Argentation chromatography, fatty acid

Argentation thin-layer chromatography is an extemely useful procedure for the separation of methyl esters of fatty acids. Saturated fatty acids have the highest Rf values, which decrease with the increasing degree of unsaturation, and for a particular acid, the trans isomer usually travels ahead of its corresponding cis isomer. The solvents most commonly used contain hexane and diethyl ether (9 1) although a mixture of 4 6 is used to separate compounds with more than two double bonds. In order to separate positional isomers of the same acid, conditions must be carefully controlled and multiple development in toluene at low temperatures is often necessary. 

As emphasized, dietary fatty acids produced drastic modification of the molecular species of brain ethanolamine phospholipids. Hargreaves and Clandinin reported similar findings in the rat (Connor et al., 1997). Using argentation thin-layer chromatography (TLC), which is unable to resolve individual molecular species, they fed fish-oil or linseed-oil diets to rats, which resulted in an increased microsomal and synaptic membrane content of phosphatidylethanolamine species containing six double bonds, and a decrease in species containing five double bonds, compared with animals fed soy or safflower oil. 

Silver ion TLC has also been used for separation of a variety of substituted unsaturated fatty acids such as epoxy, hydroxy and halohydroxy fatty acids, and these have been reviewed by Morris and Nichols (1972). Wax esters and steryl esters exhibit similar polarities and do not separate on column chromatography or normal silica TLC. Kiosseoglou and Boskou (1990) have separated the wax esters from steryl esters by using argentation TLC. They have used silica gel plates impregnated with 10% silver nitrate, with developing solvent hexane/chloroform (7 3 vol./vol.). 

Martinez-Lorenzo, M. J., Marzo, I., Naval, J. et al. (1994) Self-staining of polyunsaturated fatty acids in argentation thin-layer chromatography. Analytical Biochemistry, 220 (1), 210-12. 

Unsaturated fatty acids or their triacylglycerols, as well as unsaturated aldehydes obtained through autoxidation of lipids (cf. 3.7.2.1.5), can be separated by argentation chromatography . The separation is based on the number, position and configuration of the double bonds present. The separation mechanism involves interaction of the r-electrons of the double bond with Ag" ions, forming a reversible TT-complex of variable stability ... 

Since its introduction by Morris and others in 1962, TLC on silica gel impregnated with silver nitrate has been of enormous value to the lipid analyst. It is sometimes termed "argentation" chromatography. The basis of the separation is the facility with which the double bonds in the alkyl chains of fatty acids form polar complexes reversibly with silver compounds. Fatty acids can be separated according to both the number and the configuration of their double bonds and sometimes, with care, according to the position of the double bonds in the alkyl chain. HPLC has been slow to make a mark in this area, because of problems in preparing stable columns, but many of the major difficulties now appear to have been resolved. However, most of the data on the elution characteristics of silver complexes of unsaturated fatty acids has been obtained by TLC. 

Chloroplasts were incubated with [ " C]acetate to isolate MCTE activity from p-oxidation. Radiolabelled fatty acids were extracted and separated using successive argentation thin layer chromatography (TLC) and Cig-reverse phase TLC. The Brassica chloroplasts did not synthesise fatty acids well whereas control spinach chloroplasts incorporated [ " CJacetate into fatty acids at a rate of 100-300nmol/hour/mg chlorophyll, the Brassica chloroplasts had a specific activity of only approximately 20nmol/hour/mg chlorophyll. 

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