The use of SP2340 glass capillary columns for the estimation of thetrans fatty acid content of foods

Glass capillary gas chromatography (GCGC) on 100-m and 60-m SP2340 columns was used for quantitation of thetrans unsaturated fatty acids in shortenings and fast foods. The separation of thecis andtrans octadecenoates on GCGC was evaluated by preparatory argentation thin layer chromatography. In addition, thetrans content of shortening samples obtained by GCGC was compared totrans content determined by infrared analysis. This research conducted by the Science and Education Administration, USDA, on the commercial foods as reported in this paper was limited to analysis of their lipid compositions. The data are reported solely as factual information and are limited to the samples analyzed. No warranty or guarantee is made or implied that other samples of these products will have the same or similar composition. It is the policy of the USDA not to endorse those commercial products used in research over those that were not included in the research.     

Derivative UV spectra of lipid conjugated dienes

Zeroth and second order derivative UV spectra of some lipids and model substances having a conjugated system of double bonds were recorded. The second derivative spectra showed fine spectral structures with at least three negative peaks. The wavelengths of their minima are different fortrans,trans andcis,trans geometric isomers, which allowed determination of each individual isomer in a mixture without any separation. The derivative spectra of 2,4-hexadienes, some acetyl esters of unsaturated fatty alcohols and autoxidized linoleic acid, soybean oil and soybean lecithin are discussed.     

Thin-layer chromatographic separation of some bromo-and hydroxyderivatives of stearic acid

Addition of polar substituents to unsaturated fatty acids or their methyl esters enhanced the adsorption affinity contributed by each unsaturation site to such an extent that the resulting derivatives could be easily separated by differential adsorption on a silica gel coating. Saturated and unsaturated fatty acids could be separated by thin layer chromatography after either bromination of their methyl esters or oxidation of their mixture into unsaturated esters, monoenoates, dienoates, and trienoates. Furthermore, it was possible to separate the configurational isomers of the hydroxystearic acids derived fromcis andtrans hydroxylation of oleic and linoleic acids. Partial support of these studies by Grant No. H-1819 from National Institutes of Health.     

Identification of fatty acid polymorphic modifications by infrared spectroscopy

IR spectra in the 7.5–25.0 μ region are shown to be unique for the individual long chain odd carbon numbered saturated fatty acids and for the monounsaturated even carbon numbered fatty acids, their isomers, and polymorphic modifications. IR spectra are presented which illustrate similarites and dissimilarities resulting from chain length differences and polymorphic variations for monounsaturated acids. X-ray crystal long spacings for the saturated odd carbon numbered acids are compared with literature values. X-ray crystal long spacings for monounsaturatedtrans acids are shown to be related to carbon chain length and independent of the location of unsaturation. Diffractograms and IR spectra of the 2 polymorphic modifications of elaidic acids are presented. IR spectra illustrating band differences forcis andtrans isomers are shown. ARS, USDA.     

Monoethylenic fatty acids of a partially hydrogenated herring oil

A Canadian Atlantic herring oil hydrogenated for margarine use to an iodine value of 76 and melting point of 32.5 C was found to have 30% saturated acids and 66% monounsaturated fatty acids. The monounsaturated fatty acids could be analytically determined ascis andtrans isomers by open tubular gas liquid chromatography.Trans acids were 33% of the C₁₆ and C₁₈ monounsaturated acids, and 32 and 28%, respectively, of the C₂₀ and C₂₂ monounsaturated acids. After separation of geometric isomers by Florisil-silver nitrate chromatography the positional isomers in each class were determined by oxidative fission. The double bond positions of the originalcis fatty acids were largely retained in bothcis andtrans isomers, but additional isomers were observed, especially in thetrans fatty acids.     

Influence of stage of lactation on the triacylglycerol composition of buffalo milk fat

Milk fats obtained from colostrum and early, middle and late lactation samples of buffalo milk were analyzed for their triacylglycerol (TG) compositions. Each milk fat first separated by thin layer chromatography (TLC) into high, medium and low molecular weight TG. The TG fractions thus obtained were further segregated by argentation TLC, according to their degree of unsaturation into saturated,trans-monoene,cis-monoene, diene and polyene species. With progressive lactation, the major changes from colostral fat were an increase in lower fatty acids and decline in oleic acid. This caused, in turn, marked variations in saturated TG and diene TG and, to a smaller extent, in polyene TG. Monoene TG, bothcis andtrans, remained practically constant throughout. These trends were largely reversed toward the end of lactation. NDRI publication no. 79-297.     

Simplified method for the determination oftrans monoenes in edible fats by TLC-GLC

Several modifications of an established thin-layer chromatography-gas-liquid chromatography (TLC-GLC) procedure for quantitatingtrans unsaturated fatty acids in edible fats are presented. These refinements considerably simplify the procedure without affecting accuracy. The modifications include: i) the use of pre-coated silica sheets, dynamically impregnated with Ag⁺, which allow separated bands to be cut off with a pair of scissors; and ii) the use of stearic acid in the deliberately combined saturated andtrans monounsaturated fatty acid methyl ester bands as an (endogenous) internal standard.Trans values thereby obtained agree favorably with the results from the conventional technique.     

cis/trans isomerization of unsaturated fatty acids as possible control mechanism of membrane fluidity inPseudomonas putida P8

Exponentially growing cells ofPseudomonas putida had an increased ratio of saturated to unsaturated fatty acids in response to increased growth temperatures. Resting cells in which fatty acid biosynthesis was stopped reacted to a thermal increase by convertingcis-monounsaturated fatty acids totrans isomers.cis/trans isomerization of up to 60% of the unsaturated fatty acids was also activated by alcohols of different chain length. Their effective concentrations apparently depended on the lipophilic character of the alcohols. Also, a salt shock caused by the addition of NaCl resulted in the production oftrans fatty acids. However, cells that were adapted to growth media of high osmolarity synthesized cyclopropane fatty acids instead oftrans fatty acids. Activity ofcis/trans-isomerase was dependent on the growth phase and was significantly higher during logarithmic growth than during the stationary phase. The results of this study agree with the hypothesis that the isomerization ofcis intotrans unsaturated fatty acids is an emergency action of cells ofP. putida to adapt membrane fluidity to drastic changes of environmental conditions.     

Argentation chromatography on silver sulfamate-impregnated silica gel layers

Silver sulfamate was used in place of silver nitrate as argentation and charring reagent in thin layer chromatography (TLC). This reagent permits thin layer chromatographic separations of fatty acid esters with differing degrees of unsaturation as well as ofcis/trans isomeric fatty acids, and allows for direct charring of fractions. Examples given for separations on silica gel plates impregnated with silver sulfamate include cholesterol esters and fatty acid methyl esters.     

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.     

Determination of Geometrical Isomers of 9,12-Octadecadienoic Acid Present in Food Fats

The present work presents a method to be used in the determination of different geometrical isomers of 9,12-octadecadienoic acid present in food fats. The method is based on isotope dilution, the radioactive isomers needed being prepared by isomerization of cis,cis-9,12-octadecadienoic acid. Radioactivity is acquired through esterification of the isomers by ¹⁴C-diazomethane. For the identification of the isomers infra-red spectrophotometry, alkali isomerization combined with ultraviolet spectrophotometry, gas chromatography, and ozonolysis are used. The isomer to be determined is separated from other fatty acids by argentation thin layer chromatography. The standard deviation of the method was in 15 parallel analyses 0.04%. The sample of food fat to be examined was milk fat, the cis,cis-9,12-octadecadienoic acid content obtained being 0.83%, which corresponded to about one half of octadecadienoic acids of the sample. When a fatty acid mixture with a known composition was analyzed, the cis,cis-9,12-octadecadienoic acid yield was 99.7% on the average, the standard deviation being 0.04%.     

Evaluation of sequential methods for the determination of butterfat fatty acid composition with emphasis ontrans-18:1 acids. Application to the study of seasonal variations in french butters

The successive steps of an integrated analytical procedure aimed at the accurate determination of butterfat fatty acid composition, includingtrans-18:1 acid content and profile, have been carefully checked. This sequential procedure includes: dispersion of a portion of butter in hexane/isopropanol (2:1, vol/vol) with anhydrous Na₂SO₄, filtration of aliquots of the suspension through a microfiltration unit, subsequent preparation of fatty acid isopropyl esters (FAIPE) with H₂SO₄ as a catalyst, and analysis of total FAIPE by capillary gas-liquid chromatography (GLC). Isolation oftrans-18:1 isomers was by silver-ion thin-layer chromatography (Ag-TLC), followed by extraction from the gel of combined saturated andtrans-monoenoic acids with a biphasic solvent system. Analysis of these fractions by GLC allows the accurate quantitation oftrans-18:1 acids with saturated acids (14:0, 16:0, and 18:0) as internal standards. A partial insight in the distribution oftrans-18:1 isomers can be obtained by GLC on a CP Sil 88 capillary column (Chrompack, Middelburg, The Netherlands). All steps of the procedure are quite reproducible, part of the coefficients of variation (generally less than 3%, mainly limited to butyric and stearic acids) being associated with GLC analysis (injection, integration of peaks) and, to a lesser extent, to FAIPE preparation. FAIPE appear to be of greater practical interest than any other fatty acid esters, including fatty acid methyl esters (FAME), for the quantitation of short-chain fatty acids, because peak area percentages, calculated by the integrator coupled to the flame-ionization detector, are almost equal (theoretically and experimentally) to fatty acid weight percentages and do not require correction factors. With this set of procedures, we have followed in detail the seasonal variations of fatty acids in butterfat, with sixty commercial samples of French butter collected at five different periods of the year. Important variations occur around mid-April, when cows are shifted from forage and concentrates during winters spent in their stalls to fresh grass in pastures. At this period, there is a decrease of 4:0–14:0 acids and of 16:0 (−2 and −6%, respectively), while 18:0 andcis- plustrans-18:1 acids rise suddenly (2 and 5%, respectively). These modifications then progressively disappear until late fall or early winter. Other variations are of minor quantitative importance. Although influenced by the season, the content of 18:2n-6 acid lies in the narrow range of 1.2–1.5%.Trans-18:1 acids, quantitated by GLC after Ag-TLC fractionation, are at their highest level in May–June (4.3% of total fatty acids), and at their lowest level between January and the end of March (2.4%), with a mean annual value of 3.3%. The proportion of vaccenic (trans-11 18:1) acid, relative to totaltrans-18:1 isomers, is higher in spring than in winter, with intermediate decreasing values in summer and fall, which supports the hypothesis that the level of this isomer is linked to the feed of the cattle, and probably to the amount of grass in the feed.     

Sequential gas chromatographic procedure for microanalysis of monoenoic double bond position in hydrogenated oils

Quantitative cleavage of epoxyoctadecanoates with periodic acid (HIO₄) has been demonstrated and the technique incorporated into an all gas chromatographic system for detailed lipid analysis. The overall procedure involves three sequential gas chromatographic separations interspersed by two microreactions. By this procedure, a complete analysis forcis- andtrans-geometric isomers corresponding to each positional octadecenoate isomer is obtained. Total sample requirements are less than 10 mg, and the elapsed analysis time/sample is less than 10 hr. In this all gas chromatographic procedure, a lipid methyl ester sample is first separated by preparative gas chromatography, and the monoene fraction is collected and epoxidized. Next, the epoxidized sample is separated by gas chromatography intocis- andtrans-epoxyoctadecanoate fractions. These epoxyoctadecanoate fractions are collected and cleaved with HIO₄ into aldehyde and aldehyde-ester fragments, which are quantitatively analyzed by gas chromatography. The double bond positions are determined from the aldehyde and aldehyde-ester cleavage data, which are stored and processed by a computerized on-line gas chromatographic data acquisition system. The procedure was tested on pure octadecenoate isomers, standard mixtures, and commercially hydrogenated vegetable oils. Analyses of hydrogenated vegetable oils are compared with data acquired by reverse-phase and argentation chromatography followed by reductive ozonolysis. Presented at the AOCS Meeting, April 1973, New Orleans. ARS, USDA.     

trans Fatty acids in milkfat

Milkfat is extremely complex, with ca. 500 different fatty acids reported in the triglycerides. Seasonal feed variation results in higher unsaturated fatty acid levels in summer than in winter. Rumen microbes hydrogenate unsaturated feed lipids to yield a mixture of geometrical and positional isomers which are transmitted to the milk. Total isolatedtrans fatty acids in milkfat reported in the literature range from 2 to 11% with maximal values in summer and minimal values in winter. A study was undertaken about the use of AOCS Method Cd 14-61 for the determination of isolatedtrans levels in milkfat. The triglycerides of milkfat were analyzed using trielaidin as a standard and milkfat methyl esters were analyzed using methyl elaidate as a standard. Corrections were made, for the loss of the methyl esters of the short-chain fatty acids. The apparent levels oftrans found by using the triglycerides were considerably higher than by using the methyl esters. Infrared spectra of pure triglycerides of saturated fatty acids showed measurable absorption at 10.36 μm, whereas methyl esters of saturated andcis unsaturated fatty acids did not.     

Determination of fatty acids and some undesirable fatty acid isomers in selected Turkish margarines

The fatty acid composition and total trans fatty acid content in 10 margarines produced in Turkey were determined by capillary gas chromatography and Fourier transform‐infrared spectroscopy (FT‐IR) spectroscopy. The fatty acid composition ranged as follows: saturated fatty acids, C16:0 (palmitic) 11.3 to 31.8% and C18:0 (stearic) 5.7 to 8.7%, monounsaturated fatty acids, C18:1 (oleic) 21.8 to 35.7% and C18:1 trans isomers 0.4 to 27.4%, polyunsaturated fatty acid, C18:2 linoleic acid 5.2 to 40.2%. Some positional isomers of C18:1 as cis‐11‐octadecenoic acid varied from 0.7 to 4.6% and cis‐13 trace to 2.4%. The total trans fatty acid contents were between 0.9 and 32.0% when measured with capillary gas chromatography and between 0 and 30.2% with FT‐IR spectroscopy. Some of the margarines analyzed contained trace amount of trans fatty acids which could not be detected by FT‐IR spectroscopy.     

GLC and TLC analysis of isopropylidene derivatives of isomeric polyhydroxy acids derived from positional and geometrical isomers of unsaturated fatty acids

Gas-liquid chromatography (GLC) and thin-layer chromatography (TLC) were used to investigate the isomeric positional geometrical isopropylidene derivatives of nine isomeric dihydroxy esters, four isomeric methyl 9,10-12-trihydroxystearates, and eight isomeric methyl 9,10-12,13-tetrahydroxystearates prepared from unsaturated fatty acids. The isopropylidenes derived fromcis andtrans monounsaturated fatty acids were easily separated on both polar and nonpolar columns. Positional isopropylidenes derived from positional isomers of monounsaturated fatty acids were not separated on either liquid phase but were resolved by TLC. Four of the eight isomeric isopropylidenes derived from the four geometrical isomers of linoleic acid were resolved on the polar column; the other four isomers eluted as a single peak. The four isomeric isopropylidene-trifluoroacetate derivatives derived from ricinoleic and ricinelaidic acids were also resolved on the polar column. GLC analyses were carried out with liquid phases of ethylene glycol succinate methyl silicone polymer (EGSS-X) and methyl silicone polymer (SE-30) packed columns. Isopropylidenes, in addition to their applicability for the resolution of polyhydroxy acid mixtures, are particularly useful for the determination of double bond positions and geometrical configurations of fatty acids without cleavage. Under contract with the U. S. Atomic Energy Commission.     

Fatty acid and conjugated linoleic acid isomer profiles in human milk fat

The fatty acid composition of 39 mature human milk samples from four Spanish women collected between 2 and 18 weeks during lactation was studied by gas chromatography. The conjugated linoleic acid (CLA) isomer profile was also determined by silver‐ion HPLC (Ag⁺‐HPLC) with three columns in series. The major fatty acid fraction in milk lipids throughout lactation was represented by the monounsaturated fatty acids, with oleic acid being the predominant compound (36–49% of total fatty acids). The saturated fatty acid fraction represented more than 35% of the total fatty acids, and polyunsaturated fatty acids ranged on average between 10 and 13%. Mean values of total CLA varied from 0.12 to 0.15% of total fatty acids. The complex mixture of CLA isomers was separated by Ag⁺‐HPLC. Rumenic acid (RA, cis‐9 trans‐11 C18:2) was the major isomer, representing more than 60% of total CLA. Trans‐9 trans‐11 and 7‐9 (cis‐trans + trans‐cis) C18:2 were the main CLA isomers after RA. Very small amounts of 8‐10 and 10‐12 C18:2 (cis‐trans + trans‐cis) isomers were detected, as were different proportions of cis‐11 trans‐13 and trans‐11 cis‐13 C18:2. Although most of the isomers were present in all samples, their concentrations varied considerably.     

cis-Vaccenic acid in mango pulp lipids

A peak corresponding to a methyl octadecenoate other than oleate has been detected on the capillary gas chromatogram of the methyl esters of mango pulp fatty acids. This octadecenoate was isolated by silica gel and argentation column chromatography, high performance liquid chromatography and argentation thin layer chromatography, and then analyzed by infrared, nuclear magnetic resonance and gas chromatography-mass spectrometry, chromatographic separations and oxidative degradation. These analytical data proved that the octadecenoic acid wascis-vaccenic acid (cis-11-octadecenoic acid). The concentration of this acid in total octadecenoic acids ranged from 35% to 50% in the pulp of mangoes from Fiji, Mexico, the Philippines and Taiwan.cis-Vaccenic acid was revealed to be one of the major component fatty acids of non-polar lipids (mainly triacylglycerols), glycolipids and phospholipids in mango pulp. The glycolipids containedcis-vaccenic acid (ca. 20%) in higher concentration than oleic acid (ca. 15%). A trace amount ofcis-vaccenic acid (0.5%) was detected in the total lipids of mango seeds. Profile of fatty acid composition of mango pulp lipids (0.2–0.3 wt% of wet pulp) was characterized by the presence of n−7 acid isomers,cis-vaccenic acid and palmitoleic acid, and unusual mono- and dienoic positional isomers.     

Fatty acid composition andtrans isomer content of hardened olive oil

Cooking fats and margarines prepared from olive oil were analyzed gas chromatographically for fatty acid composition andtrans isomer content. Argentation thin layer chromatography and mass spectrometry were used to assist in identifications of minor components. Olive oil margarines and cooking fats contained 70–76% monoenoic acids, of which 30–37% was in thetrans configuration, and 5% dienoic acids. Twenty percent of the dienes were in the form oftrans-trans andcis-trans octadecadienoic acid.     

Heat treatment of vegetable oils. II. GC-MS and GC-FTIR spectra of some isolated cyclic fatty acid monomers

Gas liquid chromatography coupled with mass spectrometry (GC-MS) showed that the cyclic fatty acid monomers (CFAM) isolated from a heated linseed oil have two ethylenic bonds, while the CFAM isolated from heated sunflower oils were saturated and monoethylenic isomers. GC-MS studies also showed the presence of cyclohexenic derivatives in the case of linseed oil. GLC coupled with Fourier transform infrared spectrometry (GC-FTIR) studies indicated that the CFAM isolated from linseed oil were ofcis (Z),trans (E) structures except two components which werecis,cis (Z,Z) dienoic acids. The unsaturated CFAM isolated from sunflower oils werecis (Z) andtrans (E) monoethylenic isomers. For sunflower oils, the major CFAM were isomers having acis (Z) ethylenic bond. The saturated CFAM isolated from a heated sunflower oil had molecular weights of 296 and 294. The latter could correspond to some bicyclic isomers.