Fatty acid methyl ester technique

Gas chromatography is one of the most powerful analytical techniques available. Its only major limitation is that it can not analyse involatile compounds such as fats. The solution in this case is to make a volatile derivative, e.g. the use of fatty acid methyl esters to analyse triglycerides. 

The most widely used method for quantifying FFAs is gas chromatography (GC), which has attained widespread favor due to its versatility, high sensitivity and relatively low cost. GC complexed with a flame ionization detector is used routinely to quantify FFAs, either directly or derivatized as fatty acid methyl esters (FAME). GC with mass spectroscopic detection has become the favored technique for quantification of volatile compounds derived from lipids (esters, lactones, ketones, alcohols and acids). 

GLC and HPLC are powerful techniques for analyzing lipids. GLC is particularly useful for analysis of fatty acid mixtures or fatty acid methyl esters derived from isolated lipid classes or specific... 

Preparative TLC is still very much in use as a preliminary separation technique when there is interference of peaks in mixtures to be analysed by GC or HPLC. An example of this is the characterisation of the double-bond configurations of fatty acid methyl esters derived from hydrogenated soybean oil and margarine which required preparative TLC using silver nitrate/silica gel coated plates to separate the mixture prior to GC analysis [10],... 

In this study, we have attempted to evaluate the efficacy of a technique for the production of the methyl ester of rapeseed oil via enzyme-catalyzed transesterifications using tert-butanol, a moderately polar organic solvent. We conducted experiments involving the alteration of several reaction conditions, including reaction temperature, methanol/oil molar ratio, enzyme amount, water content, and reaction time. The selected conditions for biodiesel production were as follows reaction temperature 40 °C, Novozym 435 5% (w/w), methanol/oil molar ratio 3 1, water content 1% (w/w), and 24h of reaction time. Under these reaction conditions, a conversion of approximately 76.1% was achieved. Further studies are currently underway to determine a method by which the cost of fatty acid methyl ester production might be lowered, via the development of enzyme-catalyzed methanolysis protocols involving a continuous bioprocess. 

Of the many analytical techniques now available to the lipid chemist, mass spectrometry (MS), is probably the one that has experienced the fastest growth in the last two decades. This is due both to the development of new techniques (gas and liquid chromatography combined with MS, soft-ionization MS, field desorption MS, atmospheric pressure MS etc.) and to the refinement of more traditional methods and their successful application to very complex problems, e.g. the elucidation of glycolipid structure, or the study of structures in lipid mixtures. Much progress has been made since the pioneering work of Ryhage and Stenhagen (1963) on fatty acid methyl esters. 

Analysis of the fatty acid profile of the extracted fat is now almost exclusively carried out using gas-liquid chromatography (GLC). Standard methodology for this technique is detailed by the International Union of Pure and Applied Chemistry (lUPAC). It involves the saponification of the extracted fat to break down glycerides, with the liberated fatty acids being esterified in the presence of methanol and boron trifluoride. Fatty acid methyl esters are then extracted with heptane and analyzed using GLC with flame ionization detection. 

Methylation of fatty acids is a well-known technique. A wide range of methylation and transesterification procedures are available in the literature. Defined lUPAC methods for the preparation and GC analysis of fatty acid methyl esters (EAMEs) have been published. The methylation with diazomethane, as modified by Schlenk and Gelleman is in common use. Under the proper conditions, methylation with diazomethane gives good results. 

In a comparative study of the reproducibility of results obtained by the two techniques for the analysis of blood lipids Mares et al. (1983) concluded that the variations obtained with the TLC-FID system were much higher than those obtained by GC. However, recent improvements in the latroscan detection system are likely to increase significantly the reproducibility for this type of analyses. Sebedio and Ackman (1981) analysed a synthetic mixture of fatty acid methyl esters on silver-nitrate-impregnated Chroma-rods and compared the results with those obtained from conventional GC. Comparable results were obtained by the two methods for all methyl esters, except for methyl linolenate, the quantity of which was substantially overestimated by the TLC-FID method. 

In Chapter 8 a leading expert in the detection of adulteration of oils and fats, Rossell, explains the economic advantages which tempt unscrupulous dealers to add cheap oils to high-value oils such as olive oil. He then describes a technique which he and his coworkers at Bristol have pioneered where stable carbon isotope ratio (SCIR) measurement has simplified the task of detecting adulteration. He compares sterol and fatty acid methyl ester analysis with SCIR and shows how clearly the latter detects maize oil. 

The object of the work [72] was to produce, for the authors own use, highly polar SCOT capillary columns capable of separating isomeric fatty acid methyl esters several solid supports and techniques were tested. 

Derivatives are often used in lipid chemistry to prepare fatty acid methyl esters needed to determine the fatty acid composition of lipids. Fried et al. (1992) published a technique on the transesterification of 500-mg samples of snail tissue. Because the technique is applicable to other biological tissues and fluids, it is presented herein. Lipids were extracted from snail bodies with 10 ml of chloroform-methanol (2 1) the extracts were filtered through a plug of glass wool contained in a Pasteur pipet, and nonlipid contaminants were removed by extraction with 8 ml of Folch wash (0.88% aqueous KCl). The lipid-containing lower phase was separated and evaporated just to dryness under a stream of nitrogen at room temperature. The total lipid sample was dissolved in about 30 ml of methanol, and 0.5-1.0 ml of concentrated sulfuric acid was added. The mixture was refluxed for 1 h the formed fatty acid methyl esters were extracted with 30-40 ml of petroleum ether and the extract dried over anhydrous sodium sulfate. The fatty acid methyl esters were concentrated on a Rotoevaporator at 40°C and the volume reduced to 1 ml. The fatty acid methyl esters can be separated by TLC on silica gel impregnated with silver nitrate (Christie, 1982). 

In most respects, the standard approach taken to analyze the fatty acids of functional foods is similar to that for conventional foods. The steps are to extract the total lipids or fatty acids, convert the fatty acids to a suitable derivative (often to fatty acid methyl esters (FAME)), and analyze the derivatized fatty acids by a suitable chromatographic technique, usually GC with flame ionization detection (FID), Other chromatographic techniques, including gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC), may be required. Nonchromatographic techniques such as infrared (IR) spectroscopy may be used in some simations, perhaps because of the speed of analysis. 

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