Methods for the study of fatty acid composition

Methods for the study of fatty acid composition of lipids Prepared: Alibekova Alina

the separation of the major classes of lipids using thin layer chromatography as a system suitable solvent system hexane-diethyl etherformic acid (in the ratio 80: 2 by volume) Thin layer chromatography performed on glass plates coated with a thin layer of the slurry of adsorbent, usually silica gel. After drying of the suspension plate is calcined in a furnace a predetermined time at a predetermined temperature. Further chilled the "activated" plate is applied a mixture of lipids in a suitable solvent. After evaporation of solvent the edge of the plate nearest the applied spot immersed in a mixture of appropriate solvents, then the plate "show" in a closed chamber until the solvent reaches the opposite edge of the plate. Then the plate is dried to remove the solvent and determine the position of the spots or by "charring" (treatment with sulfuric acid followed by heating) or by fluorescence (after treatment with dichlorofluorescein), or by treatment with vapors of iodine

Method of low-temperature crystallization. Fractionation of fatty acids based on their different solubility, is used not only to separate saturated and unsaturated compounds, but in many cases further separation of the mixture of unsaturated fatty acids. In a series of normal saturated and unsaturated fatty acids with the same number of carbon atoms the solubility increases with increasing degree of unsaturation. When conducting a crystallization, you must take into account the mutual solubility of the acids and the possible formation of mixed crystals. The disadvantages of the method of low temperature crystallization are the long duration of the process because of the slow onset of equilibrium at low temperatures, the difficulty of complete separation of the uterine liquor and precipitate. In many cases, especially in the allocation of polyunsaturated acids, this process is used as a preliminary stage for cleaning and separation.

Separation of higher fatty acids using complexes with urea. One of the effective methods of separation and identification is to obtain crystalline inclusion complexes of organic compounds with urea or thiourea, commonly called adducted or molecular compounds. The inclusion complexes, in particular complexes with urea, are molecular compounds in which one component is contained in the lattice of the other component. The ability of fatty acid molecules to form complexes with urea were used to separate them from animal fats and vegetable oils. For best results, the length of the chain of shared connections shall not be less than 4 carbon atoms. Separation of acids by degree of unsaturation based on the fact that with increasing degree of unsaturation of compounds with long chain ability to complexation decreases and the solubility of the complexes increases.

Chromatographical methods Lately for separation of fatty acids using various types of chromatography: adsorption and distribution. Adsorption chromatography on flinty acid, silica gel or alumina is used as a method of isolation and purification of unsaturated acids, mainly in the form of esters. For the separation of acids using liquid-liquid distribution chromatography, and the best separation is achieved using reversed-phase systems. Common to all chromatographic distribution processes is the difficulty of separating "critical pairs". Partially this problem is solved by obtaining the derivatives of higher fatty acids, mainly by means of specific reactions of double bonds: bromation, the oxidation, the accession of the acetate of mercury, etc. .

One of the known methods of qualitative and quantitative analysis of higher fatty acids is reversed-phase partition chromatography on paper. Required for this method hydrophobization paper is dipping its silicone and paraffin oil, the solution by hydrophobic agents. To identify the acids on the chromatogram use their reactions at the carboxyl group (formation of colored salts with metals, the colour change of the indicators) and addition reactions and oxidation of double bonds. Quantitative analysis of acids is carried out by photoretrieval spots by using a densitometer or extraction of individual spots of different solvents and subsequent quantification of the acids in the eluates obtained by conventional methods (titration, etc. ). When the chromatography on the paper in the reversed-phase system, the possibility of the separation of saturated and unsaturated acids, oxyacids, as well as the fractionation of polyen acids with varying unsaturation and chain length. The method is applied to quantitative analysis of acids of vegetable oils and animal fats.

One of the most effective methods of analysis and separation of higher fatty acids is partition chromatography in a thin layer of silica gel or silicic acid. This is achieved by the separation of acids (esters) according to the chain length and degree of unsaturation

The most effective method of separating unsaturated acids using thin layer chromatography based on the use of adsorbents impregnated with silver nitrate. It is possible not only the separation according to the degree of unsaturation, but also the separation of positional and geometrical isomers. Using gas-liquid chromatography of methyl esters of fatty acids is carried out quantitative and qualitative analysis of complex mixtures of acids and their preparative separation. The advantages of this method are rapidity of analysis, clarity of division of components, the possibility of a large number of tests (100 and more) without regeneration phases, the high sensitivity (up to 1%), possibility of automatic control of the process, a sufficient accuracy of the results. Using this method achieved separation of fatty acids according to chain length and degree of unsaturation, it is also possible the separation of structural and geometric isomers. Accurate quantitative analysis of fatty acids has become available due to improvement of technology GLC and with the introduction of mass spectrometric identification of extracted components.

Currently, this method is the basic method of determining the fatty acid composition of individual lipid classes isolated from natural objects. In some cases, use a combination of methods thin-layer chromatography on silica impregnated with Ag. NOs, and gas-liquid chromatography. Using this method we studied the distribution of fatty acids in separate organs and tissues of the animal organism, in individual elements of the cell (mitochondria, microsome, etc. ) and other lipid systems. It is widely used to study the changes of fatty acid composition of lipids in various pathological conditions of the body.