Fatty acids, determination

Procedures for determining fatty acids in sediments involved liquid-liquid extraction, liquid-solid adsorption chromatography followed by gas liquid chromatographic analysis [10-12], Liquid extractions have been performed with methanol-chloroform [13], methylene chloride [14] and benzene-methanol [15, 16]. Typical liquid-solid adsorbents are silicic acid. Standard gas chromatographic separations for complex mixtures employ non-polar columns packed with OV-1, OV-17, OV-101, SE-30, or glass capillary columns containing similar phases. 

In the second round of fatty acid synthesis, butyryl ACP condenses with malonyl ACP to form a C5-P-ketoacyl ACP. This reaction is like the one in the first round, in which acetyl ACP condenses with malonyl ACP to form a C4-P-ketoacyl ACP. Reduction, dehydration, and a second reduction convert the C5-P-ketoacyl ACP into a C5-acyl ACP, which is ready for a third round of elongation. The elongation cycles continue until Ci5-acyl ACP is formed. This intermediate is a good substrate for a thioesterase that hydrolyzes C 15-acyl ACP to yield palmitate and ACP. The thioesterase acts as a ruler to determine fatty acid chain length. The synthesis of longer-chain fatty acids is discussed in Section 22.6. 

Antonis (A3) has estimated phospholipids by a procedure for determining fatty acids. This technique requires a total serum extract and a phospholipid-free extract for the measurement of both total and free fatty acids, the difference between them being a measure of the phospholipid content. Free fatty acids (A4) are determined on a phospholipid-free extract by a procedure based on partitioning the fatty acids as copper soaps into chloroform, and subsequent photometric determination of the copper with diethyldithiocarbamate. Phospholipids, as well as the free fatty acids present in the total lipid-extract, are measured by the same method, since they also form a complex with copper that is soluble in chloroform. A criticism of this technique is that equal response is not given by dipalmitoyl lecithin, dipalmitoyl cephalin, or beef brain sphingomyelin. 

The Codex Alimentarius standard includes limits for acidity, volatile matter, insoluble impurities, peroxide value, colour, odour, taste, iron, copper, K27o, AK, permitted additives, contaminants (lead, arsenic, halogenated solvents), refractive index, saponification value, iodine value and unsaponifiable matter. Physical and chemical constants, such as iodine value and saponification value, are not found in the EU regulation. This is explained by the fact that more definite information is obtained by determining fatty acid composition, sterol and wax composition, trans fatty acid content, stigmastadiene, and so on. 

Gas-liquid (GLC) and high-performance liquid (HPLC) chromatography are extremely useful techniques and are fully described in a later chapter. They are primarily used for quantitative analysis, GLC for more volatile and HPLC for less volatile substances. GLC can determine, for example, hydrocarbons, alcohols and esters, and HPLC can determine fatty acids and high-molecular-weight materials. Both can determine chain-length distributions of both hydrocarbon and ethylene oxide chains. It is frequently necessary to prepare derivatives of the materials to be separated by GLC, but this is not usually the case for HPLC. Neither is very useful as an aid to identification of unknowns. 

However, direct titration of the liberated acidity is not really practicable, because of the high level of acid reagent. Also, the liberated hydrogen ion is neutralised by protonation of the alkanolamine, and titration to bromocresol green or methyl orange (ASTM method D 1570, ref. 6) would not measure it. On the other hand, titration to phenolphthalein (with alkali in alcoholic solution) would titrate not only that proton but the liberated fatty acid as well. It is therefore necessary to extract the fatty acid and to determine fatty acid and alkanolamine separately. 

Neubeller, J. and Buchloh, G. (1971) Tests to determine fatty acid patterns in the seeds of different plants, in particular fruit sorts. 2, Lehrstuhl fflr Obstbau der Universitat Hohenheim, Stuttgart-Hohenheim. Versuch. Mitt. 21, 469-483. 

GC-MS coupled with chemometric techniques has been used to characterize roasted coffees [54], to detect adulterants in olive oils [55], and to determine fatty acids in fish oils [56], GC-MS data have also been used in toxicology assessments to reveal patterns in complex chemical mixtures with the help of multivariate analyses [57,58],... 

Animal fats and vegetable oils are triacylglycerols, or triesters, formed from the reaction of glycerol (1,2, 3-propanetriol) with three long-chain fatty acids. One of the methods used to characterize a fat or an oil is a determination of its saponification number. When treated with boiling aqueous KOH, an ester is saponified into the parent alcohol and fatty acids (as carboxylate ions). The saponification number is the number of milligrams of KOH required to saponify 1.000 g of the fat or oil. In a typical analysis, a 2.085-g sample of butter is added to 25.00 ml of 0.5131 M KOH. After saponification is complete, the excess KOH is back titrated with 10.26 ml of0.5000 M HCl. What is the saponification number for this sample of butter ... 

Smoke, Flash, and Fire Points. These thermal properties may be determined under standard test conditions (57). The smoke poiat is defined as the temperature at which smoke begias to evolve continuously from the sample. Flash poiat is the temperature at which a flash is observed whea a test flame is appHed. The fire poiat is defiaed as the temperature at which the fire coatiaues to bum. These values are profouadly affected by minor coastitueats ia the oil, such as fatty acids, moao- and diglycerides, and residual solvents. These factors are of commercial importance where fats or oils are used at high temperatures such as ia lubricants or edible frying fats. 

Infrared spectra of fats and oils are similar regardless of their composition. The principal absorption seen is the carbonyl stretching peak which is virtually identical for all triglyceride oils. The most common appHcation of infrared spectroscopy is the determination of trans fatty acids occurring in a partially hydrogenated fat (58,59). Absorption at 965 - 975 cm is unique to the trans functionaHty. Near infrared spectroscopy has been utilized for simultaneous quantitation of fat, protein, and moisture in grain samples (60). The technique has also been reported to be useful for instmmental determination of iodine value (61). 

Proton chemical shift data from nuclear magnetic resonance has historically not been very informative because the methylene groups in the hydrocarbon chain are not easily differentiated. However, this can be turned to advantage if a polar group is present on the side chain causing the shift of adjacent hydrogens downfteld. High resolution C-nmr has been able to determine position and stereochemistry of double bonds in the fatty acid chain (62). Broad band nmr has also been shown useful for determination of soHd fat content. 

Unfortunately, excess consumption of fatty foods has been correlated with serious human disease conditions. Effects on cardiovascular disease (95), cancer (96), and function of the immune system (97) have been shown. Numerous studies have been conducted to determine the effects of saturated, monounsaturated, and polyunsaturated fatty acids on semm cholesterol and more recently high density Hpoprotein (HDL) and low density Hpoprotein... 

To determine the phosphoHpid and fatty acid compositions chromatographic methods (28) like gas chromatography (gc), thin-layer chromatography (tic), and high performance Hquid chromatography (hlpc) are used. Newer methods for quantitative deterrnination of different phosphoHpid classes include P-nmr (29). 

Phospholipids. Phospholipids, components of every cell membrane, are active determinants of membrane permeabiUty. They are sources of energy, components of certain enzyme systems, and involved in Hpid transport in plasma. Because of their polar nature, phosphoUpids can act as emulsifying agents (42). The stmcture of most phosphoUpids resembles that of triglycerides except that one fatty acid radical has been replaced by a radical derived from phosphoric acid and a nitrogen base, eg, choline or serine. 

These oxazolines have cationic surface-active properties and are emulsifying agents of the water-in-oil type. They ate acid acceptors and, in some cases, corrosion inhibitors (see Corrosion). Reaction to oxazoline also is useful as a tool for determination of double-bond location in fatty acids (2), or for use as a protective group in synthesis (3). The oxazolines from AEPD and TRIS AMINO contain hydroxyl groups that can be esterified easily, giving waxes (qv) with saturated acids and drying oils (qv) with unsaturated acids. 

Cocoa butter is composed mainly of glycerides of stearic, palmitic, and oleic fatty acids (see Eats AND FATTY oils). The triglyceride stmcture of cocoa butter has been determined (11,12) and is as foUows ... 

Properties are furthermore determined by the nature of the organic acid, the type of metal and its concentration, the presence of solvent and additives, and the method of manufacture. Higher melting points are characteristics of soaps made of high molecular-weight, straight-chain, saturated fatty acids. Branched-chain unsaturated fatty acids form soaps with lower melting points. Table 1 Hsts the properties of some soHd metal soaps. 

Potentiometry is another useful method for determining enzyme activity in cases where the reaction Hberates or consumes protons. This is the so-called pH-stat method. pH is kept constant by countertitration, and the amount of acid or base requited is measured. An example of the use of this method is the determination of Hpase activity. The enzyme hydroly2es triglycerides and the fatty acids formed are neutralized with NaOH. The rate of consumption of NaOH is a measure of the catalytic activity. 

Consumer acceptance of milk is strongly determined by its sensory characteristics. The development of off-flavor in milk as a result of lipolysis can reduce the quality of milk. The enzymatic release, by milk lipase, of free fatty acids (FFA) from triglycerides causes a flavor defect in milk described as rancid . Triglycerides in milk contain both long chain and short chain fatty acids, which are released at random by milk lipase. The short chains FFA, like butyric acid, are responsible for the off-flavor. 

The purity of the product was determined by the checkers by GLC analysis using the following column and conditions 3-nm by 1.8-m column, 5% free fatty acid phase (FFAP) on acid-washed chromosorb W (60-80 mesh) treated with dimethyldichlorosilane, 90 C (1 min) then 90 to 200 C (15°C per rain). The chromatogram showed a major peak for methyl 2-methyl-l-cyclohexene-l-carboxylate preceded by two minor peaks for methyl 1-cyclohexene-l-carboxylate and l-acetyl-2-methylcyclohexene. The areas of the two impurity peaks were 5-6% and 0.5-2% that of the major peak. The purity of the product seems to depend upon careful temperature control during the reaction. The total amount of the two impurities was 14-21% in runs conducted at about -15 to -20°C or at temperatures below -23°C. 

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