Free fatty acids, HPLC analysis

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). 

Norlen, L. et al., A new HPLC-based method for the quantitative analysis of inner stratum corneum lipids in vivo with special reference to the free fatty acid fraction, Arch. Dermatol. Res., 290, 508, 1998. 

Garcia, H.S., Reyes, H.R., Malcata, F.X., Hill, C.G., Amundson, C.H. 1990. Determination of the major free fatty acids in milkfat using a three-component mobile phase for HPLC analysis. Milchwissenschaft 45, 757-759. 

The CB used was a standard factory product originating from the Ivory Coast. Its composition, determined by gas chromatography and HPLC for TAG analysis and by atomic absorption for phospholipid content, was as follows TAG 97% diacyl-glycerols 1.1% monoacylglycerols 0.2% free fatty acids 1.3% phosphatides 0.15% and others 0.25% (4). The TAG composition which is given in detail elsewhere (4) is for main TAG, POP 17.3%, POS 37.3%, and SOS 27.3% saturated TAG represented 2.3%. 

The preferred method for analysis of free fatty acids is GC, while TLC and HPLC can be used to isolate the fatty acid fraction. The alternative determination using HPLC needs fatty acid derivatives to be prepared using a chromophore to allow detection using the most sensitive fluorescent detector. Many examples of different derivatives can be found in the literature. 

Fig. 3.11. HPLC-analysis of soy raw lethicin (according to Sotirhos et al. 1986) 1 Triacylglyc-erols, 2 free fatty acids, 3 phosphatidyl glycerol, 4 cerebrosides, 5 phytosphingosine, 6 diphosphatidyl glycerol, 7 digalactosyldiacyl glycerol, 8 phosphatidyl ethanolamine, 9 phosphatidyl inositol, 10 lysophos-phatidyl ethanolamine, 11 phosphatidic acid, 12 phosphatidyl serine, 13 phosphatidyl choline, 14 lysophos-phatidyl choline...

The use of pre-column HPLC method for the analysis of fatty acid with 2-nitrophenylhydrazine hydrochloride was reported (Miwa Yamamoto 1990). After alkaline hydrolysis of coconut oil free fatty acids are reacted with 2-nitrophenylhydrazine hydrochloride and then derivatized to corresponding fatty acid hydrazides. Each of the derivatives was separated on reversed-phase HPLC with isocratic elution and detected at VIS 400 nm (Miwa Yamamoto 1996). In Table 2 is presented the typical fatty acid composition of coconut oil. 

FIG. 8 Normal phase HPLC analysis of a crude ester quat based on quatemized tallow triethanolamine esters. Peak identies 1, fatty acid methyl ester 2, free fatty acid 3, unquatemized triesteram-ine 4, tetraoctylammonium bromide internal standard 5, diesteramine 6, triesterquat 7, diesterquat 8, monoesteramine 9, monoesterquat. ELS detection. (Reprinted with the author s permission from Ref. 288.)... 

The sterols include that group of compounds which contain an alcoholic hydroxyl group at C3 and a branched aliphatic chain of at least eight carbon atoms at C17. These compounds, which include cholesterol and its derivatives, can occur as free alcohols or as long-chain fatty acid esters. A number of HPLC techniques have been used in the analysis of the sterols including reversed phase, non-aque-ous reversed phase, normal phase, argentation and combinations of the above. 

Fatty acids are easily determined by gas chromatography, either high temperature GC of the free acids or lower temperature analysis of ester derivatives. All vendors of GC col-unrns will provide application information. The alkyl chain length may also be determined by reversed-phase HPLC, as described in Chapter 7. 

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