The Analysis of Fatty Acids
The Preparation of Derivatives of Fatty Acids
Before the fatty acid components of lipids can be analysed by GC, it is necessary to convert them to low molecular weight nonpolar derivatives, such as methyl esters. In addition, it may be advisable to mask other polar functional groups in a similar manner, or to prepare specific derivatives as an aid to identification. Peak shape and resolution are greatly improved at the same time. It is only possible to identify fatty acids tentatively by GC retention times alone, but GC used in combination with derivatization and chemical degradative or spectroscopic procedures, especially mass spectrometry, can be an extremely powerful means of characterisation. Of course, such derivatives are also of value in isolating specific fatty acids by other chromatographic methods. Therefore, before any chromatographic procedure is undertaken, it is necessary to consider what derivatives should be prepared and what method should be employed for the purpose.
Because of the high sensitivity of chromatographic analysis procedures, small amounts of material (usually less than 1 mg, and certainly less than 10 mg) may be all that is required, and most of the procedures described below are on this scale. Any conventional Pyrex glassware with ground-glass joints can be used for the reactions, but for many the author has found it convenient to use test tubes of about 15 mL capacity with a standard ground-glass joint and stopper. Condensers and other equipment can be connected to these when required. Organic and aqueous layers can be separated efficiently in such tubes with the aid of Pasteur pipettes, perhaps after brief centrifugation to ensure a clean separation of the layers. Usually it is necessary to dry the solvents prior to evaporation by allowing them to stand over anhydrous sodium sulfate, but if hexane-containing layers are separated with care, this step can frequently be omitted. If an internal standard is to be added, it should be introduced into the sample at the earliest possible stage. Precautions should be taken at all times to prevent autoxidation of lipids (see Chapter 2). Methods of preparing derivatives in general have been reviewed [111,230].
B. Hydrolysis (Saponification) of Lipids
Lipids can be hydrolysed by heating them under reflux with an excess of dilute aqueous ethanolic alkali and the fatty acids, diethyl ether-soluble nonsaponifiable materials and any water-soluble hydrolysis products recovered for further analysis. When the water-soluble components (such as glycerol, glycerophosphorylcholine, etc.) are required, special procedures must be used and most of these are outside the scope of this book. The free fatty acids and the nonpolar nonsaponifiable components are separately recovered in the following procedure:
The lipid sample (10 mg) is refluxed with a 1 M solution of potassium hydroxide in 95% ethanol (2 mL) for 1 hour; alternatively, reaction at room temperature overnight is equally effective. The solution is cooled, water (5 mL) is added and the mixture is extracted thoroughly with hexane-diethyl ether (1:1, v/v; 3 × 5 mL). It may be necessary to centrifuge to break any emulsions that form. The solvent extract is washed with water, dried over anhydrous sodium sulfate and the nonsaponifiable materials are recovered on removal of the solvent in a rotary evaporator. The water washings are added to the aqueous layer, which is acidified with 6 M hydrochloric acid and extracted with diethyl ether-hexane (1:1, v/v; 3 × 5 mL). The free fatty acids are recovered after washing the extract with water, drying them over anhydrous sodium sulfate and removing the solvent by evaporation.
The nonsaponifiable layer will contain any hydrocarbons, long-chain alcohols and sterols originally present in the lipid sample in the free or esterified form. If the sample contained any glycerol ethers or plasmalogens, the deacylated residues will also be in this layer. With single lipid classes, isolated by chromatographic means, the preliminary extraction of the alkaline medium may not be required, and this step can be omitted from the procedure.
When short-chain fatty acids (C12 or less) are present in lipids, it is necessary to extract the acidified solution much more exhaustively, and even then it may be almost impossible to recover the fatty acids of shortest chain length, such as butyric, quantitatively. Epoxyl groups and cyclopropene rings in fatty acids are normally disrupted by acid, but with care they will survive the above procedure if the exposure to the acidic conditions is short.
Cholesterol esters are hydrolysed very slowly by most reagents, and they may not react completely under the conditions above. Therefore, when they are major components of the lipid sample, longer reflux times are necessary.
Similarly, N-acyl derivatives of long-chain bases are saponified only slowly by alkali, but hydrolysis is virtually complete when sphingomyelin, for example, is refluxed for 10 hours in 1M KOH in methanol-water (9:1, v/v) . An alternative procedure has been described in which acidic hydrolysis conditions are employed, i.e. the sphingolipids are hydrolysed with 0.5 M hydrochloric acid in acetonitrile-water (9:1, v/v) for 45 minutes at 100°C or for 4 hours at 70°C . Long-chain bases can also be isolated by procedures of this kind, although some precautions are necessary to prevent any degradation occurring (see Chapter 10).
The nonsaponifiable 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 .
DEAE-Sephadex™ (1 g; type A-25; capacity 3.2 m-equiv./g; Pharmacia, Sweden) on a Büchner funnel is washed successively with small amounts of 1 M hydrochloric acid, water, 1 M potassium hydroxide and water again (the procedure is repeated three times), the last wash until neutral. It is then washed twice with methanol (25 mL), and with 25 mL of diethyl ether-methanol-water (89:10:1 by volume). It is slurried in the latter solvent mixture, left to equilibrate overnight and packed into a small column. The sample, containing up to 100 mg of fatty acids, is washed through with 25 mL of the solvent mixture; the neutral materials are eluted while the acids remain on the column. The latter can be recovered with a mobile phase of diethyl ether-methanol (9:1, v/v) saturated with carbon dioxide."
Polyunsaturated fatty acids are not altered by the mild hydrolysis conditions described above. On the other hand, if the reaction time is prolonged unduly or if too strong an alkaline solution is used, some isomerization of double bonds can occur.
C. The Preparation of Methyl and Other Esters of Fatty Acids
The preparation of the methyl ester derivatives of fatty acids must be by far the commonest chemical reaction performed by lipid analysts, yet it is often poorly understood; the topic has been comprehensively reviewed [160,205,839]. (Further information is available on this web site here..). There is no need to hydrolyse lipids to obtain the free fatty acids before preparing the esters as most lipids can be transesterified 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 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.
1. Acid-catalysed esterification and transesterification
Free fatty acids are esterified and O-acyl lipids transesterified by heating them with a large excess of anhydrous methanol in the presence of an acidic catalyst.
If water is present, it may prevent the reaction going to completion. The commonest and mildest reagent is 5% (w/v) anhydrous hydrogen chloride in methanol. It is most often prepared by bubbling hydrogen chloride gas (which is commercially available in cylinders or can be prepared by dropping concentrated sulfuric acid slowly onto fused ammonium chloride or into concentrated hydrochloric acid) into dry methanol. A simpler procedure is to add 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. It is usual to heat the lipid sample in the reagent under reflux for about 2 hours, but them may also be heated together 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).
A solution of 1-2% (v/v) concentrated sulfuric acid in methanol transesterifies lipids in the same manner and at much the same rate as methanolic hydrogen chloride. It is very easy to prepare, and it is thus the author's preferred reagent for esterification of free fatty acids, but utilised at a temperature below reflux. If the reagent is used carelessly, some decomposition of polyunsaturated fatty acids may occur.
Boron trifluoride in methanol (12-14% w/v) has also been used as a transesterification catalyst and in particular as a rapid means of esterifying free fatty acids. 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. In view of the large amount of acid catalyst used in comparison with other reagents and the many known side reactions, it is the author's opinion that boron trifluoride in methanol has been greatly overrated, and that it is best avoided.
Boron trichloride in methanol does not appear to have been much used for transesterification of lipids, but is almost as effective as boron trifluoride-methanol and does not appear to bring about the same unwanted side reactions [129,485].
Nonpolar lipids, such as cholesterol esters or triacylglycerols, are not soluble in reagents composed predominantly of methanol, and will not react in a reasonable time, unless a further solvent is added to effect solution. 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.
Methanolic hydrogen chloride (5%) or sulfuric acid (1%) are then probably the best general-purpose esterifying agents. They methylate free fatty acids very rapidly and can be employed to transesterify other O-acyl lipids efficiently; they are generally used as follows:
The lipid sample (up to 50 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. The mixture is left overnight in a stoppered tube at 50°C (or is refluxed for 2 hours), then water (5 mL) containing sodium chloride (5%) is added and the required esters are extracted with hexane (2 × 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 and the solvent 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 (also only 20 minutes at reflux, or 2 hours at 50°C, is required), or if polar lipids such as phospholipids are to be transesterified. The reaction can be scaled up considerably; for example, 50 g of lipid in 100 mL of toluene can be transesterified with 200 mL of methanol containing 4 mL of concentrated sulfuric acid. N-Acyl lipids are transesterified very slowly with these reagents (see below). If acidic reagents are permitted to superheat in air, some artefact formation is possible.
The same method is used to prepare dimethylacetals from aliphatic aldehydes or plasmalogens (see Chapter 10), and when it is used on lipid samples containing such compounds, acetals are formed which may contaminate the methyl esters. The two classes of compound can be separated by saponification or better by means of preparative TLC with toluene  or dichloroethane  as the solvent for development (esters migrate ahead of acetals). Acetals are not formed during base-catalysed transesterification.
With lipid samples from animal tissues, it is sometimes necessary to purify methyl esters after transesterification has been carried out in order to eliminate cholesterol, which can be troublesome when the esters are subjected to gas chromatography. 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. Methyl esters can also be purified by preparative TLC, with hexane-diethyl ether (9:1, v/v) as the mobile phase.
2. Base-catalysed transesterification
O-Acyl lipids are transesterified 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 as a result of hydrolysis of lipids. 0.5 M 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 has also been used as acatalyst. The reagent is stable for some months at room temperature, especially 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. It is commonly performed as follows:
The lipid sample (up to 50 mg) is dissolved in dry toluene (1 mL) in a test tube, 0.5 M 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 × 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 nonpolar lipids such as cholesterol esters or triacylglycerols, but is not required if they are not present in the sample. Chloroform should not be used in this way, because it contains ethanol as a stabiliser, and because dichlorocarbene, which can react with double bonds, is generated by reaction with sodium methoxide. Again, cholesterol esters are transesterified very slowly and may require twice as long a reaction time as that quoted. The quantities of lipid used can be scaled up considerably; 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 . Under the conditions described above, no isomerization of double bonds in polyunsaturated fatty acids occurs, though prolonged or careless use of basic reagents can cause alterations to fatty acids.
The author has made extensive use of the following convenient microscale procedure, in which methyl acetate is added to the medium to suppress the competing hydrolysis reaction .
The lipids (up to 2 mg) are dissolved in sodium-dried diethyl ether (0.5 mL) and methyl acetate (20 μL). 1 M Sodium methoxide in dry methanol (20 μL) is added, and the solution is agitated briefly to ensure thorough mixing. The solution immediately becomes cloudy as sodium-glycerol derivatives are precipitated. After 5 min at room temperature, the reaction is stopped by the addition of acetic acid (2 μL), the solvent is evaporated in a stream of nitrogen (taking care not to blow out the solid precipitate), hexane (1 mL) is added and the mixture is centrifuged at about 1500g for 2 min. The supernatant layer is decanted, and an aliquot is taken directly for GC analysis.
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 such lipids. Also, aldehydes are not liberated from plasmalogens with basic reagents, in contrast to when acidic conditions are employed.
Although free fatty acids are not esterified under the basic conditions described above, methyl esters can be prepared by exchange with N,N-dimethylformamide dimethyl acetal in the presence of pyridine . Similarly, methyl iodide reacts with sodium or potassium salts of fatty acids in the presence of a polar aprotic solvent such as dimethylacetamide to form methyl esters [41,186].
Quaternary ammonium salts of fatty acids are converted to methyl ester derivatives pyrolytically in the injection port of a gas chromatograph. Of a number of reagents which have been described for the purpose, it appears that trimethylsulfonium hydroxide is the most powerful and exhibits fewer side reactions; it can be used for the simultaneous transesterification of lipids and esterification of free acids .
Diazomethane reacts rapidly with unesterified fatty acids in the presence of a little methanol, which catalyses the reaction, to form methyl esters. The reagent is generally prepared as a solution in diethyl ether by the action of alkali on a nitrosamide, e.g. N-methyl-N-nitroso-p-toluene-sulfonamide (Diazald™, Aldrich Chemical Co., Milwaukee, U.S.A.). Solutions of diazomethane are stable for short periods if stored refrigerated in the dark over potassium hydroxide pellets. If they are kept too long, polymeric by-products form which may interfere with the subsequent GC analysis.
Diazomethane is highly toxic, carcinogenic and potentially explosive, so great care must be exercised in its preparation; in particular, strong light and apparatus with ground glass joints must be avoided, and the reagent should only be prepared in an efficient fume cupboard. In addition, the intermediate nitrosamines are among the most potent carcinogens known. Accordingly, diazomethane should only be used when no other reagent is suitable.
The procedure of Schlenk and Gellerman  is particularly convenient for the preparation of small quantities of diazomethane for immediate use. In this instance, there is very little by-product formation and, if sensible precautions are taken, the risk to health is minimal.
A simple apparatus is required that can be quickly assembled by a glassblower. It consists of three tubes with side arms that are bent downwards and arranged so that the arm of each projects into and is near the bottom of the next tube. A stream of nitrogen is saturated with diethyl ether in the first tube and carries diazomethane, generated in the second tube, into the third tube where it esterifies the acids. The flow of nitrogen through diethyl ether in tube 1 is adjusted to 6 mL per min. Tube 2 contains 2-(2-ethoxyethoxy)ethanol (0.7 mL), diethyl ether (0.7 mL) and 1 mL of an aqueous solution of potassium hydroxide (600 g/L). The fatty acids (5-30 mg) are dissolved in diethyl ether-methanol (2 mL; 9:1 by volume) in tube 3. About 2 mmole of N-methyl-N-nitroso-p-toluene-sulfonamide per mmole of fatty acid in ether (1 mL) is added to tube 2 and the diazomethane which is formed is passed into tube 3, until the yellow colour persists. Excess reagent is then removed in a stream of nitrogen."
An alternative small-scale procedure has recently been described .
4. Special cases
(i) Short-chain fatty acids: Short-chain acids are completely esterified in all of the procedures described above, but quantitative recovery of the esters from the reaction medium can be very difficult because of their high volatility and partial solubility in water. As short-chain acids are major components of such commercially important fats and oils as milk fats or coconut oil, a great deal of attention has been given to the problem. Diazomethane can be used to esterify free fatty acids quantitatively in ethereal solution, and a portion of the reaction medium may then be injected directly onto the GC column so that there are no losses. On the other hand, if the free acids have to be obtained by hydrolysis of lipids, it is not easy to ensure that there are no losses at this stage. The best methods are those in which there are no aqueous extraction or solvent removal steps and in which the reagents are not heated. The alkaline transesterification procedure of Christopherson and Glass , on which the following method is based, meets these criteria better than most.
The oil (20 mg) is dissolved in hexane (2.5 mL) in a stoppered test tube, and 0.5 M sodium methoxide in methanol (0.1 mL) is added. The mixture is shaken gently for 5 min at room temperature then acetic acid (5 μL) is added followed by powdered anhydrous calcium chloride (about 1 g). After 1 hour, the mixture is centrifuged at 700g for 2 to 3 minutes to precipitate the drying agent. An aliquot of the supernatant liquid is taken for GC analysis."
The method described above,or variations upon it are widely used, and can give excellent results if care is also exercised during the chromatography stage [72,78]. Others have argued that more reproducible gas chromatographic analyses are obtained by preparing butyl ester derivatives [419,420].
If the sample contains both O-acyl bound and unesterified fatty acids, the latter can be esterified with diazomethane first, before the former are transesterified. Alternatively, for safety reasons, the procedures of Thenot et al.  or of Martinez-Castro et al.  might be used.
(ii) Unusual fatty acids: The methods described above can be used to esterify all fatty acids of animal origin without causing any alteration to them. Many fatty acids from plant sources and certain of bacterial origin are more susceptible to chemical attack. For example, cyclopropene, cyclopropane and epoxyl groups in fatty acids are disrupted by acidic conditions, and lipid samples containing such acids are best transesterified with basic reagents; the free fatty acids can be methylated safely with diazomethane.
Conjugated polyenoic fatty acids such as α-eleostearic acid undergo cis/trans-isomerization and double bond migration when esterified with methanolic hydrogen chloride, and all acidic reagents can cause addition of methanol to conjugated double bond systems. Similar reactions occur under acidic conditions with fatty acids containing a hydroxyl group immediately adjacent to a conjugated double bond system (e.g. dimorphecolic acid, 9-hydroxy,10-trans,12-trans-octadecadienoic acid), and dehydration and other unwanted side reactions may also take place (reviewed elsewhere [160,163]). However, no side effects occur when basic transesterification is used.
Diazomethane can add to double bonds and keto groups in 2,3-unsaturated and 2-keto acids respectively .
(iii) Amide-bound fatty acids: Sphingolipids, which contain fatty acids linked by N-acyl bonds, are not easily transesterified under acidic or basic conditions. If the fatty acids alone are required for analysis, the lipids may be refluxed with methanol containing concentrated hydrochloric acid (5:1, v/v) for 5 hours or by maintaining the reagents at 50°C for 24 hours, and the products worked up as described above for the anhydrous reagent [160,895]. Unfortunately, a small proportion of free fatty acid is also formed. As an alternative, the specific hydrolysis methods, described above (Section B), can be used to generate the free acids quantitatively, and these can then be methylated by an appropriate procedure. Traces of degradation products of the bases that might interfere with subsequent analyses can be removed by adsorption chromatography (see Section C.1 above). If the long-chain bases are also required for analysis, suitable procedures are available (see Chapter 10) . With N-acylphosphatidylserine and related lipids, the O-acyl bound fatty acids can be released by mild alkaline methanolysis and so distinguished from the N-acyl components which require much more vigorous hydrolytic conditions.
(iv) Esterification on TLC adsorbents: After lipids have been separated by TLC, the conventional procedure is to elute them from the adsorbent before transesterifying for GC analysis. A number of methods have been described for transesterifying lipids on silica gel without prior elution, with the objective of simplifying the methodology and of reducing the opportunities for contamination. Regrettably, it has been the author's experience that poor recoveries of esters are obtained when basic transesterification reagents are used, probably because water bound to the silica gel causes some hydrolysis. Acid-catalysed procedures give better results, but when the ratio of silica gel to lipid is very high (>4000:1), poor recoveries are again the rule. In practice, such high ratios may not often be obtained and satisfactory methylation is achieved by direct transesterification.
The favoured technique is to scrape the band of adsorbent containing the lipid into a test tube, then to add the reagent (e.g. 2% methanolic sulfuric acid) with efficient mixing, and to carry out the reaction as if no adsorbent were present. On working up the aqueous mixture obtained when the reaction is stopped, it is necessary to centrifuge to precipitate all the silica gel and to extract with a more polar solvent than hexane, for example diethyl ether, to ensure quantitative recovery of the methyl esters. Unfortunately, cholesterol esters are not transesterified readily in the presence of silica gel, and it is still necessary to elute these from the adsorbent prior to reaction.
(v) Side reactions: Methyl esters are the derivatives of choice for gas chromatography but in choosing an appropriate reagent, it is necessary to consider its effect on lipid components other than fatty acids and on gas chromatography stationary phases since artefacts may be produced which interfere with subsequent analyses (see also Section C.4.ii above). For example, BHT may be partially methylated by boron trifluoride-methanol reagent giving rise to an extraneous peak which tends to emerge from a GC column together with or just in front of the C16 fatty acid derivatives .
If cholesterol esters are esterified directly and the free cholesterol is not removed prior to GC analysis, it may dehydrate to form cholestadiene on the column and this may obscure some of the C22 components . Similarly, cholestadiene and cholesterol methyl ether are generated to some extent when most acidic reagents are used for transesterification, and analogous by-products are formed from plant sterols [376,494,626,835]. This does not occur with base-catalysed transesterification. Other hydrolysis products of low molecular weight from lipids, such as phytol and aldehydes, can be troublesome in some circumstances. If need be, the methyl ester derivatives can be purified by adsorption chromatography as discussed in Section C.1 above.
If the methyl ester derivatives are not cleaned up properly, traces of the transesterification reagents injected onto the column will bring about degradation of the stationary phase, and cause spurious peaks to emerge.
5. Preparation of esters other than methyl
(i) Other alkyl and aromatic esters: Esters other than methyl may be required for a variety of reasons, for example to diminish the volatility of short-chain fatty acids or to introduce aromatic groupings into molecules so that the UV-detection systems can be used in HPLC. The latter aspect has been reviewed elsewhere . Many of the methods described above can be adapted to the preparation of alternative alkyl esters simply by substituting the appropriate alcohol for methanol; for example, either 1 M sodium butoxide or 2% sulfuric acid in butanol may be utilised for the preparation of butyl esters. Where the alcohol has a high boiling point or is comparatively expensive, the acid chloride or anhydride can be prepared and reacted with a slight excess of the appropriate alcohol in the presence of a base such as pyridine [160,205]. Section (iii) below contains an example of this. Analogous methods have been used for the preparation of fluorinated esters for GC with electron-capture detection .
Trimethylsilylesters of unesterified fatty acids have been prepared for GC analysis by the same methods used to prepare the trimethylsilylether derivatives of hydroxyl groups (see Section D.3 below) . Similarly, t-butyldimethylsilyl esters have proved of value for the analysis of saturated (including deuterated) fatty acids by mass spectrometry [692,709,1004].
Figure 4.1. Derivatives of fatty acids. (a) Pyrrolidide; (b) picolinyl ester; (c) trimethylsilylether; (d) isopropylidene derivative; (e) butylboronate derivative; (f) mercuric acetate adduct; (g) dimethyldisulfide adduct.
(ii) Pyrrolidides: Amide derivatives of fatty acids can be prepared by reaction of acid chlorides or anhydrides with an amine. N-Acylpyrrolidines (Fig. 4.1(a)) are prepared for mass spectrometric analysis by reaction of methyl esters with pyrrolidine and acetic acid (see Chapter 7). (Methods of preparing these derivatives are discussed at greater length on our mass spectrometry pages here..). The reaction is carried out as follows:
The fatty acid methyl ester (10 mg) is dissolved in freshly distilled pyrrolidine (1 mL), acetic acid (0.1 mL) is added, and the mixture is heated at 100°C for 1 hour. On cooling, the amide is taken up in dichloromethane (8 mL) and is washed repeatedly with 2 M hydrochloric acid then water (4 mL portions). After drying over anhydrous sodium sulfate, the required product is obtained on evaporation of the solvent.
(iii) Picolinyl esters: It is increasingly being recognised that picolinyl ester derivatives (Fig. 4.1(b)) offer distinctive mass spectrometric fragmentation patterns of particular value in the location of double bonds (see Chapter 7). (Methods of preparing these derivatives are discussed at greater length on our mass spectrometry pages here..) They are prepared by the following procedure:
Unesterified fatty acids (5 mg) are dissolved in trifluoroacetic anhydride (0.5 mL) and are left for 30 min at 50°C. The excess reagent is blown off in a stream of nitrogen. As soon as it has gone, 3-hydroxymethylpyridine (20 mg) and 4-dimethylaminopyridine (4 mg) in dichloromethane (0.2 mL) are added. (This reagent can be made up in bulk; for 1 mg or less, simply add half the volume). The mixture is left in a stoppered tube for 3 hours at room temperature, then the solvent is removed in a stream of nitrogen, and hexane (8 mL) and water (4 mL) are added. After thorough shaking and vortex mixing, the hexane layer is washed twice more in the same way, before the solvent is evaporated.
To remove any residual free fatty acids, with small samples especially, the products are taken up in diethyl ether (1 mL), a few mg of a silica-NH2 bonded phase (Bond Elut™ or Sep-Pak™) is added and the mixture is shaken briefly. After 10 min, the mixture is centrifuged, then the solvent is decanted carefully and evaporated.
Purification of picolinyl esters can also be successfully accomplished by elution from a small column of Florisil™ with hexane-diethyl ether (1:4, v/v).
D. Derivatives of Hydroxyl Groups
Many fatty acids with hydroxyl substituents exist in nature and the polar functional moieties must be masked by derivatization prior to analysis by GC. In addition, the free hydroxyl groups of long-chain alcohols, glycerol ethers, sterols, and mono- and diacylglycerols are frequently converted to the same nonpolar derivatives for chromatographic analysis. Acyl migration of fatty acids in partial glycerides is prevented, and more symmetrical peaks are obtained on gas chromatography than could be obtained with the native compounds. As essentially the same procedures are used for these classes of lipids, they are all considered for convenience here. The choice of derivative will depend on the nature of the compound and the separation to be attempted, and on occasion it may be necessary to prepare several types of derivative to confirm identifications. With hydroxy fatty acids, it is normal practice to derivatize the carboxyl group first. The following types of derivative are among the more useful.
Acetyl chloride and pyridine at room temperature, or prolonged heating with acetic anhydride, can be used to acetylate lipids, but the mildest reagent is probably acetic anhydride in pyridine (5:1, v/v), which is used as follows :
The lipid (up to 50 mg) is dissolved in acetic anhydride in pyridine (2 mL, 5:1. v/v), and is left at room temperature overnight. The reagents are then removed in a stream of nitrogen with gentle warming and the acetylated lipid is purified, if necessary, by preparative TLC on silica gel layers, generally with hexane-diethyl ether (80:20, v/v) as the mobile phase.
Free amino groups are also acetylated with this reagent. N-Acetylation without simultaneous O-acetylation (e.g. of long-chain bases) can be accomplished by reaction with acetic anhydride in methanol (1:4, v/v) at room temperature overnight (see Chapter 10 for details) . Acetylmethanesulfonate in microcolumns of celite acetylates alcohols very rapidly and is claimed to be suited to the routine analysis of large number of samples . It is also possible to acetylate lipids with <[1-14C]-acetic anhydride in order to make use of the high sensitivity and precision of liquid scintillation counting for quantification following chromatographic separation .
Trifluoroacetate derivatives of hydroxy acids, monoacylglycerols and glycerol ethers are comparatively volatile and are sufficiently temperature-stable to be subjected to gas chromatographic analysis. They are prepared simply by dissolving the hydroxy compound in excess of trifluoroacetic anhydride, leaving for 30 minutes and then removing most of the excess reagent on a rotary film evaporator. Diacylglycerol trifluoroacetates are not sufficiently stable at high temperatures for gas chromatography, however . Trifluoroacetates hydrolyse very rapidly, even in inert solvents such as hexane, and it is necessary to store them and to inject them onto the gas chromatographic column in a solution containing some trifluoroacetic anhydride. Column packings must be conditioned by repeatedly injecting trifluoroacetic anhydride into them before being used, but they are rendered acidic and may no longer be suitable for other analyses. Such a procedure is not recommended with capillary columns. Trifluoroacetic acid also appears to attack the methylene group between double bonds in some circumstances, causing losses of polyunsaturated components .
3. Trimethylsilyl ether and related derivatives
Trimethylsilyl (often abbreviated to TMS) ether derivatives (Fig. 4.1(c)) are a useful alternative to acetates for gas chromatographic analysis. They are much more volatile than the latter, but are not as stable, particularly to acidic conditions, and will hydrolyse slowly on TLC adsorbents. The preparation and properties of TMS and related derivatives have been reviewed [230,727,729]. Probably the most popular reagent for the preparation of TMS ethers in the earlier literature consists of a mixture of hexamethyldisilazane, trimethylchlorosilane and pyridine (3:1:10 by volume), and it is used as follows.
To the hydroxy-compound (up to 10 mg) is added pyridine (0.5 mL), hexamethyldisilazane (0.15 mL) and trimethylchlorosilane (0.05 mL). The mixture is shaken for 30 seconds and then is allowed to stand for 5 min. An aliquot can then be injected directly into a gas chromatography column; alternatively, the reaction mixture can be taken to dryness on a rotary evaporator, the products extracted with hexane (5 mL), the hexane layer washed with water (1 mL) and dried over anhydrous sodium sulfate, before the solvent is removed. The derivatives are stored in fresh hexane and are stable at -20°C for long periods.
More recently, many simpler more-powerful silylating reagents have become available, and that used most frequently in lipid analysis is probably bis(trimethylsilyl)acetamide ("BSA"). Reaction is carried out simply by dissolving the lipid in a solvent such as acetone or acetonitrile and adding a tenfold excess by weight of BSA; with the normal range of unhindered alcohols likely to be encountered, the reaction is complete in 10 minutes at room temperature. While some analysts inject an aliquot of the reaction mixture directly onto the GC column, the author prefers to clean the sample up, as described above, immediately prior to analysis in order to prolong column life.
t-Butyldimethylsilyl (t-BDMS) etherification has been proved to be of particular value for the preparation of lipid derivatives of higher molecular weight. They are approximately 104 times more stable than the corresponding TMS ethers and only hydrolyse at an appreciable rate under strongly acidic conditions. The following method of preparation is recommended .
The silylation reagent consists of t-butyldimethylsilyl chloride (1 mmole) and imidazole (2 mmole) in N,N-dimethylformamide (10 mL). The reagent (0.5 mL) is added to the lipid (up to 10 mg) and heated at 60°C for 30 min. After rapid cooling, hexane (5 mL) is added, and the mixture is washed with water (3 × 1 mL). The solvent is dried over anhydrous sodium sulfate, filtered or decanted and evaporated in a stream of nitrogen to yield the required derivative.
In contrast to TMS derivatives, it is possible to purify t-BDMS derivatives by preparative TLC.
Recently, nicotinates [96,364,950] and certain hybrid derivatives, i.e. picolinyldimethylsilyl ethers  have been shown to be of value for the identification of alcohols by means of GC/mass spectrometry (see Chapter 10).
4. Isopropylidene compounds
Isopropylidene derivatives of vicinal diols, for example glycerol ethers, 1-monoacylglycerols or dihydroxy acids, are prepared by reacting the diol with acetone in the presence of a small amount of an acidic catalyst (Fig. 4.1(d)). Anhydrous copper sulfate is probably the mildest catalyst and is used as follows:
The esters (10 mg) are dissolved in dry acetone (3 mL) and anhydrous copper sulfate (50 mg) is added. After 24 hr at room temperature (or 3 hr at 50°C), the solution is filtered, the copper salts are washed with dry ether and the combined solutions are evaporated in vacuo.
The use of perchloric acid as a catalyst permits a more rapid reaction, but is potentially much more hazardous . Isopropylidene compounds are stable under basic conditions, but are hydrolysed by aqueous acid; the original diol compound can be regenerated by shaking with a 1 M solution of concentrated hydrochloric acid in 90% methanol.
5. n-Butylboronate derivatives
Alkyl-boronic acids, such as n-butylboronic acid, react with 1,2- or 1,3-diols or with α- or β-hydroxy acids to form 5- or 6-membered ring nonpolar boronate derivatives (Fig. 4.1(e)). They are prepared simply by adding n-butylboronic acid to a solution of the hydroxy-compound in dimethylformamide. The reaction is complete in 10-20 minutes at room temperature and the reaction mixture can be injected directly into a gas chromatographic column for analysis [55,133]. As alternatives, cyclic di-tert-butylsilylene derivatives have been shown to be of value in the analysis of diols and hydroxy acids . The preparation and use of cyclic derivatives for the analysis of bifunctional compounds have been reviewed .
E. Derivatives of Double Bonds
The double bonds of unsaturated fatty acids may be reacted to form various addition compounds, as an aid to the isolation of individual fatty acids or as part of a method for establishing the configuration or location of the double bonds in the aliphatic chain.
1. Mercuric acetate derivatives
Mercuric acetate in methanol solution reacts with the double bonds of unsaturated fatty acids to form polar derivatives (Fig. 4.1(f)), which are more easily separated by adsorption or partition chromatography, according to degree of unsaturation, than are the parent fatty acids. They can be prepared by the following procedure:
The lipid sample is refluxed with a 20% excess of the theoretical amount of mercuric acetate in methanol (2 mL per g of mercuric acetate) for 60 min. After cooling to room temperature, a volume of diethyl ether 2.6-fold that of the methanol is added to precipitate inorganic mercury salts, and the solution is filtered. The mercuric acetate adducts are obtained on evaporation of the solvent.
When fatty acids with five or six double bonds are present, it has been recommended that the reaction mixture should be heated at 100°C for two hours in a sealed tube [827,830]. Although silver ion chromatography has superseded the use of mercuric acetate derivatives for most analytical purposes, the latter may still be useful for the bulk preparation of concentrates of single fatty acids, for example.
The original double bond is regenerated by reaction with aqueous acid with no double bond migration or cis/trans isomerization if the following method is used:
10 g of the adduct in methanol (20 mL) is mixed with concentrated hydrochloric acid (50 mL) and a stream of hydrogen chloride gas is bubbled into the solution for a few minutes. The solution is extracted twice with hexane (50 mL portions), and the extracts are combined and washed with water. If a test of the organic layer with a solution of diphenylcarbazone in methanol reveals that mercury is still present, a single repeat of the process will remove it entirely.
Acetylenic groups react with 2 moles of mercuric acetate to form adducts, but the original bonds cannot be regenerated and a keto derivative is formed on acidic hydrolysis. This reaction has been turned to advantage to locate the position of the original triple bond in a fatty acid derivative by mass spectrometry [140,480].
Mercuric acetate derivatives can be further reacted with inorganic halides yielding methoxyhalogenomercuri-derivatives, which are less polar and more volatile than the original mercury compounds and are more easily separated by chromatography [972,973] (see Chapter 6). The reaction is accomplished simply by adding a 10% excess of sodium bromide in methanol to the mercuration mixture whenever the adduct formation is complete. After about 2 minutes, the new derivative is formed and the mixture is worked up as before.
Mercury adducts can also be reacted with sodium borohydride and converted to methoxy compounds (oxymercuration-demercuration) [321,613], or reacted with halogens in methanol to form methoxyhalogen compounds . Such derivatives have been utilised for locating double bonds in fatty acids by mass spectrometry.
Double bonds can be oxidised to vicinal diols by a variety of reagents of which the most useful are alkaline potassium permanganate and osmium tetroxide. With both reagents, cis-addition occurs to yield diols of the erythro configuration from cis-double bonds, and diols of the threo configuration from trans-double bonds. The following procedure utilises alkaline permanganate and is particularly suited to reaction with monoenes and dienes .
The fatty acid (0.1-100 μmole) is dissolved in 0.25 M sodium hydroxide solution (0.2 mL) and diluted with ice water (1 mL). 0.05 M Potassium permanganate (0.2 mL) is added and after 5 min, the solution is decolorized by bubbling sulfur dioxide into it. The fatty acid derivatives are extracted with chloroform methanol (7 mL, 2:1, v/v), and the lower layer is collected and dried over anhydrous sodium sulfate before the solvent is evaporated.
Osmium tetroxide gives higher yields of multiple diols from polyunsaturated fatty acids .
The fatty acid (0.1-100 μmoles) is dissolved in dioxane-pyridine (1 mL, 8:1, v/v), and a 5% solution of osmium tetroxide in dioxane (0.1 mL) is added. After 1 hour at room temperature, methanol (2.5 mL) and 16% sodium sulfite in water (8.5 mL) are added, and the mixture is allowed to stand for 1 hour more. After centrifuging to precipitate and compact the sodium sulfite, the supernatant solution is diluted with 4 volumes of methanol and filtered. The filtrate is evaporated to dryness and suspended in methanol (2 mL). Chloroform (4 mL) is added, the suspension is filtered once more and the solvent evaporated."
Such hydroxylated fatty acids in the form of less polar derivatives, for example isopropylidene, TMS ether or methoxy compounds, have been used in conjunction with mass spectrometry to locate the positions of the original double bonds (see Chapter 7). Because the isopropylidene derivatives of threo and erythro compounds are separable by GC, the configuration of the original double bonds can also be established [572,985].
Epoxides are formed from olefins by the action of certain per-acids. Cis-addition occurs, so cis-epoxides are formed from cis-olefins and trans-epoxides from trans-olefins. The following procedure is based on that of Gunstone and Jacobsberg .
The monoenoic ester (20 mg) is reacted with m-chloroperbenzoic acid (16 mg) in chloroform (2 mL) at room temperature for 4 hr. Potassium bicarbonate solution (5%, 4 mL) is added, and the product is extracted thoroughly with diethyl ether. After drying the organic layer over anhydrous sodium sulfate and removing the solvent, the required epoxy ester is obtained by preparative TLC on silica gel G layers with hexane-diethyl ether (4:1, v/v) as the mobile phase.
Mass spectrometry of epoxy derivatives has been used to locate the position of double bonds in fatty acids, and as cis- and trans-isomers can be separated by GC, as a means of estimating fatty acids with trans-double bonds (see Chapter 5) [243,244].
4. Dimethyl disulfide addition
One of the most convenient methods for the location of double bonds by mass spectrometry involves the addition of dimethyl disulfide across the double bond, a reaction catalysed by iodine (Fig. 4.1(g)) . It is carried out as follows:
The monoenes (1 mg) are dissolved in dimethyl disulfide (0.2 mL) and a solution (0.05 mL) of iodine in diethyl ether (60 mg/mL) is added. The mixture is stirred for 24 hours, then hexane (5 mL) is added, and the mixture is washed with dilute sodium thiosulfate solution, dried over sodium sulfate and evaporated to dryness. The product is taken up in fresh hexane for injection directly onto the GC column.
Some residual starting material may remain, but it elutes substantially ahead of the product when this is subjected to GC analysis. By using a higher temperature, the reaction can be taken to completion but some by-product formation may occur (see Chapter 7). The author (unpublished) observed excessive by-product formation when the dimethyl disulfide was evaporated off before the washing step. (Methods of preparing these derivatives are discussed at greater length on our mass spectrometry pages here...)
Hydrogenation of lipids is undertaken prior to confirming the chain lengths of aliphatic moieties, or to protect lipids (and simultaneously simplify the chromatograms) during high-temperature GC analysis of intact lipids. Many of the published hydrogenation procedures are needlessly complex and the following method is adequate for most purposes.
The unsaturated ester (1-2 mg) in a test tube is dissolved in methanol (1 mL) and Adams' catalyst (platinum oxide; 1 mg) is added. The tube is connected via a two-way tap to a reservoir of hydrogen (e.g. in a balloon or football bladder) at or just above atmospheric pressure and to a vacuum pump. The tube is alternatively evacuated and flushed with hydrogen several times to remove any air, then it is shaken vigorously while an atmosphere of hydrogen at a slight positive pressure is maintained for 2 hr. At the end of this time, the hydrogen supply is disconnected, the tube is flushed with nitrogen and the solution is filtered to remove the catalyst. The solvent is evaporated under reduced pressure, and the required saturated ester is taken up in hexane or diethyl ether for GC analysis.
Hexane may be used as the solvent for the hydrogenation reaction if the fatty acid is still esterified to glycerol as in a triacylglycerol or diacylglycerol acetate, but the hydrogenated compounds must later be recovered from the catalyst with a more polar solvent such as chloroform.
Deuterium can be added across double bonds to assist in their location by mass spectrometry, by means of deuterohydrazine reduction . Oxygen is required to generate D2-diimine for reaction to occur, so the procedure must be carried out in air.
The unsaturated ester (0.5 mmole) is dissolved in anhydrous dioxane (5 mL) at room temperature, and deuterohydrazine (5 mmole) in twice its volume of deuterium oxide is added. The mixture is stirred at 55-60°C in air, but in the absence of atmospheric moisture, for 8 hr when the reagents are evaporated in vacuo. The product is purified by preparative silver ion chromatography (see Chapter 6).
Others recommend a procedure in which diimine is generated by the reaction of acetic acid with potassium azodicarboxylate [472,484,809].
2H4-acetic acid (0.3 mL) is added in small aliquots to a constantly stirred slurry of potassium azodicarboxylate (500 mg) and the fatty acid (up to 10 mg) in 2H-methanol (5 mL) over a period of 3 to 5 hours. The progress of the reaction can be checked by gas chromatography. The methanol is evaporated in a stream of nitrogen, and the reaction is repeated twice more. The product is purified as above.
Potassium azodicarboxylate is not available commercially, but can be prepared by reaction of 40% aqueous potassium hydroxide on azodicarbonamide . Neither procedure is entirely satisfactory, and occasionally can refuse to work for no apparent reason. A further approach has still to be tried with fatty acids per se, but does appear to give excellent results with intact lipids. It involves catalytic deuteration with deuterium gas and Wilkinson's catalyst (tris(triphenylphosphine)rhodium (I) chloride) .
The following abbreviations are employed at various points in the text of these chapters:
amu, atomic mass units; BDMS, tert-butyldimethylsilyl; BHT, 2,6-di-tert-butyl-p-cresol; CI, chemical ionisation; DNP, dinitrophenyl; ECL, equivalent chain-length; ECN, equivalent carbon number; EI, electron-impact ionisation; FCL, fractional chain-length; GC, gas chromatography; GLC, gas-liquid chromatography; HPLC, high-performance liquid chromatography; IR, infrared; MS, mass spectrometry; NMR, nuclear magnetic resonance; PAF, platelet-activating factor; ODS, octadecylsilyl; TLC, thin-layer chromatography; TMS, trimethylsilyl; UV, ultraviolet.
This document is part of the book Gas Chromatography and Lipids by William W. Christie and published in 1989 by P.J. Barnes & Associates (The Oily Press Ltd), who retain the copyright.
Updated June 27, 2011