Free fatty acids methods

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). 

Acylglycerols can be hydrolyzed by heating with acid or base or by treatment with lipases. Hydrolysis with alkali is called saponification and yields salts of free fatty acids and glycerol. This is how soap (a metal salt of an acid derived from fat) was made by our ancestors. One method used potassium hydroxide potash) leached from wood ashes to hydrolyze animal fat (mostly triacylglycerols). (The tendency of such soaps to be precipitated by Mg and Ca ions in hard water makes them less useful than modern detergents.) When the fatty acids esterified at the first and third carbons of glycerol are different, the sec-... 

The hydrolysis is performed as a continuous countercurrent reaction in tall reaction towers (height 15-20 m, diameter 0.7 m). The reaction time amounts to 60-90 min. Reaction products are as well obtained an aqueous glycerin solution (about 15%) as on a mixture of raw fatty acids [50]. The free fatty acids are carefully distilled with the aid of a thin film evaporator (2-10 mbar, 260°C maximum) [51]. Crystallization and transwetting are additional methods for fractionation of fatty acid mixtures. 

Only a very small proportion of the fatty acids are present in the free, unester-ified form and the vast majority are components of other lipids. Nevertheless it is important to be able to measure and identify the free fatty acids present in either form and for this they must be first extracted into an organic solvent and then usually converted to their methyl ester. The simplest method of methyla-tion, which is applicable to both esterified and non-esterified fatty acids, is to heat the lipid sample for 2 h under a current of nitrogen at 80-90°C with 4% sulphuric acid in methanol. After cooling and the addition of water, the resulting methyl esters are extracted several times into hexane and the combined extracts are dried over sodium carbonate and anhydrous sodium sulphate. The solvent fraction is then reduced in volume by a stream of nitrogen. 

Traditional column chromatography has also been employed for the extraction of carotenoids from palm oil. Separations were carried out on silica columns, carotenoids were eluted with n-hexane while the free fatty acids of the oil were removed from the stationary phase with ethyl acetate. The recovery of the method was 45 per cent and the purity of the cartotenoid fraction about 20 per cent w/w [23],... 

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. 

Harper, W. J., Schwartz, D. P. and El-Hagarawy, I. S. 1956B. A rapid silica gel method for measuring total free fatty Acids in milk. J. Dairy Sci. 39, 46-50. 

Koops, J. and Klomp, H. 1977. Rapid colorimetric determination of free fatty acids (lipol-ysis) in milk by the copper soap method. Neth. Milk Dairy J. 31, 56-74. 

Novak, M. 1965. Colorimetric ultramicro method for the determination of free fatty acids. J. Lipid Res. 6, 431-433. 

Shipe, W. F., Senyk, G. F. and Fountain, K. B. 1980B. Modified copper soap solvent extraction method for measuring free fatty acids in milk. J. Dairy Sci. 63, 193-198. 

Kwon, D.Y. and Rhee, J.S. 1986. A simple and rapid colorimetric method for determination of free fatty acids for lipase assay. J. Am. Oil Chem. Soc. 63 89-92. 

In this method, lipid samples are first dissolved in dry toluene, then treated with sodium methoxide, an alkaline catalyst. Fatty acids in complex lipids are then transesterified to form their corresponding methyl esters. This is a relatively mild reaction and does not produce isomerization of conjugated dienes. The limitation of this method is that, unlike the BF3 method (see Basic Protocol 1), free fatty acids are not methylated and the procedure requires more time and glassware. 

This is another alkaline-catalyzed transesterification technique. Similar to the sodium methoxide method (see Basic Protocol 2), only one step is required for the methylation reaction, followed by FAME extraction and isolation. As opposed to the sodium methoxide method, the investigator of the original publication for this method claimed that the tetramethylguanidine (TMG) works on both esterified and free fatty acids however, in the authors experience, and in published studies (see Background Information), TMG does not satisfactorily convert free fatty acids to corresponding methyl esters. 

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. 

American Oil Chemists Society. 1993a. AOCS Official Method Ca5a-40. Free Fatty Acid. Champaign, 111. 

Chromarod FID peaks of sterols, diglycerides, monoglycerides, and polar lipids are narrower and sharper than peaks of triglycerides and free fatty acids when analyzed using either method described in this unit (see Basic Protocol and Alternate Protocol). Hydrogenation of total lipids (see Support Protocol) results in much sharper and narrower peaks, which in turn substantially improves the resolution between lipid classes. The accuracy and precision in quantitating lipid classes of vegetable oils and animal fats are expected to be better than those from marine lipids. 

Low Molecular Weight Acids. The method devised for analyzing free fatty acids will resolve Ci to CB acids as shown in Figure 1, except for formic and propionic acids which are poorly resolved under the conditions used. Propionic acid, however, has been shown to be absent in all mixtures of oxidation products, and thus it presents no problem in this study. Acetophenone, shown in the chromatogram, was a convenient and reliable internal standard for this technique. Detection by thermal conductivity was selected because the flame ionization detector is insensitive to formic acid and, as noted, the high volatility of methyl formate and acetate precludes their quantitative determination by reasonably simple esterification procedures. 

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