Methylation of fatty acids a beginner's guide
Abstract: Preparation of methyl esters of fatty acids for analysis by chromatographic means is one of the most important procedures in the analysis of lipids. Here the principles of the more important methods are described together with a brief summary of the problems that may be encountered.
The preparation of the methyl ester derivatives of fatty acids for analysis by gas chromatography and other means must be by far the commonest chemical reaction performed by lipid analysts. Although it should be a relatively simple operation, it is often poorly understood and reaction conditions that are unnecessarily vigorous are often applied. I have reviewed the topic on a number of previous occasions, and the last of these  is now available online here.. Detailed protocols are not given in this article, but they are available with further discussion in my online book 'Gas Chromatography and Lipids' and and in our mass spectrometry pages here... My book (coauthored with Xianlin Han) ‘Lipid Analysis. 4th Edition’ (Oily Press, 2010) is of course more up-to-date.
It is a fallacy that it is necessary to hydrolyse lipids to obtain the free fatty acids before preparing the esters, although some standardization bodies recommend this, and most lipids are best transesterified directly. I am also dubious about recommending any single general-purpose transesterification reagent. Many good methods are available and some are better than others for specific tasks. Better results may be obtained if one is chosen that is appropriate to the circumstances. It is also necessary to consider the scale of the preparation; methods suitable for gram amounts of fats and oils in quality control applications may be less suitable for microgram quantities, for example those obtained from lipid classes isolated by thin-layer chromatography.
Free fatty acids can be esterified and O-acyl lipids, such as triacylglycerols, can be transesterified by heating them with a large excess of anhydrous methanol and an acidic reagent as catalyst.
If any water is present, it inhibits the reaction and may prevent it going to completion. As the scale of the preparation diminishes, this aspect of the problem becomes more relevant. The commonest and mildest reagent is 5% (w/v) anhydrous hydrogen chloride in methanol, and it is most often prepared by bubbling hydrogen chloride gas (which is commercially available in cylinders or can be prepared by dropping concentrated sulphuric acid slowly onto fused ammonium chloride or into concentrated hydrochloric acid) into dry methanol. This method is best suited to bulk preparation of the reagent. A simpler and safer procedure, more suited to small-scale preparations, is to add acetyl chloride (5 mL) slowly to cooled dry methanol (50 mL). Some methyl acetate is formed as a by-product, but it does not interfere with methylations at the concentration here.
In common practice, the lipid sample is dissolved in the reagent and the mixture is heated under reflux for about 2 hours, but it may also be heated in a sealed tube at higher temperatures for a shorter period. Alternatively, equally effective esterification is obtained if the reaction mixture is heated in a stoppered tube at 50°C overnight (also incidentally reducing the glassware requirements). This is the approach that I prefer when time is not an important factor. Free acids are esterified in much less time than is required for transesterification.
As an alternative to methanolic hydrogen chloride, a solution of 1-2% (v/v) concentrated sulphuric acid in methanol transesterifies lipids in the same manner and at much the same rate. It is very easy to prepare, and it is then my preferred reagent for esterification of free fatty acids. Again, it is probably advisable to carry out transesterification reactions overnight at 50°C, and not to reflux the mixture, since some decomposition of polyunsaturated fatty acids may occur if the solvent is inadvertently allowed to evaporate, for example.
One of the most popular of all transesterification catalysts is boron trifluoride in methanol (12-14% w/v), and in particular it is often utilised as a rapid means of esterifying free fatty acids. It is even recommended in certain official methods. On the other hand, the reagent has a limited shelf life, even when refrigerated, and the use of old or too concentrated solutions often results in the production of artefacts and the loss of appreciable amounts of polyunsaturated fatty acids. Again, it is approved by some standardization bodies, but I do not recommend it.
In my opinion then, the best acidic general purpose esterifying agents are methanolic hydrogen chloride (5%) or sulphuric acid (1%). They methylate free fatty acids very rapidly and can be employed to transesterify other O-acyl lipids efficiently. The reaction can be carried out on the microgram to 50-gram scale with very little modification to the methodology. Similar methods are used to prepare dimethyl acetals from aldehydes or plasmalogens. N-Acyl lipids are transesterified very slowly with these reagents, and in contrast to the situation with O-acyl lipids a small amount of water is usually required to assist the reaction (see below). Acid-catalysed procedures are better avoided for short-chain acids bound to triglycerides, as in milk fat.
Base-catalysed procedures are even simpler and are much more rapid than those that are acid-catalysed in many circumstances. O-Acyl lipids such as triglycerides and phospholipids are transesterified very rapidly in anhydrous methanol in the presence of a basic catalyst, usually sodium methoxide, which facilitates the exchange between glycerol and methanol, and the methyl esters required are soon obtained in quantitative yield.
On the other hand, if we replace methanol with water in the equation, the product is a free fatty acid, i.e. hydrolysis occurs. Free fatty acids are not esterified under such conditions, so care must be taken to exclude water from the reaction medium to prevent the occurrence of this unwanted and irreversible side reaction.
0.5 M Sodium methoxide in anhydrous methanol, prepared simply by dissolving fresh clean sodium in dry methanol, is the most popular reagent. It is stable for some months at refrigeration temperature, especially if oxygen-free methanol is used in its preparation, but it will deteriorate slowly as carbon dioxide is absorbed from the air and sodium carbonate is precipitated. I use it myself more than any other reagent, and I find it equally useful on a gram scale or with a few micrograms of a purified lipid. Potassium methoxide is an even better catalyst, but metallic potassium reacts very vigorously with methanol and great care must be taken in preparing the reagent. In addition, potassium hydroxide can be used as a catalyst, but it cannot be made water-free so is best reserved for large-scale preparations.
The reaction between sodium methoxide in methanol and a glycerolipid is very rapid; both triacylglycerols and phosphoglycerides, for example, are completely transesterified in a few minutes at room temperature. A stoppered tube is the only glassware needed. Cholesterol and wax esters only are transesterified very slowly and may require reaction times as long as an hour. The methodology can be used on quite a large scale if need be. For example, 50 g of lipid is transesterified in toluene (50 mL) and methanol (100 mL) containing fresh sodium (0.5 g) in 10 minutes at reflux, and a related procedure has been used to transesterify litre quantities of oils . Conversely, the reaction can be scaled down to 5 to 10 micrograms of lipid very easily.
Under these conditions, no isomerisation of double bonds in polyunsaturated fatty acids occurs, though it would be unwise to assume that alterations to fatty acids can never happen. With excessively prolonged or careless use of basic reagents, some degradation might occur. Aldehydes are not liberated from the plasmalogen forms of phospholipids with basic reagents, in contrast to when acidic conditions are employed. This can be of advantage in simplifying the interpretation of GC traces.
The Requirement for an Additional Solvent
Nonpolar lipids, such as cholesterol esters or triglycerides are not soluble in reagents composed predominantly of methanol, and will not be transesterified in a reasonable time, unless a further solvent is added to effect solution. This is true of all approaches to methylation. Benzene was once employed regularly to this end, but because of its great toxicity, it is advisable to use some other solvent such as toluene or tetrahydrofuran. Chloroform is also better avoided as it can contain ethanol as a stabiliser, and this may result in the formation of a proportion of ethyl esters, which will complicate GC chromatograms. Also, dichlorocarbene, which can react with double bonds, may be generated by the reaction of chloroform with sodium methoxide.
No solvent other than methanol is necessary if free fatty acids alone are to be methylated (also a reaction time of only 20 minutes at reflux, or 2 hours at 50°C, is required), or if polar lipids such as phospholipids are to be transesterified.
Lipids Other Than Glycerolipids
Amide-bound fatty acids, as in sphingolipids, are not affected by alkaline transesterification reagents under such mild conditions, and this fact is sometimes used in the purification of lipids of this kind. Methanol containing aqueous acid is usually recommended for preparing methyl esters from sphingolipids .
Although free fatty acids are not esterified with alkali as described above, methyl esters can be prepared under mild basic conditions in some circumstances. For example, free acids react with N,N-dimethylformamide dimethyl acetal in the presence of pyridine to form methyl esters . Similarly, the sodium or potassium salts of fatty acids react with methyl iodide in the presence of a polar aprotic solvent such as dimethylacetamide to form methyl esters . It is no longer possible to even consider toxic reagents such as diazomethane, as the starting materials are no longer available from commercial sources. Trimethylsilyldiazomethane is occasionally recommended as an alternative, but a number of analysts have reported artefact formation (especially trimethylsilyl ester formation) with this reagent.
Impurities, Artefacts and Other Problems
With lipid samples from animal tissues, it is sometimes necessary to purify methyl esters after transesterification has been carried out in order, for example, to eliminate cholesterol, which can be troublesome when the esters are subjected to gas chromatography. Other impurities can also interfere from time to time. This can be accomplished by adsorption chromatography with a short column (approx. 2 cm) of silica gel or Florisil™ in a Pasteur pipette plugged with glass wool, and eluted with hexane-diethyl ether (95:5, v/v; 10 mL). The cholesterol and other polar impurities remain on the column. Commercial pre-packed columns (Bond Elut™ or Sep-Pak™) can be used in a similar way, and they can be re-utilised repeatedly if dry diethyl ether is employed for elution. Methyl esters can also be purified by preparative thin-layer chromatography, with hexane-diethyl ether (9:1, v/v) as the mobile phase.
Note that if acidic reagents are permitted to superheat in air through carelessness, some artefact formation is possible. Care should be taken in the evaporation of solvents as appreciable amounts of esters up to C14 can be lost if this step is performed carelessly. In particular, an over-vigorous use of nitrogen to blow off solvents must be avoided. Esters other than methyl may be required from time to time for specific purposes, and the methodology just described is easily adapted by substituting the appropriate alcohol. In summary then, acid-catalysed transesterification methods can be excellent means of preparing methyl ester derivatives of fatty acids, especially when free acids are present in the sample.
It has often puzzled me why analysts still prefer to use the two-step procedure of hydrolysing lipids and then esterifying them with an acidic reagent, as in at least one of the internationally approved methods, when direct transesterification with a basic catalyst is so simple and rapid. I am not the only one to say this . I suppose that such methodology may have some advantages if high proportions of free acids are present in samples, but then direct transesterification with an acidic catalyst is simpler.
- Christie, W.W. Preparation of ester derivatives of fatty acids for chromatographic analysis. In: Advances in Lipid Methodology - Two, pp. 69-111 (edited by W.W. Christie, Oily Press, Dundee)(1993). Available here...
- Nadenicek, J.D. and Privett, O.S. Preparation of pure polyunsaturated fatty acids 1. Linolenic acid. Chem. Phys. Lipids, 2, 409-414 (1968).
- Gaver, R.C. and Sweeley, C.C. Methods for methanolysis of sphingolipids and direct determination of long-chain bases by gas chromatography. J. Am. Oil Chem. Soc., 42, 294-298 (1965).
- Thenot, J.-P., Horning, E.C., Stafford, M. and Horning, M.G. Fatty acid esterification with N,N-dimethylformamide dialkyl acetals for gas chromatography analysis. Anal. Lett., 5, 217-223 (1972).
- Ciucanu, I. and Kerek, F. Rapid and simultaneous methylation of fatty acids and hydroxy fatty acids for gas chromatography analysis. J. Chromatogr. A, 284, 179-185 (1984).
- Ackman, R.G. Remarks on official methods employing boron trifluoride in the preparation of methyl esters of the fatty acids of fish oils>. J. Am. Oil Chem. Soc., 75, 541-545 (1998).
This article is based on two previous publications (Lipid Technology, 2, 48-49 (1990) and Lipid Technology, 2, 79-80 (1990)) (by kind permission of P.J. Barnes & Associates (The Oily Press Ltd), who retain the copyright to the original articles). When amalgamating the two, they were substantially updated.
Updated: July 20, 2011