Free fatty acid analysis

Greco, A. V., Mingrone, G., Gasbarrini, G. Clin. Chim. Acta 239, 1995, 13-22. Free fatty acid analysis in ascitic fluid improves diagnosis in malignant abdominal tumors. 

The samples were analysed by capillary gas chromatography GC 6000 (Carlo Erba) with FFAP-CB phase (Chrompack) that is special for free fatty acid analysis. The FID temperature was 235°C, the injector was an on-column system and the oven gradient was 40°C during 2 min, then increasing to 210°C at 45°C/min. The injected quantity was 2pl with no dilution. 

Composition of fatty acids. The saturated and unsaturated fatty acids with 8 to 24 carbon atoms in animal fats, vegetable oils, marine oils, and fatty acids are quantitatively determined by gas chromatography (GC) after conversion to their methyl ester forms. However, free fatty acid analysis is also possible by using specially selected stationary solid... 

Rozes, N., C. Gargia-Jares, F. Larue, and A. Lonvaud-Funel. 1992. Differentiation between fermenting and spoilage yeast in wine by total free fatty acid analysis. /. Sci. Food Agric. 59 351-357. 

In addition to sensory evaluation, free fatty acid analysis (the content is usually below 0.05%) and analysis of possible contaminants are carried out. The data given in Table 14.15 illustrate the amounts of pesticides and polycyclic aromatic compounds removed hy deodorization. However, this refining step also removes the highly desirable aroma substances which are characteristic of some cold-pressed oils such as olive oil. 

Aldehydes, enals, dienals, ketones, and hydrocarbons, which are responsible for disagreeable odors, generally bok at lower temperatures than fatty acids. Analysis showkig a free fatty acid concentration of less than 0.05% is an kidication that deodorization is sufficientiy complete. Some of the dienals have very low odor thresholds and sensory evaluation of the finished ok is a judicious quaHty assurance step. 

Oils are mixtures of mixed esters with different fatty acids distributed among the ester molecules. Generally, identification of specific esters is not attempted instead the oils are characterized by analysis of the fatty acid composition (8,9). The principal methods have been gas—Hquid and high performance Hquid chromatographic separation of the methyl esters of the fatty acids obtained by transesterification of the oils. Mass spectrometry and nmr are used to identify the individual esters. It has been reported that the free fatty acids obtained by hydrolysis can be separated with equal accuracy by high performance Hquid chromatography (10). A review of the identification and deterrnination of the various mixed triglycerides is available (11). 

The purity of the product was determined by the checkers by GLC analysis using the following column and conditions 3-nm by 1.8-m column, 5% free fatty acid phase (FFAP) on acid-washed chromosorb W (60-80 mesh) treated with dimethyldichlorosilane, 90 C (1 min) then 90 to 200 C (15°C per rain). The chromatogram showed a major peak for methyl 2-methyl-l-cyclohexene-l-carboxylate preceded by two minor peaks for methyl 1-cyclohexene-l-carboxylate and l-acetyl-2-methylcyclohexene. The areas of the two impurity peaks were 5-6% and 0.5-2% that of the major peak. The purity of the product seems to depend upon careful temperature control during the reaction. The total amount of the two impurities was 14-21% in runs conducted at about -15 to -20°C or at temperatures below -23°C. 

Poly(3HAMCL)s have also been produced from free fatty acid mixtures derived from industrial by-products which are potentially interesting low-cost renewable resources. Isolation and analysis of the polymer allowed the identification of 16 different saturated, mono-unsaturated and di-unsaturated monomers [46]. Except for the presence of diene-containing monomers and a large number of minor components, the composition of the fatty acid mixture derived PHA did not differ significantly from oleic acid derived PHAs. 

In addition to these numerous results, two other points are discussed by the authors fatty acid speciation and oil identification. These two aspects are developed in another publication written by the same authors [Keune et al. 2005]. The fatty acid speciation is based on the positive ion ToF-SIMS analysis and aims to prove if the fatty acids detected exist as free fatty acids, ester bound fatty acids or metal soaps. On account of the study of different standards, it is shown that when free fatty acids are present, the protonated molecular ion and its acylium ([M-OH]+) ion are detected. In cases of ester-bound fatty acid only the... 

Lipids can be identified and quantified using thin-layer chromatography (TEC) and gas chromatography (GC) (Galliard, 1968). Extraction of lipids is achieved by homogenizing potato tubers with isopropanol in a blender, followed by a series of filtrations and extractions with chloroform-methanol (2 1). Chloroform is removed by rotary evaporation and the residue is redissolved in benzene-ethanol (4 1). This extract is passed through a DEAE-cellulose column, and the fractions collected are subjected to TEC on 250 p,m layers of silica gel G, using three solvent systems. Fatty acid methyl esters for GC analysis are prepared by transmethylation of the parent lipids, or by diazomethane treatment of the free fatty acids released by acid... 

Klausner RD, Kleinfeld AM, Hoover RL, Kamovsky MJ Lipid domains in membranes evidence derived from structural perturbations induced by free fatty acids and lifetime heterogeneity analysis. J Biol Chem 1980 255 1286-1295. 

As always in the analysis of milk fat, the short chain fatty acids cause problems. A major difficulty has not been the GLC separation of these acids but their transfer from the esterification mixture to the GLC instrument without loss of the volatile esters. A widely used procedure is a slight modification of the method developed by Chris-topherson and Glass (1969) which uses sodium methoxide for transesterification. This technique can be employed with other fats, but not with those containing appreciable amounts of free fatty acids where HCl-methanol is required. 

B 7. Why are FAMEs rather than free fatty acids used for gas chromato graphic analysis ... 

One method that uses none of the above catalysis is worthy of mention, the diazomethane method. This method is very useful if there is only a very limited amount of free fatty acids as the starting material, since no extraction is needed before the resulting FAME can be injected into the GC for analysis. The authors of this unit have never performed this method. Interested readers can learn more from information presented elsewhere (Christie, 1989b). In addition, esters other than methyl may be required from time to time for specific purposes, and this is beyond the scope of the present discussion. 

This unit defines three different tests that are used to evaluate lipid systems. The first two, i.e., iodine value (IV see Basic Protocol I) and saponification value (SV see Basic Protocol 2), are used to determine the level of unsaturation and the relative size (chain length) of the fatty acids in the system, respectively. The free fatty acid (FFA) analysis (see Basic Protocol 3) is self-explanatory. Each of these analyses provides a specific set of information about the lipid system. The IV and SV provide relative information this means that the data obtained are compared to the same data from other, defined lipid systems. In mixed triacylglyceride systems there is no absolute IV that indicates the exact number of double bonds or SV that indicates the exact chain length. The data from the FFA analysis is an absolute value however, the meaning of the value is not absolute. As a quality indicator, ranges of FFA content are used and the amount that can be tolerated is product and/or process dependent. 

This analysis measures the amount of free acid, and is used to reflect the level of FFA in the sample. Free fatty acids are significant for the quality of the oil because they increase the oil s susceptibility to oxidation, can contribute bitter/soapy flavors, and can cause a decrease in the oil s smoke and flash points. 

The sample is now ready for analysis using the procedures described above (see Basic-Protocol and Alternate Protocol). Figure D1.6.6 shows chromatograms of cod muscle lipids before and after hydrogenation. Note the single peaks for steryl esters and free fatty acids in panel C compared to the double peaks in panels A and B. 

Free fatty acid (FFA) analysis definition of FFA, 471 lipid composition, 471 -472, 476-477 Free induction decay (FID), 22 (fig.), 23 Free radicals lipid oxidation, 535-536 lipids, 525... 

Free fatty acids are separable by GC by the inclusion of phosphoric acid in the packing so, for HPLC analysis, the phosphoric acid or other equivalent strong acid is included in the mobile phase. On a SUPELCOSIL LC 18 column, a model mixture of free fatty acids was separated with a mobile phase containing tetrahydrofuran, acetonitrile, water, and phosphoric acid (6 64 30 0.1) at pH 2 (Fig. 1) (15). Oleic and elaidic acids, palmitoleic and palmitelaidic acids, and linoleic and linoelaidic acids were well separated, but margarine fatty acids presented a difficult problem. Ultraviolet detection of 220 nm was used to prepare this chromatogram. 

With samples containing a wide range of components, such as hydrogenated fats, the column was eluted with solvent A for 13 min, then changed in one step to A-B (75 25), with a gradient to 100% B over 20 min. The free fatty acids were converted into the phenacyl derivatives and, prior to HPLC analysis, were purified by elution from a BOND ELUT NH2 column with hexane-diethyl ether (9 1). 

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