Two-column gas-liquid chromatography of fatty acid methyl esters

Fatty acid methyl esters were separated into fractions according to chain length on a nonpolar gas-liquid chromatographic column. These fractions were collected and rechromatographed on a polar column. Temperature programming was used in both cases. Data are given for the accuracy of the double procedure applied to a synthetic mixture.     

Preparation of fatty acid methyl esters for gas-liquid chromatography

A convenient method using commercial aqueous concentrated HCl (conc. HCl; 35%, w/w) as an acid catalyst was developed for preparation of fatty acid methyl esters (FAMEs) from sterol esters, triacylglycerols, phospholipids, and FFAs for gas-liquid chromatography (GC). An 8% (w/v) solution of HCl in methanol/water (85:15, v/v) was prepared by diluting 9.7 ml of conc. HCl with 41.5 ml of methanol. Toluene (0.2 ml), methanol (1.5 ml), and the 8% HCl solution (0.3 ml) were added sequentially to the lipid sample. The final HCl concentration was 1.2% (w/v). This solution (2 ml) was incubated at 45°C overnight or heated at 100°C for 1–1.5 h. The amount of FFA formed in the presence of water derived from conc. HCl was estimated to be <1.4%. The yields of FAMEs were >96% for the above lipid classes and were the same as or better than those obtained by saponification/methylation or by acid-catalyzed methanolysis/methylation using commercial anhydrous HCl/methanol. The method developed here could be successfully applied to fatty acid analysis of various lipid samples, including fish oils, vegetable oils, and blood lipids by GC.     

Liquid chromatography/mass spectrometry of fatty acids as their anthrylmethyl esters

The mass spectra of a series of saturated and unsaturated fatty acids have been recorded as their anthrylmethyl esters using a liquid chromatographic mass spectrometric interface. The spectra show an intense peak for the aromatic nucleus, and a molecular ion. The liquid chromatographic/mass spectrometric separation was performed on a reverse phase column using a solvent system of acetone + acetonitrile. While a complete separation of the fatty acids known to occur in man was not achieved, the recognition of all of these acids is possible using a scanning mode or by ion monitoring.     

Purification of fatty acid methyl esters by high-performance liquid chromatography

The determination by gas chromatography (GC) of fatty acid methyl esters (FAMEs) prepared from complex biological samples is subject to interference from cholesterol. During sample injection on the GC system of FAMEs prepared from tissues that contain cholesterol, we observed a major contaminant that co-eluted with docosahexaenoic acid (DHA, 22:6n-3). To address this problem, FAMEs were purified on an amino-phase high-performance liquid chromatography (HPLC) column using a hexane–isopropanol gradient. The HPLC retention times for both the FAME fraction and cholesterol were stable and reproducible when the amino column was used for sample purification. The purified extracts were analyzed by GC without artifacts or impurity peaks after 50 analytical runs. The method described here will be useful for measurement of 22:6n-3 and other fatty acids important for studies of nutrition or pathology.     

Characterization of Thiobacillus species by gas-liquid chromatography of cellular fatty acids

Summary Fatty acids of 18 strains representing 10 species of Thiobacillus were extracted from whole cells and examined as methyl esters by gas-liquid chromatography. Both visual and quantitative comparison of the resulting chromatograms for the presence and relative amounts of major peaks allowed rapid differentiation between such closely related species as Thiobacillus neapolitanus and T. thioparus and of eight other species. Except for a feature common to all thiobacilli tested, T. thiooxidans, T. neapolitanus and T. thioparus each possessed a characteristic fatty acid methyl ester profile that was exhibited by all the strains of that species. Hence, the thiobacilli could be divided into three distinct groups. It was possible to use the gas-liquid chromatographic patterns of the cellular fatty acids for rapid identification or grouping of these microorganisms since the fatty acid composition of the genus Thiobacillus thus appeared to be of taxonomic significance.Non-standard abbreviations GLC Gas-liquid chromatography - FAME Fatty acid methyl ester(s)     

Rapid microdetermination of fatty acids in biological materials by gas-liquid chromatography

Total and free fatty acids in general ranging from lauric to nervonic acid were separated and quantitated based on an internal standard method as methyl esters by “on column” methylation with trimethyl-(α,α,α-trifluoro-m-tolyl) ammonium hydroxide (TMTFTH) in a gas chromatographic system. This study represents an application of a method published by MacGee and Allen and a change to an internal standard technique. For the determination of the total fatty acids the sampls were saponified with KOH-CH₃OH, acidified with H₂PO₄, and then the fatty acids were extracted into hexane. An aliquot of the hexane extract was then extracted with TMTFTH and chromatographed. For determination of free fatty acids the sample was acidified with H₃PO₄, immediately extracted with hexane and processed as described earlier. The relative standard deviation of 1.4 to 4.2% illustrates the precision of the method and the recovery of the fatty acids ranged from 88.5 to 100.5%. This method was applied to the determination of fecal fatty acids in conjunction with an interdepartmental study on “High protein diet in colon cancer” at the University of Missouri. In addition, the applicability of the analytical procedure (with small modifications) was shown for a wide variety of biological materials (serum, milk, skin tissue, fungal spores, food homogenates, beef tissues, and tumor cell cultures). The analyses were performed on different gas chromatographs by different analysts.     

Separation of bile acids as their phenacyl esters by high-pressure liquid chromatography

Bile acids have been separated by high-pressure liquid chromatography. The free acids were derivatized to their phenacyl esters by treatment with triethylamine and α-bromoacetophenone. The stationary phase was a C₁₈, Partisil ODS column. A dual-solvent, stepwise gradient system was used for the mobile phase. The method is applied to a human bile sample and shows excellent resolution of the dihydroxy bile acid phenacyl esters. Detection limits for pure derivatized bile acids are 10–20 pmol (5–10 ng), except for the cholic acid derivative, which has a detection limit of 265 pmol.     

A reliable analysis of tissue free fatty acids by gas-liquid chromatography

A convenient and reliable gas-liquid chromatographic method for determining the free fatty acids in biological specimens is described. The free fatty acids were extracted with hexane in the presence of H3PO4 and then back-extracted from the hexane phase into a very small volume of trimethyl (alpha, alpha, alpha-trifluoro-m-tolyl)ammonium hydroxide solution. Direct injection of the resultant quaternary ammonium salts of the fatty acids into a gas-liquid chromatograph unit gave their methyl esters, with a high recovery. The presence of triglycerides, phospholipids, or cholesterol esters did not interfere with the determination of free fatty acids. This method was applied to determination of free fatty acids in the samples of serum or brain. The results were more precise and reliable than those reported with the conventional methods with TLC separation. This method should be a useful aid for providing precise information about the physiological or pathological roles of free fatty acids.     

Determination of enthalpy of formation of methyl and ethyl esters of fatty acids

Biofuels composed by fatty acid methyl esters are widely used as partly substituting fuels for diesel fossil fuels. Additionally, it is expected that the diesel biofuel norms will be extended to ethyl esters produced from bioethanol in the upcoming years. A precise knowledge of the standard enthalpy of formation is necessary for the calculation of some parameters useful for the analysis of the combustion process and emissions of a diesel engine operating with different fuels, such as the heating value, the adiabatic flame temperature or the kinetic mechanisms. However, experimental data for this property are scarce, and only available for short-chain, saturated methyl esters. In this work, four estimation methods for the calculation of the enthalpy of formation are examined and compared. Three of them are simple methods based on groups or bonds contribution, and another one is a computational method (with Gaussian 03 software). After presenting the implementation rules for each of them, conclusions are stated based on the results attained. Gaussian and Benson-Groups methods seem to be more accurate in predicting the actual values of the enthalpy of formation, both methods considering the separation between double bonds and the edge effects in the molecule. However, only the Gaussian method considers the effect of the position of the double bond in the molecule for all the unsaturated esters.     

Free fatty acids from the pasture grass Brachiaria humidicola and one of their methyl esters as inhibitors of nitrification

The tropical pasture grass, Brachiaria humidicola (Rendle) Schweick, produces nitrification inhibitory compounds (termed biological nitrification inhibitors or BNIs) in its shoot and root tissues and releases BNIs from its roots. In the present study, two BNI compounds were isolated and identified from the shoot tissue of B. humidicola using activity-guided fractionation. The recombinant Nitrosomonas europaea containing luxAB genes derived from the bioluminescent marine gram-negative bacterium Vibrio harveyi, were used to determine BNI activity. The BNI compounds in the shoot tissue were identified as linoleic acid (LA) and linolenic acid (LN) using authentic-chemicals obtained from ©Sigma (ED₈₀ 16.0 μg ml^?1 for both LA and LN) for verification. None of the other tested free fatty acids namely stearic acid, oleic acid, arachidonic acid, and cis-vaccenic acid showed any inhibitory effect on nitrification. Among the fatty acid methyl esters (FAME) evaluated [methyl oleate, methyl linoleate (LA-ME) and methyl linoleneate (LN-ME)], only LA-ME showed an inhibitory effect (ED₈₀ 8.0 μg ml^?1). The inhibitory effect of LA, LN and LA-ME in the soil was stable for 120 days at 20°C. Soil treated with LA, LN and LA-ME showed a very low accumulation of NO₃ ^? and the maintenance of soil inorganic N in the NH₄ ⁺ form. The inhibitory effect of LA-ME on soil nitrification was greater than that of LA or LN. In addition to BNI activity, both LA and LA-ME showed a suppressive effect on urea hydrolysis in soil. Both LA and LN blocked the AMO (ammonia monooxygenase) and HAO (hydroxylamino oxidoreductase) enzymatic pathways in Nitrosomonas. Since LA and LN can be produced from vegetable oils such as soybean, flax or sunflower, they have the potential for use as nitrification inhibitors in production agriculture.     

Automated glass capillary gas-liquid chromatography of fatty acid methyl esters with reference to cis and trans isomers.

The availability of an excellent separation method for fatty acid methyl esters, including separation of cis and trans isomers and of isomers that differ only in the position of double bonds, has become more and more important. The present glass capillary chromatography system combines high separation power with high precision and easy handling. Moreover, the system is completely automated and therefore provides a time saving method. As compared to a conventional packed column, the glass capillary column provides about one hundred fold more theoretical plates (227,000), as well as narrower peaks, thus giving rise to less error when integrating with electronic integrators. The reproducibility for relative retention time is better with the capillary column (0.26%) and reproducibility of the weight percent values is at least similar to that of the packed column (1.53%). When handling only small sample amounts the capillary provides better values because of its low capacity. This powerful system should open up new possibilities in the field of fatty acid investigation.     

Analysis of all protein amino acids as their tert.-butyldimethylsilyl derivatives by gas-liquid chromatography

A gas chromatographic method for the separation and quantitation of the 20 protein amino acids is described using N-methyl-N(tert.-butyldimethylsilyl)trifluoroacetamide, with 1% tert.-butyldimethylchlorosilane as catalyst, to prepare the tert.-butyldimethylsilyl amino acid derivatives. Alkylsilylation of amino acids proceeds at 140 degrees C in 20 min. The derivatives formed in the one-step reaction are used directly for gas-liquid chromatographic analysis, using a flame-ionization detector, without prior isolation or purification. Complete separation and quantitation of all protein amino acids are readily achieved using a 15-m DB-5 capillary column. Strict linearity extends from less than 15 to about 100 ng for all amino acids except Arg, which has a linear range from 50 to 300 ng. The limits of detection, however, range from one to several hundred nanograms. The method was used to analyze the free amino acid pool in carnation petals.     

Assessment of fatty acids in cardiac tissue as 9-anthryldiazomethane esters by high-performance liquid chromatography

A high-performance liquid chromatographic technique for the rapid assessment of fatty acids in cardiac tissue is described. A level of 50.4 ± 14.9 nmol fatty acids per g wet weight of rat myocardial tissue could be monitored. The content of the individual fatty acids C_14:0, C_16:0, C_16:1, C_18:0, C_18:1, C_18:2 and C_20:4 amounted to 1.9, 13.5, 0.6, 14.4, 6.1, 6.5 and 7.2 nmol/g wet weight, respectively. A comparison of this method with a well established gas chromatographic technique yielded good agreement. In contrast with time-consuming gas chromatographic techniques, there is no need to isolate (unesterified) fatty acids from the other lipid classes with column chromatography or thin-layer chromatography, because the derivatizing reagent 9-anthryldiazomethane reacts highly specifically with fatty acids.