The GLC and TLC resolution of diastereoisomeric polyhydroxystearates and assignment of configurations

Trifluoroacetate (TFA) derivatives of methyl 12-hydroxystearate, methyl ricinoleate, five positional isomers of methylthreo- anderythro-dihydroxystearate, four diastereoisomeric methyl 9,10–12-trihydroxystearates, and four racemic diastereoisomeric methyl 9,10–12,13-tetrahydroxystearates were prepared and analyzed by gas-liquid chromatography (GLC). The isomericthreo- anderythro-dihydroxystearates that had not previously been resolved by GLC were separated. Excellent resolution of the diastereoisomeric pairs of methylthreo- anderythro-9,10–12-triand and methylerythro, erythro- andthreo, threo-9,10–12,13-tetrahydroxystearates was obtained by GLC of their TFA derivatives. Analyses of these high-molecular-weight compounds were carried out on polar and nonpolar packed columns used routinely for methyl ester analysis. The various methyl mono-, di-, tri-, and tetrahydroxysterate esters were also analyzed by thin-layer chromatography (TLC) on Silica Gel G adsorbent layers and on Silica Gel G impregnated with sodium arsenite. Relative and absolute configurations were assigned to the various diastereoisomeric tri- and tetrahydroxysterates based on information obtained from GLC, TLC, synthetic ratios, and molecular-models. A micro hydroxylation method that gives quantitative yields ofthreo- anderythro-dihydroxy acids from various concentrations of C₁₈ monoene geometrical isomers was developed. Subsequent GLC analysis of the isomeric methyl dihydroxy TFA derivatives allows the quantitative determination of double-bond configuration on small samples without expensive or specialized equipment. Presented at the AOCS Meeting, Los Angeles, April 1966. under appointment from Oak Ridge Associated Universities. An operating unit of the Oak Ridge Associated Universities, under contract with the US Atomic Energy Commission.     

High performance liquid chromatography and glass capillary gas chromatography of geometric and positional isomers of long chain monounsaturated fatty acids

Positional and geometrical isomers of monounsaturated long chain fatty acids were analyzed by the combination of high performance liquid chromatography (HPLC) and glass capillary gas chromatography (GC). A preparative group separation ofcis andtrans isomers of the monounsaturated fatty acid methyl esters was achieved according to chain length by reversed-phase HPLC, and using a highly sensitive interference refractive index detector. After collection of the different fractions containingcis andtrans forms of the monounsaturated fatty acid methyl esters, the fractions were analyzed for their content of positional isomers using glass capillary GC with Silar-5 CP as stationary phase. The preparative step in the HPLC was also used analytically for the determination of the ratio between thecis andtrans monounsaturated fatty acids. A comparison was made between the results obtained with the HPLC technique and the results of a GLC technique with a packed OV-275 column. There was a good correlation between the 2 techniques with a tendency to highertrans values with the HPLC technique (4%). It was shown with reference substances that 18∶1ω6-cis to ω11-cis and 18∶1ω5-trans to ω12-trans, the most common monounsaturated fatty acid isomers in partially hydrogenated vegetable oils, could be almost quantitatively recovered in the HPLC step. Most of the individual positional isomers of monounsaturated fatty acids of varying chain length could be separated and determined in the glass capillary GC step with the exception of those isomers containing the double bond in a relatively high ω-position. The relative standard deviation of the technique as determined with reference substances was better than 4%. The described technique was applied to the analysis of the isomeric monounsaturated fatty acid content in partially hydrogenated vegetable and marine oils, and about 5 samples a day could be executed. Part of this work has been presented at the ISF/AOCS World Congress, New York (1980)JAOCS 58, (4), 1981, abstr. no. 184.     

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     

Zur gaschromatographischen Analytik trans-isomerer Fettsäuren auf gepackten Säulen (Silar 10 C,Silar 9 CP,SP 2340, OV 275) sowie auf der mit SP 2340 beschichteten Glaskapillare

Gas Chromatographic Analysis of trans-Isomeric Fatty Acids on Packed Columns (Silar 10C, Silar 9CP, SP 2340, OV 275) and in Glass Capillary Coated with SP 2340 High polar and temperature-resistant cyanopropylsiloxane phases such as Silar 9CP, Silar 10C, SP 2340 and OV 275 have a high selectivity for double bonds and make it possible, for the first time, to separate cis/trans isomeric fatty acid methyl esters on packed columns. The possibilities and limitations for identifying and separating the positional and geometric isomers of monoene, diene and triene fatty acid methyl esters on packed columns are demonstrated by numerous examples and compared with the application range of the SP 2340 glass capillary column. In addition to fatty acid methyl esters, non-derivated fatty acids and alditol acetates resulting from analysis of the sugar moiety of glycolipids and glycoproteins can also be gas chromatographed on these phases.     

A comparison of pyrrolidide and picolinyl ester derivatives for the identification of fatty acids in natural samples by gas chromatography-mass spectrometry

The pyrrolidide and picolinyl ester derivatives of the fatty acids in two natural lipid samples rich in unsaturated fatty acids, pig testis lipids and cod liver oil were satisfactorily resolved on capillary columns of fused silica coated with stationary phases of varying polarity. The picolinyl esters, in particular, when subjected to gas chromatography-mass spectrometry on a column containing a cross-linked methyl silicone, gave distinctive mass spectra, which could be interpreted in terms of both the numbers and positions of the double bonds.     

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.     

Gas-liquid chromatography of bile acids

Methods are reviewed for the preparation, gasliquid chromatographic (GLC) separation, identification, and quantitative estimation of the trifluoroacetyl derivatives of bile acid methyl esters. Of the stationary phases tried (SE-30, QF-1 and XE-60) methylfluoroalkyl silicone (QF-1) was best suited for analysis of the trifluoroacetates. This phase (1–2% QF-1 on 100–120 mesh Gas Chrom P) allowed an orderly resolution of conformational isomers and consistently gave GLC columns (stainless steel tubes, 1/8 in. O.D. × 3 ft) from which the bile acid derivatives could be recovered in high yield. Applications to biological materials are illustrated with bile acid samples from animal biles and from human duodenal drainage and feces. Identifications of the major bile acids made by the GLC of the trifluoroacetates were confirmed by results obtained with bile acid methyl esters and bile acid methyl ester acetates on QF-1 and the other liquid phases investigated. For most mixtures of bile acids, however, it appears that GLC of methyl esters and methyl ester trifluoroacetates on QF-1 is sufficient for a reliable recognition of common bile acids. Overall accuracy of the estimates was of the order of ± 5%, but it varied with the nature and concn of the component.     

Application of methoxy-bromomercuri-adduct fractionation to the analysis of fatty acids of partially hydrogenated marine oils

The fatty acids of a refined and of a partially hydrogenated menhaden oil, iodine value (IV) 84.5, were separated into different classes (e.g., monoene, diene, including pentaene and hexaene) by thin layer chromatography (TLC) of their methoxy-bromomercuri-adducts (MBM). In the solvent system hexane: dioxane, the separation of fatty acids occurred according to the degree of unsaturation. No influence was exerted by either the geometry or the position of the ethylenic bonds. The effect of the various chain lengths (C₁₄−C₂₂) was to broaden the bands, but no overlap occurred among the chain lengths. A wide range of C₂₀ unsaturated fatty acids were prepared by the hydrazine reduction of 20∶5-Δ5,8,11,14,17. These were separated into groups as MBM adducts and identified by comparison of their experimental and calculated equivalent chain lengths (ECL) in gas liquid chromatography (GLC) on SILAR-5CP and SILAR-7CP columns. This confirmed that GLC did not totally separate all groups of isomers of different degrees of unsaturation. The quantitative analysis of both refined and partially hydrogenated (IV-84.5) menhaden oils by GLC was effected by the recovery of the fatty acid methyl esters from the MBM adduct TLC bands with the addition of methyl heptadecanoate (17∶0) as an internal standard, followed by analysis of the different fractions on open-tubular columns coated with SILAR-5CP. For methylene- and nonmethylene-interrupted unsaturated acids, 100% recovery from the MBM adducts was achieved, but in the case of the conjugated dienes the maximal recovery was 70%.     

Rapid determination of double-bond positions in monoenoic fatty acids by periodate-permanganate oxidation

Tetramethylammonium hydroxide has been used in the extraction and pyrolysis methylation of the carboxylic acids produced by periodate-permanganate oxidation of monounsaturated fatty acid methyl esters. This modification of the von Rudloff procedure allows rapid determination of double-bond positions and analysis of mixtures of positional isomers of monoenoic fatty acids.     

2-alkenyl-4,4-dimethyloxazolines as derivatives for the structural elucidation of iosmeric unsaturated fatty acids

Several types of unsaturated fatty acid methyl esters were converted into 4,4-dimethyloxazoline (DMOX) derivatives and analyzed by mass spectrometry to further evaluate the feasibility of using this derivative for locating the positions of double bonds in isomeric fatty acids. Five isomeric 20-carbon tetraenoic acids were analyzed in which the fourcis double bonds were systematically moved from the 4,7,10,13- to the 8,11,14,17-positions. It was possible to locate the positions of all four double bonds in the 7,10,13,16- and 8,11,14,17-isomers by appropriate ions differing by 12 atomic mass units. In a similar way the three terminal double bonds in the 4,7,10,13-, 5,8,11,14- and 6,9,12,15-isomers could be assigned. Odd-numbered ions atm/z 139, 153 and 167 which are accompanied by an even mass ion at 138, 152 and 166, respectively, are diagnostic for DMOX derivatives of acids with their first double bond, respectively, at positions 4, 5 and 6. It was thus possible to assign the location of all four double bonds in these three isomers. A comparison of the spectra of the DMOX derivatives of 17,17,18,18-d ₄ vs. 9,10,12,13-d ₄ linoleic acid suggests that double bonds preferentially migrate toward the polar end of the molecule prior to fragmentation. The merit of using DMOX derivatives to locate double-bond positions in mono- and dicarboxylic acids, produced during β-oxidation of polyunsaturated fatty acids, was evaluated. The spectra of 3-cis- and 4-cis-decenoic acids differ as do the spectra of 8-carbon dicarboxylic acids with their double bonds at positions 3 and 4.     

Cyclopropane fatty acid metabolism: Physical and chemical identification of propane ring metabolic products in the adipose tissue

Twelve male weanling rats were distributed equally into 3 groups and placed on fat-free diets. The diets of groups 1 and 2 were supplemented with 0.54% of recemic methylcis-9,10-methylene octadecanoate (CMO) and racemic methyltrans-9,10-methylene octadecanoate (TMO), respectively. Group 3 served as a control. Gas liquid chromatography (GLC) analyses of the adipose tissue methyl esters indicated at the level fed, that cyclopropane fatty acids do not affect normal fatty acid metabolism as has been shown for cyclopropene fatty acids. GLC analyses of groups 1 and 2 revealed the presence of a different unidentified fatty acid for each of the acids fed in addition to the CMO and TMO acids themselves. Each of the unidentified acids and the CMO and TMO acids were isolated and purified by preparative GLC. The absolute identity of the CMO and TMO acids fed and isolated from body fat was established by IR, NMR, and mass spectra. The biodegradation products of the CMO and TMO esters were shown to becis- andtrans-3,4-methylene dodecanoic acid, respectively. Unequivocal proof of structure was established through synthesis followed by comparison of IR, NMR, and mass spectra and melting points, GLC retention times, and elemental analyses with those obtained for the degradation products. Neither member of the racemic mixtures of either thecis or thetrans cyclopropane acids was preferentially utilized by the rat as shown by the lack of octical activity in the degradation products and the CMO and TMO acids isolated from the body fat. The accumulations of the 3,4-methylene dodecanoic acids in the adipose tissue of the rats fed CMO and TMO cyclopropane fatty acids suggest the inability of the beta oxidation enzyme system to proceed past the cyclopropane ring in a fatty acid chain. The synthesis ofcis- andtrans-3-dodecenoic acids, intermediates in the synthesis of the 3,4-methylene dodecanoic acids, and the geometrical cyclopropane isomers are discussed. This work to be submitted in partial fulfillment of the requirement for Ph.D.     

Studies on the preparation and analysis of glyceryl ether derivatives and the isolation and reductive ozonolysis of unsaturated glyceryl ethers

Glyceryl ethers were identified and quantified by the GLC analysis of their alkyl iodide (special conditions described), acetonide and allyl alkyl ether derivatives. The acetoxy-mercuri-methoxy derivatives of unsaturated glyceryl ether swere separated from saturated glyceryl ethers by TLC and the fractions were analyzed as alkyl iodides. Monoiodides from saturated glyceryl ethers and diiodides from unsaturated glyceryl ethers were also separated by TLC and then analyzed. Allyl alkyl ethers had small retention volumes and these derivatives were separated into pure fractions by preparative GLC. Ozonides were reduced either to alcohols with lithium aluminum hydrodie or to aldehydes with dimethyl sulfide. The alcohols were converted to mono- and diiodides which were separated by TLC and analyzed by GLC. The recovery of n-C₆ and n-C₇ monoiodides was not quantitative. Diiodides and aldehydes were both identified and quantified by GLC. Ozonolysis data suggest that the 16∶1 glyceryl ethers from dogfish liver oil contain 7,8 and 9,10 isomers and the 18∶1 glyceryl ethers contain 9,10 and 11,12 isomers. Presented in part at the AOCS Meeting, Philadelphia, October 1966.     

Analysis of C18:1 cis and trans fatty acid isomers by the combination of gas-liquid chromatography of 4,4-dimethyloxazoline derivatives and methyl esters

Trans fatty acids in foods are usually analyzed by gas-liquid chromatography (GLC) of fatty acid methyl esters (FAME). However, this method may produce erroneously low values because of insufficient separation between cis and trans isomers. Separation can be optimized by preceding silver-ion thin-layer chromatography (Ag-TLC), but this is laborious. We have developed an efficient method for the separation of 18-carbon trans fatty acid isomers by combining GLC of FAME with GLC of fatty acid 4,4-dimethyloxazoline (DMOX) derivatives. We validated this method against conventional GLC of FAME, with and without preceding Ag-TLC. Fatty acid isomers were identified by comparison with standards, based on retention times and mass spectrometry. Analysis of DMOX derivatives allowed the 13t, 14t, and 15t isomers to be separated from the cis isomers. The combination of the GLC analyses of FAME and DMOX derivatives gave results comparable with those obtained by GLC of FAME after preceding Ag-TLC, while saving about 100 h of manpower per 25 samples. It allowed the identification and quantitation of 11 trans and 8 cis isomers and resulted in 25% higher values for total C_18:1 trans, compared with the analysis of FAME alone. The combination of DMOX and FAME analyses, as applied to the analysis of 14 foods that contained ruminant fat and partially hydrogenated vegetable and fish oils, indicated that the most common isomers were 11t in ruminant fats, 9t in partially hydrogenated fish fats, and either 9t or 10t in partially hydrogenated vegetable fats. The combination of GLC analyses of FAME and DMOX derivatives of fatty acids improves the quantitation of 18-carbon fatty acid isomers and may replace the laborious and time-consuming Ag-TLC.     

Bleaching of vegetable oils. II. Conversions of methyl oleate and linoleate

Methyl oleate and linoleate were treated with 10% acid activated clay at 90–100 C for 1–50 hr with and without admission of air. Positional and geometric isomers of fatty acid esters were found. Polar compounds were detected having one or more functional groups with respect to the starting esters. Preparative thin layer chromatography and gas liquid chromatography were used in isolating the compounds, while IR, NMR, mass spectroscopy, and gas liquid chromatography analysis were employed for identification. The unsaturation of the isolated isomers was present at carbon atoms 5–11. The polar compounds were, among others, 9- and 10-keto octadecanoic methyl esters, isomeric keto octadecenoic methyl esters, isomeric epoxy octadecanoic methyl esters, 9- and 10-hydroxy octadecanoic methyl esters, and some mono methyl ester dicarbonic acids. It may be concluded that geometric, as well as positional, isomerization occurs and that small amounts of compounds with one or more functional groups are formed when unsaturated fatty acids were treated with acid-activated clay.     

The analysis ofCis-trans fatty acid isomers using gas-liquid chromatography

The geometric isomers of many unsaturated fatty acid methyl esters can be separated using high-resolution gas-liquid chromatography on polyester or Apiezon columns. Separations reported for the geometric isomers of monounsaturated, ricinoleic, linoleic, conjugated, and epoxy fatty acids are reviewed here. New data is presented on the resolution of linolenate geometric isomers on both polyester and Apiezon columns. The separation of methyl oleate and methyl elaidate on a polyester column has also been accomplished. Techniques for preparing and using the high-resolution columns necessary for these separations are reviewed. Presented at the AOCS meeting in New Orleans, La., 1962. Supported in part by a grant from the National Institutes of Health (A-6011).     

Retention behavior of trans isomers of eicosapentaenoic and docosahexaenoic acid methyl esters on a polyethylene glycol stationary phase

A polyethylene glycol (PEG) stationary phase was evaluated for the separation of mono‐trans isomers of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) methyl esters. The resolution patterns were compared to patterns achieved with previously applied conditions on a cyanopropyl phase. There were no overlaps between all‐cis EPA/DHA and their mono‐trans isomers on the PEG phase. Because of overlap between 22:0 and 22:1 isomers, the PEG column is not a good choice for analyses of EPA trans isomers in crude fish oils. However, if the saturated and monounsaturated fatty acids are not present in significant amounts, PEG can be a better choice than cyanopropyl columns.     

Hydrazine reduction in the gas liquid chromatographic analysis of the methyl esters of cyclopropenoic fatty acids

Hydrazine was used to hydrogenate the double bond of cyclopropenoic fatty acids (CPEFA) without cleaving the cyclic system. Using gas liquid chromatography (GLC), quantitation studies showed that it was possible to accurately determine CPEFA at concentrations as low as 0.1%. Studies with kapok seed oil methyl esters illustrated the potential application of such a technique to selectively hydrogenate the CPEFA to cyclopropanoic fatty acids (CPAFA), rendering them stable on the column, with a minimum effect on the degree of unsaturation of the remaining double bonds. Hydrazine reduction was found to be a simple and accurate method of measuring CPEFA in seed oils. Decomposition of pure methyl sterculate (methyl 9,10-methylene-9-octadecenoate) on GLC columns of various polarities also was examined.     

Analysis of conjugated octadecatrienoic acids inmomordica balsamina seed oil by GLC and13C NMR Spectroscopy

Separation of conjugated octadecatrienoic acids by open tubular gas liquid chromatography (GLC) was performed using glass capillary columns coated with Carbowax 20 M and with OV-1. The equivalent chain length of geometrical isomers of the conjugated octadecatrienoic acids belonging to the two series C_18:3Δ^8.10.12 and C_18:3Δ^9.11.13 were determined. The application of these results to the study of theMomordica balsamina seed oil shows that this oil contains two conjugated octadecatrienoic fatty acids in appreciable amounts, punicic acid (50%) and α-eleostearic acid (13%). The isomerization of conjugated acids inM. balsamina seed oil was followed for one year. Quantitation of octadecatrienoic acids using GLC gave results similar to those obtained with¹³C NMR.     

Effect of temperature on the separation of some Δ9-cis-trans isomers of methyl esters of fatty acids by open tubular gas chromatography

The influence of temperature on the gas chromatographic separation ofcis-trans isomers of the methyl esters of some monounsaturated fatty acids was studied on capillary columns coated with Apiezon L, BDS and DEGS. As far as methyl oleate and methyl elaidate are concerned, the separation is better at lower temperatures on Apiezon L (180–210 C) and at higher temperatures on polyester phases (BDS, DEGS; 150–180 C). The influence of temperature on the separation ofcis-trans isomers on the three stationary phases under study is explained by the higher values of δECL/δt forcis isomers. The variation of the equivalent carbon chain length with temperature can be used for the identification ofcis-trans isomers in natural mixtures.     

Identification and characterization of fully deuterated fatty acids isolated fromScenedesmus obliquus cultured in deuterium oxide

The algaScenedesmus obliquus cultured in deuterated water, synthesized fully deuterated saturated and unsaturated long chain fatty acids. Their methyl esters were purified and their equivalent chain lengths were determined by gas liquid chromatography (GLC). They were characterized by mass spectroscopy, IR and near-IR spectroscopy, and NMR spectroscopy. Hexadeca-3,6-dienoic acid was identified. The fatty acid compositions of the total lipid and of individual lipid classes were measured. The melting point of methyl perdeuteriohexadecanoate was lower than that of its hydrogen counterpart. Methyl esters of perdeuterio fatty acids had shorter retention times in GLC chromatography on polar and nonpolar phases. Presented before ISF-AOCS Congress, Chicago, September 1970.