Free fatty acids isolation procedure

Fatty acids, soaps and fatty acid methyl esters are the most extensively used oleochemicals [13]. The free fatty acids are prepared by hydrolysis of oils and fats by alkalis. This procedure named saponification proceeds at high temperature and pressure and leads to crude soaps the acidification of which gives fatty acids. They are subjected to various purification procedures, and finally isolated individually or as mixtures of carboxylic acids of the general formula RCOOH or as soaps RCOOM. The carboxylic acid methyl esters can be produced in two ways by esterification of the isolated carboxylic acid with methanol or by low-temperature... 

The non-saponifiable materials and the free fatty acids can be obtained by separating the total acidified extract by adsorption chromatography techniques (TLC or HPLC), as described in Chapter 3 and elsewhere [163,168], eliminating the step in which the alkaline solution is extracted. The free fatty acids are easily separated from the other products of hydrolysis, which can be individually isolated and identified. As an alternative, acidic and neutral materials can be separated by ion-exchange chromatography using the following procedure [1014],... 

The lower, chloroform-rich phase is separated carefully from the protein-containing interface, and then it is washed twice with methanol-water (10 9, v/v) and the washes are discarded. The chloroform layer contains the phosphatidic acid (as a sodium salt) and can be isolated by acetone precipitation. The yields can be of the order of 90-95%. One alternative route to identification of the chloroform-soluble material is to analyze it for total phosphorus and total fatty acid ester (see procedures described earlier). In the case of diacylphosphatidylcholine as the substrate, the fatty acid ester/P molar ratio should be 2.0. Another approach is to subject the chloroform-soluble fraction to preparative thin-layer chromatography on silica gel H (calcium ion free) in a two-dimensional system with a solvent system of chloroform-methanol-28% ammonium hydroxide (65 35 6, v/v) in the first direction and a solvent system of chloroform-acetone-methanol-glacial acetic acid-water (4.5 2 1 1.3 0.5, v/v) in the second direction. The phosphatidic acid will not migrate far in the basic solvent Rf 0.10) and will show an Rf value one-half of that of any remaining starting substrate (fyO.40) in the second solvent. Of course with a simple substrate system, one can use the basic solvent in one dimension only... 

In a separate study of Acinetobacter calcoaceticus it was indeed demonstrated that 6-hydroxy fatty acids from LPS almost exclusively occur in the -H - fraction when the mentioned isolation procedure is used (38). Using this approach we have studied several recent sediments. One study is discussed here. The fractions "-Free-", "-0H -" and were obtained from a 225,000 yr old Mediterranean... 

Alcohols exhibit a slightly younger age than ketones and sterols. However, hydroxy fatty acids present in sediments could retain free hydroxyl group after methylation into esters and could have eluted here along with alcohols. It should be noted that since hydroxyl acids were not isolated in our procedure, their contribution to carbon isotope data of the alcohol fraction can not be excluded. 

A typical procedure for the isolation of the free bile acids included precipitation of the bile protein with alcohol and hydrolysis of the conjugated acids for long periods of time in from 5 to 10 percent aqueous sodium hydroxide. Different methods were then applied for the isolation of each individual acid from the resulting crude mixture of free bile acids. Cholic and deoxycholic acid could be separated from each other by the addition of concentrated barium chloride to an ammoniacal solution of the hydrolyzed acids, since most of the barium deoxycholate is precipitated with the fatty acids, whereas barium cholate remains in solution (34). The greater solubility of chenodeoxycholic acid in ether was used by Wieland for its isolation lithocholic acid, the weakest of the acids, was obtained by fractional precipitation... 

Fatty alcohols may be separated and analysed by many of the procedures described in detail in Chapter 6 for fatty acid derivatives. For example, silver ion chromatography has been much used as an aid to the isolation and identification of specific components. Saturated and monoenoic fractions were isolated as the acetates from mullet roe by a column procedure [422], but TLC has been used more often hexane-diethyl ether (9 1, v/v) as the mobile phase in silver ion TLC will separate these fractions [344]. The effect of double bond position on the migration of all the isomeric c/s-octadecenols and the corresponding acetates on TLC with layers impregnated with silver nitrate was examined [327]. Like the methyl ester derivatives of the analogous fatty acids, the Rf values of isomeric acetates fall on a sinusoidal curve with a minimum around the 5- and 6-isomers, but the free alcohols hardly show this effect. Similarly, HPLC in the reversed-phase mode has been employed for the isolation of fatty alcohols, such as the insect sex hormones, under conditions... 

Yet another synthetic approach to PHAs is in vitro synthesis by an isolated PHA synthases.The advantages of this process include the ability to obtain polyesters in water at room temperature with a much easier purification procedure and free of biologically related impurities, which is of interest for biomedical applications. There is also a fundamental interest in this synthetic approach which allows mechanistic studies. Moreover, the possibility of synthesizing block copolymers is opened by this in vitro synthetic route. However, some recent studies have proven the possibility of also obtaining block copolymers in a living systems. In this synthesis procedure, for one-enzyme systems, the substrates are coenzyme A thioesters of hydroxy fatty acids, the prices of which greatly limits further development of this process. The transfer to the more readily available substrates requires the addition of other enzymes, adding further demands and complexity to this approach to PHA production. ... 

Methods of isolating intact sarcolemma free from admixture with other cell structures are necessary for precise study of its chemical and enzymic composition and for the investigation of possible changes in these in disease. A number of procedures have been devised to this end [97, 98], but it appears that there are difficulties still to be overcome. The sarcolemma may be expected to be rich in phospholipids which are important constituents of cell membranes. Changes have been reported in the fatty acid composition of phospholipids in muscle in human muscular dystrophy [99], in the autosomal dominant form of myotonia congenita [100], after denervation [101], and in muscular dystrophy in the mouse [102, 103]. Further work will be required to show whether the phospholipids of the sarcolemma are concerned in these abnormalities. The increased turnover of proteolipids [104] and increased biosynthesis of gangliosides [105] in denervated muscle may also represent changes in the characteristics of the sarcolemma. 

Alcohols may be released from the esterified form by any of the hydrolytic or transesterification procedures described in Chapter 4. If a pure wax ester fraction is hydrolysed, the alcohols are obtained simply by solvent extraction of the alkaline solution. On the other hand, when other lipids are present, it is advisable to isolate them as a class by adsorption chromatography. TLC on layers of silica gel G with the elution system described for simple lipid separations in Chapter 2, i.e. with hexane-diethyl ether-formic acid (80 20 2 by volume) as the mobile phase, is usually used. With such a system, any secondary alcohols migrate ahead of primary alcohols, which in turn are slightly less polar than cholesterol diols migrate just in front of monoacylglycerols. If cholesterol is present in an extract, it may be necessary to re-run the plate in the same direction to obtain additional resolution and ensure that primary alcohols and cholesterol are fully separated. Procedures of this kind were utilised to isolate trace levels of fatty alcohols from animal tissues, for example [108,662,904]. When wax esters are transesterified, the methyl esters and free alcohols can be separated on a mini-column of... 

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