Free fatty acid separation

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). 

FIGURE 12.4 (A) Diagrammatic representation of the separation of major simple lipid classes on silica gel TLC — solvent system hexane diethylether formic acid (80 20 2) (CE = cholesteryl esters, WE = wax esters, HC = hydrocarbon, EEA = free fatty acids, TG = triacylglycerol, CHO = cholesterol, DG = diacylglycerol, PL = phospholipids and other complex lipids). (B) Diagrammatic representation of the separation of major phospholipids on silica gel TLC — solvent sytem chloroform methanol water (70 30 3) (PA = phosphatidic acid, PE = phosphatidylethanolamine, PS = phosphatidylserine, PC = phosphatidylcholine, SPM = sphingomyelin, LPC = Lysophosphatidylcholine). 

Although not very commonly used in the separation of nentral hpids, two-dimensional systems have been nsed to separate hydrocarbons, steryl esters, methyl esters, and mixed glycerides that move close to each other in one-dimensional systems. Complex neutral lipids of Biomphalaria glabrata have been first developed in hexane diethyl ether (80 20), dried, and the plates have been turned 90°, followed by the second development in hexane diethyl ether methanol (70 20 10) for complete separation of sterol and wax esters, triglycerides, free fatty acids, sterols, and monoglycerides [54]. 

Chloroform-methanol extracts of Borrelia burgdorferi were used for the identification of lipids and other related components that could help in the diagnosis of Lyme disease [58]. The provitamin D fraction of skin lipids of rats was purified by PTLC and further analyzed by UV, HPLC, GLC, and GC-MS. MS results indicated that this fraction contained a small amount of cholesterol, lathosterol, and two other unknown sterols in addition to 7-dehydrocholesterol [12]. Two fluorescent lipids extracted from bovine brain white matter were isolated by two-step PTLC using silica gel G plates [59]. PTLC has been used for the separation of sterols, free fatty acids, triacylglycerols, and sterol esters in lipids extracted from the pathogenic fungus Fusarium culmorum [60]. 

Using PTLC six major fractions of lipids (phospholipids, free sterols, free fatty acids, triacylglycerols, methyl esters, and sterol esters) were separated from the skin lipids of chicken to smdy the penetration responses of Schistosoma cercaria and Austrobilharzia variglandis [79a]. To determine the structure of nontoxic lipids in lipopolysaccharides of Salmonella typhimurium, monophosphoryl lipids were separated from these lipids using PTLC. The separated fractions were used in FAB-MS to determine [3-hydroxymyristic acid, lauric acid, and 3-hydroxymyristic acids [79b]. 

It is usual to convert fatty acids to their methyl ester derivatives before separation by GLC, although it may be possible to analyse those with short chain lengths (two to eight carbon atoms) as the free fatty acids. Polar or non-polar stationary phases can be used and capillary (open-tubular) or SCOT columns will separate positional and geometric isomers. The cis isomers have shorter retention times than the corresponding trans isomers on a non-polar phase and visa versa on a polar phase. 

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],... 

The stratum corneum consists of separated, nonviable, cornified, almost nonpermeable corneocytes embedded into a continuous lipid bilayer made of various classes of lipids, for example, ceramides, cholesterol, cholesterol esters, free fatty acids, and triglycerides [6], Structurally, this epidermis layer is best described by the so-called brick-and-mortar model [7], The stratum corneum is crucial for the barrier function of the skin, controlling percutaneous absorption of dermally applied substances and regulating fluid homeostasis. The thickness of the stratum corneum is usually 10-25 /an, with exceptions at the soles of the feet and the palms, and swells several-fold when hydrated. All components of the stratum corneum originate from the basal layer of the epidermis, the stratum germinativum. 

Here, we review two organic acid separations the first being citric acid from fermentation broth and the second separates saturated from unsaturated free fatty acids. 

The process involves reacting the degummed oil with an excess of methyl alcohol in the presence of an alkaline catalyst such as sodium or potassium methoxide, reaction products between sodium or potassium hydroxide and methyl alcohol. The reaction is carried out at approximately 150°F under pressure of 20 psi and continues until trans-esterification is complete. Glycerol, free fatty acids and unreacted methyl alcohol are separated from the methyl ester product. The methyl ester is purified by removal of residual methyl alcohol and any other low-boiling-point compounds before its use as biodiesel fuel. From 7.3 lb of soybean oil, 1 gallon of biodiesel fuel can be produced. See FIGURE 12-5. 

A gas chromatograph of the Hewlett-Packard 5890 series with a HP 7673A injector and a flame ionisation detector can be used. A capillary free fatty acid phase (Hewlett-Packard FFAP 19091F-105) column (50 m x 0.20 mm x 0.33 pm) is cut into two equal 25-m lengths, which are used for the separation, provided that a capillary pre-column (J W Scientific Altech 93493) with a 50% phenyl silicone DB 17 coating (1.35 m of a 15 mx0.25 mmx0.25 pm column) is installed. 

Paprika can be extracted to recover carotenoids, not only with CO2 but also with other gases. For example, by using ethane or ethylene, better results were obtained for the yield, extraction time, and quality of product. The solubilities of carotenoids are better in these gases, which is why the consumption of solvent and the extraction time were reduced. Practically water-free dye-concentrate was recovered by supercritical fluid ethane (under the conditions extraction 250 bar, 45°C separation 46 bar, 45 °C). The separation of pungent substances (capsaicinoids, free fatty acids) from the pigments can be carried out effectively in a continuous, counter-current extraction column with a large number of theoretical plates. 

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. 

Figure D1.6.2 TLC-FID separation of lipids recovered from the gastric contents of a hooded seal pup. The mobile phase was 91 6 3 1 (v/v/v/v) hexane/ethyl acetate/diethyl ether/formic acid. Time refers to scanning time of the Chromarod. Abbreviations DG, 1,2-diglyceride FFA, free fatty acid MG, monoglyceride IS, internal standard TG, triglyceride. Reproduced from Ackman and Heras (1997) with permission from AOCS Press.

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. 

The separation of safflower oil (SFO)-linseed oil (LSO) methyl esters is shown in Fig. 16. Free fatty acid methyl ester elution reproducibility, resolution, and baseline stability were maintained at sample sizes of 17-170 /zg, although capacity factors (k) decreased approximately 25% between the 17- and 170-/zg sample sizes. The trend of longer retention times with smaller sample sizes was consistent throughout their studies. Peak distortion, such as observed when gas chromatographic columns are overloaded, was not observed in their system. Perhaps larger FAME samples compete for silver ion sites the same way the ACN cosolvent competes for those sites. Excellent peak shapes were obtained, even with sample elution times of 1.5-2.0 h. 

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