Preparation of Methyl Esters


Although methyl esters are not the best derivatives for the mass spectrometric analysis of lipids, they are so simple in structure and in such wide-spread use for fatty acid analysis in general that they are inevitably much used for mass spectrometry. Without further derivatization, they are of little worth for structural analysis of mono- or dienoic fatty acids, but they can often be of value for polyunsaturated fatty acids, and those with functional groups other than double bonds, especially when spectra of authentic compounds are available for comparison purposes. Following a brief description of the methodology, experimental protocols for the two most important methods are given here.

More detailed discussion of the mechanisms of esterification and alternative methods are given in a substantial review article [1] that is now available on this website here..., or in my recent book [2]. Beginners to the subject may prefer some shorter articles on this website here... or the appropriate chapter in our online book 'Gas Chromatography and Lipids'. Other useful derivatization techniques, e.g. hydrogenation, deuteration, etc., are described in the section of these pages dealing with 'Mass spectrometry of methyl esters - further derivatization'.

Full practical details are given elsewhere on this site for preparation of the nitrogen-containing derivatives, i.e. picolinyl esters, 4,4-dimethyloxazoline (DMOX) derivatives and pyrrolidides, which are most useful for mass spectrometric analysis of fatty acids here...

There is no need to hydrolyse lipids to obtain the free fatty acids before preparing esters, as most lipids can be trans-esterified directly. No single reagent will suffice, however, and one must be chosen that best fits the circumstances. Esters prepared by any of the following methods can be purified if necessary by adsorption chromatography (see below). Care should be taken in the evaporation of solvents as appreciable amounts of esters up to C14 (or even C16) can be lost if this step is performed carelessly, for example by an over vigorous use of a nitrogen stream. The main procedures are acid- or base-catalysed esterification/trans-esterification. Diazomethane was once used frequently as a mild method for esterification of free acids, but concerns over the toxicity of reagents now limit its use. The methods described below are suitable for the common esterified lipids and their fatty acid constituents. Special methods are required for amide-bound fatty acids, and those with of short chain-length or with sensitive functional groups, for which the review and books cited here should be consulted.


Acid-Catalysed Esterification and Transesterification

Free fatty acids are esterified and O-acyl lipids trans-esterified by heating them with a large excess of anhydrous methanol in the presence of an acidic catalyst.

Acid-catalysed methylation

If water is present, it may inhibit the reaction. One popular reagent is methanolic hydrogen chloride (not methanolic hydrochloric acid), and it is most easily prepared by adding acetyl chloride (5 mL) slowly to cooled dry methanol (50 mL). Methyl acetate is formed as a by-product, but it does not interfere with methylations at this concentration. A solution of 1-2% (v/v) concentrated sulfuric acid in methanol trans-esterifies lipids in the same manner and at much the same rate; it is very easy to prepare whenever it is required (fresh reagent is best). It is usual to heat the lipid sample in the reagent under reflux for about two hours, but 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). Free acids are esterified much more rapidly (~1hr at 50°C). While boron trifluoride in methanol (12-14% w/v) has also been much used as a transesterification catalyst and for esterifying free fatty acids, I have considerable reservations about its use.


Laboratory protocol:
The lipid sample (up to 5 mg) is dissolved in toluene (1 mL) in a test tube fitted with a condenser, and 1% sulfuric acid in methanol (2 mL) is added, before the mixture is refluxed for 2 hours (or alternatively the mixture can be left overnight in a stoppered tube at 50°C). Water (5 mL) containing sodium chloride (5%) is added and the required esters are extracted with hexane (2 x 5 mL), using Pasteur pipettes to separate the layers. The hexane layer is washed with water (4 mL) containing potassium bicarbonate (2%) and dried over anhydrous sodium sulfate. The solution is filtered to remove the drying agent, and the solvent is removed under reduced pressure in a rotary film evaporator or in a stream of nitrogen.


No solvent other than methanol is necessary if free fatty acids alone are to be methylated, or if polar lipids such as phospholipids are to be trans-esterified. The same method is used to prepare dimethylacetals from aliphatic aldehydes or plasmalogens (simultaneously with methyl ester formation).


Base-Catalysed Trans-Esterification

O-Acyl lipids are trans-esterified very rapidly in anhydrous methanol in the presence of a basic catalyst. Free fatty acids are not normally esterified, however, and care must be taken to exclude water from the reaction medium to prevent their formation by hydrolysis of lipids.


0.5M Sodium methoxide in anhydrous methanol, prepared simply by dissolving fresh clean sodium in dry methanol, is the most popular reagent, but potassium methoxide or hydroxide have also been used as catalysts. The reagent is stable for some months at room temperature, if oxygen-free methanol is used in its preparation. The reaction is very rapid; phosphoglycerides, for example, are completely transesterified in a few minutes at room temperature, but cholesterol and wax esters take longer. It is commonly performed as follows:


Laboratory protocol:
The lipid sample (up to 10 mg) is dissolved in dry toluene (1 mL) in a test-tube, 0.5M sodium methoxide in anhydrous methanol (2 mL) is added, and the solution is maintained at 50°C for 10 min. Glacial acetic acid (0.1 mL) is then added, followed by water (5 mL). The required esters are extracted into hexane (2 x 5 mL), using a Pasteur pipette to separate the layers. The hexane layer is dried over anhydrous sodium sulfate and filtered, before the solvent is removed under reduced pressure on a rotary film evaporator.


As with acid-catalysed transesterification procedures, an additional solvent, such as toluene or tetrahydrofuran, is necessary to solubilize non-polar lipids like cholesterol esters or triacylglycerols. Aldehydes are not liberated from plasmalogens with basic reagents.


Clean-Up of Methyl Esters

It is sometimes necessary to purify methyl esters after transesterification has been carried out in order to eliminate troublesome impurities prior to analysis by gas chromatography. For example, cholesterol should be removed in this way from animal tissue preparations. 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 pre-conditioned with hexane. Methyl esters are eluted with hexane-diethyl ether (95:5, v/v; 10 mL), while cholesterol and other polar impurities remain on the column. Commercial solid-phase extraction columns can be used in the same way. Methyl esters can also be purified by preparative thin-layer chromatography, with hexane-diethyl ether (9:1, v/v) as the mobile phase.



  1. Christie, W.W. Preparation of ester derivatives of fatty acids for chromatographic analysis. In: Advances in Lipid Methodology - Two, pp. 69-111 (1993) (Ed. W.W. Christie, Oily Press, Dundee). 
  2. Christie, W.W. and Han, X. Lipid Analysis - Isolation, Separation, Identification and Lipidomic Analysis (4th edition), 446 pages (Oily Press, Bridgwater, U.K. and now Elsevier) (2010) - see Science Direct.

Updated May 5, 2014