chain TG. To detect foreign fats in goat milk fat, two multiple regression equations based on TG content of the goat milk fat were proposed. Analysis of known mixtures of tallow, palm oil, and cow milk fat with goat milk fat have experimentally confirmed the accuracy of the equations.     

Gas-liquid chromatography of triglyceride mixtures containing both odd and even carbon number fatty acids

Quantitative GLC of triglycerides has been extended to natural fats containing both odd and even carbon number fatty acids. A 1.83-m glass column containing 3.0% JXR silicone on 100/120 mesh Gas-Chrom Q resolved triglycerides differing by only one carbon number. Peak resolution was significantly improved by hydrogenating each triglyceride sample prior to GLC analysis. The triglycerides of four fish oils (mullet, tuna, menhaden, and pilchard) and one seed fat (Acanthosyris spinescens) containing odd carbon number fatty acids were analyzed by this technique. The method was also useful for determining the triglyceride composition of the cyclopentene fatty acid oil fromHydnocarpus wightiana seeds. Presented at the AOCS meeting, New Orleans, May, 1967     

Determination of the fully saturated triglyceride composition of fats

A method is described for the quantitative determination of the fully saturated triglyceride composition of fats. The method involves a quantitative oxidation of the unsaturated triglycerides in the fat to a mixture of α ketol and dihydroxy compounds, using aqueous alkaline potassium permanganate and a phase transfer catalyst in a two phase reaction. The fully saturated triglycerides are fractionated from the oxidation products by column chromatography on silicic acid and their composition is quantified by gas liquid chromatography (GLC) using a flame ionization detector. Analyses of samples of lard, cocoa butter, olive oil, soybean oil, crude palm oil (ex Malaysia) and the stearin fraction from palm oil are given as examples of the method. Presented as a poster presentation at the 14th World Congress for Fat Research in Brighton, England (1978).     

Influence of the position of unsaturated fatty acid esterified glycerol on the oxidation rate of triglyceride

The influence of the position of unsaturated fatty acid esterified glycerol on the oxidation rate of triglyceride was investigated at 50 C. Randomized triglycerides used were prepared by random interesterification between saturated and unsaturated monoacid triglycerides using sodium methoxide as catalyst. The monoacid triglycerides used were tripalmitin, tristearin, triolein and trilinolein. The molecular species of the randomized triglycerides were analyzed by high performance liquid chromatography (HPLC) in combination with gas liquid chromatography (GLC) and enzymatic hydrolysis. From the results of oxygen absorption measurement by GLC, the randomized triglycerides were more stable towards oxidation than the triglyceride mixtures which were prepared by mixing the equivalent quantities of the same monoacid triglycerides as used in the random interesterification. This may be due to the decrease in the contents of most unstable unsaturated monoacid triglycerides by random interesterification with saturated monoacid triglycerides. Furthermore, from the results obtained with the detailed analysis of the randomized triglycerides at different stages of oxidation, it became clear that the triglycerides having unsaturated fatty acids linked at the 2-position of glycerol are more stable towards oxidation than those linked at the 1(or 3)-position. The carbon chain length of saturated fatty acids has essentially no influence on the oxidation rates of unsaturated fatty acids esterified in the same glycerol.     

Zusammensetzung und Aufbau der Triglyceride von Muttermilch und einiger Rohstoffe für Säuglingsnahrung

Composition and Structures of Triglycerides of Human Milk and Some Base Components for Infant Milk Formulas Following HPLC separation of human milk triglycerides with a silver-loaded ion exchange and an RP-18-phase column, the 50 substantial fractions obtained were characterized by gas chromatography of triglycerides and fatty acid methyl esters as well as by the analysis of intramolecular distribution on a reduced scale using ethyl magnesium bromide. Exploitation of all data available resulted in the detailed structures of 106 triglycerides, representing 81 g/100 g human milk fat. A substitute for producing infant milk formulas should be adapted to the composition and fatty acid distribution of those 14 substantial triglycerides which are present in human milk fat with more than 1 g/100 g, amounting to a total of 42 g/100 g. Among several fats and oils analyzed, only lard and a recent product were found to contain palmitic acid also predominantly in the 2-position. Therefore, other base components would require a directed fractionation and/or interesterification to be best adapted to human milk fat.     

Trennung der Triglyceride von Kuhmilch und Humanmilch durch HPLC und Untersuchung der Fraktionen durch GC

HPLC Separation of Triglycerides in Bovine and Human Milk and GLC Analysis of the Isolated Fractions Reversed-phase HPLC using acetonitrile-propionitrile mixtures and a very simple temperature programming device (temperature rising from 10–15°C up to 60°C) yielded optimum separation of the triglycerides in bovine milk (winter and summer) and mature human milk. The components in the isolated fractions were analysed by GLC for triglycerides and fatty acid methyl esters.     

Glyceride analysis of palm oil after solvent fractionation

A procedure is described for determing the triglyceride composition of palm oil and its fractions by the use of silver nitrate thin layer chromatography (TLC) and gas liquid chromatography (GLC). The triglycerides separated by silver nitrate TLC according to the number of double bonds are quantified using infrared spectroscopy before further analysis by GLC according to carbon number. The results from the two techniques enable the composition of the oil and fractions to be computed on a molecular basis in relation to fatty acid types. The potential application of this procedure is to analyze fractions obtained from the fractionation of oils and fats to which the 1,3-Random-2-Random distribution theory is not applicable.     

Triglyceride analysis by consecutive liquid-liquid partition and gas-liquid chromatography.Ephedra nevadensis seed fat

The triglyceride composition ofEphedra nevadensis seed fat, which contains 16 different fatty acids, has been analyzed by a combination of liquid-liquid partition and gas-liquid chromatography. Triglycerides were first separated by liquid-liquid partition chromatography. The recovered fractions were then analyzed by gas-liquid chromatography to determine the molecular weights of the triglycerides present. Consecutive separation by these two techniques resolved this complex seed fat into 30 different triglyceride groups. A method for preparative liquid-liquid partition chromatography of triglycerides is described in detail. Highly unsaturated triglyceride mixtures are easily resolved on the basis of “partition number” by using a hexadecane/nitroethane partition system. Presented at the AOCS Meeting, Chicago, October, 1967.     

Unterscheidung von Palmölen und Palmöl-Fraktionen durch HPLC der Triglyceride

Analytical Characterisation of Palm Oil Fractions by HPLC of Triglycerides The increasing importance of palm oils and palm oil fractions in international trade makes it necessary to develop analytical criteria for the characterisation of these products. One problem is the correct identification of palm oleins, which are often difficult to distinguish from palm oils. These products frequently exhibit similar fatty acid by GLC and similar iodine values. The correct identification of palm stearins by GLC is comparatively more easy. Palm oleins and palm oils can be characterized by HPLC using conditions that permit not only the separation of triolein from less unsaturated triglycerides of the same carbon number, but also, at the same time, a separation of tripalmitin from triglycerides containing oleic acid. By establishing the peak area ratios for a number of selected triglycerides a clear differentiation between palm oils and palm oleins, as well as various other palm oil fractions and their mixtures is obtained.