Isolation of Fatty Acids and Identification by Spectroscopic and Chemical Degradative Techniques

Sections A and B.  Isolation of fatty acids


A.  Introduction

As cautioned in Chapter 5, GC alone cannot give unequivocal identifications of the compounds separated. Ideally for this purpose, individual pure fatty acids (usually in the form of the methyl ester derivatives, prepared as described in Chapter 4) should be isolated by a combination of complementary chromatographic methods and examined first by nondestructive spectroscopic techniques before chemical degradative procedures are applied. For example, adsorption chromatography will separate normal fatty acids from those containing polar functional groups. Silver ion chromatography can be used to segregate fatty acids according to the number and geometrical configurations of their double bonds; a portion of each fraction should be hydrogenated so that the lengths of the carbon chains of the components can be confirmed. Finally, some form of partition chromatography must be utilised to separate components of different chain lengths in each so that the position and configuration of the double bonds may be determined by spectroscopic methods (principally infrared (IR) and nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry) or by chemical oxidative degradation procedures. Appropriate spectroscopic and chemical techniques can also be used to detect and locate other functional groups in the fatty acyl chains. With many of these techniques, GC will be necessary to monitor separations or as an aid to identification of the products of reactions. It is rarely possible for one technique on its own to give all the structural information necessary on an unknown.

Of these techniques, gas chromatography coupled with mass spectrometry has become of such importance that it merits a separate treatment, and this forms the subject of  Chapter 7. As the interface between the GC column and the mass spectrometer and the need to prepare derivatives of high molecular weight may limit the resolution attainable by GC alone, silver ion chromatography and HPLC in the reversed-phase mode especially are important complementary techniques. They permit isolation of simpler fractions, more readily analysed by GC-MS and other techniques. It should, however, be noted that mass spectrometry cannot normally give evidence as to the stereochemistry or configuration of functional groups. Other spectroscopic and chromatographic techniques together with GC retention data are required in this instance.

The chromatographic procedures used for the isolation of fatty acid derivatives may be based on either TLC or HPLC, and the latter is increasingly being favoured. Certainly, the author has now virtually abandoned TLC, because of the convenience and cleanliness of HPLC and the high reproducible resolution attainable. Nevertheless, TLC still has much to offer in terms of flexibility and economy, and an enormous amount of data has been obtained by this means; it is not neglected in this Chapter. Reversed-phase TLC and preparative GC are now little used and are not discussed here; they have been reviewed elsewhere [163].


B.  Isolation of Individual Fatty Acids for Structural Analysis

1.  Concentration techniques

(i) Urea adduct formation:  When urea is permitted to crystallise in the presence of certain long-chain aliphatic compounds, it forms hexagonal crystals with a channel, into which the aliphatic compounds may fit, provided they do not contain functional groups that increase their bulk, and thence they are removed from solution. Such crystals are known as urea inclusion complexes. Saturated straight-chain acids (as the methyl ester derivatives) form complexes readily. On the other hand, the double bonds of unsaturated fatty acids increase their bulk so that monoenoic fatty acids do not form complexes easily, but tend to form them more readily than dienes, which in turn form them somewhat more easily than compounds with three or more double bonds. Fatty acids with double bonds of the trans-configuration form complexes before the analogous compounds with cis-double bonds. The effect of double bond configuration and number on urea complex formation has been studied systematically [883]. Unfortunately, the separations are complicated by the fact that shorter chain length compounds do not complex as readily as do higher fatty acids; methyl oleate, for example, is adducted with approximately the same facility as methyl laurate [421]. For this reason among others, the procedure has never been developed as an analytical technique per se. The following method can be applied to obtain a concentrate of, for example, polyunsaturated fatty acids from a natural mixture (as the methyl esters).

The esters (10 g) are dissolved in methanol (100 mL) to which urea (20 g) is added. The mixture is warmed until all the urea is in solution, when it is allowed to cool to room temperature with occasional swirling. After a minimum of 4 hours, the material is filtered through a Büchner funnel to remove the urea complexes, which are washed twice with 2.5 mL portions of methanol saturated with urea. The solution, which is greatly enriched in polyunsaturated esters, is then poured into 1% aqueous hydrochloric acid (60 mL) and extracted alternately with hexane (50 mL) and diethyl ether (50 mL). The combined organic layers are washed with water (2 × 50 mL) and dried over anhydrous sodium sulfate, before the solvent is removed under reduced pressure.

The procedure can be scaled up or down considerably. As an example, a GLC trace of material obtained in this way from 0.25 g of the standard mixture described in Chapter 5 (Fig. 5.1) is illustrated in Figure 6.1. The fraction obtained which represents 20% of the original esters is enriched in the polyunsaturated components, but also contains some branched-chain esters, not readily apparent earlier, and some constituents of shorter chain length.

GC chromatogram of non-adducted esters

Figure 6.1. GLC recorder trace (the EGSS-X™ column of Table 5.1 and Fig 5.1) of methyl esters that did not form urea adducts from the natural mixture illustrated in Figure 5.1 (see Chapter 5).


The adducted esters can be recovered, when required, by breaking up the complexes with water and extracting the esters into hexane or diethyl ether.

Another particularly valuable application consists in the isolation of concentrates of branched-chain and cyclic esters from natural mixtures, and many useful separations of the former especially have been described [232,238,668,853,854]. iso-Branched esters complex more readily than the corresponding anteiso-compounds, so some change in the ratio of these may occur during processing. Urea fractionation procedures have also been used to separate ω-hydroxy fatty acids (after acetylation to increase their bulk) from related compounds with the substituent elsewhere in the chain [178].

Attempts to use urea in thin-layer adsorbents or in columns on an analytical scale have not been entirely convincing, and the following simplified procedure is of value when small amounts only of esters are available [552].

The methyl esters (up to 100 mg) are dissolved in hexane (4 mL), and urea (1.5 g) moistened with methanol (15 drops) is added. After standing overnight, the solid is filtered off and thoroughly washed with hexane; the washings and the hexane filtrate are combined, washed with water, dried over anhydrous sodium sulfate and evaporated, yielding a branched-chain and/or polyunsaturated fraction.

The main advantage of methods using urea are that large quantities of esters can be separated and that with care there is little chance for harm to come to polyunsaturated esters.

(ii) Partition in the form of metal ion complexes:  Although silver ion complexation is usually used in conjunction with chromatography to separate unsaturated compounds (see below), Peers and Coxon [702] have described a simple solvent partition procedure which permits the isolation of a concentrate of polyunsaturated fatty acids.

The fatty esters (up to 1 g) in 2,2,4-trimethylpentane (10 mL) are partitioned with vigorous shaking with the same volume of ethanol-water (1:1) containing silver nitrate (2.5 g). The upper organic layer, which contains mainly saturated and monoenoic components, is removed. The lower layer is diluted with water (10 mL), and is extracted with hexane (3 × 10 mL), which is dried over anhydrous sodium sulfate and evaporated to yield the polyunsaturated esters.

Take care to prevent spillages, and do not allow the silver ion solution to come in contact with the skin.

Concentrates of polyunsaturated fatty acids (as the methyl esters) can be obtained from suitable starting materials, by preparing the mercuric acetate adducts (see Chapter 4 for practical details of the preparation of adducts and regeneration of the original double bonds) and partitioning them between methanol and pentane; the methanol layer retains the adducts of the more unsaturated esters which can be regenerated unchanged. For example, methyl linoleate of 95% purity and methyl linolenate of 90% purity have been produced on the 50-100 g scale by this method from the esters of safflower and linseed oils, respectively. The method could no doubt also be adapted to the preparation of concentrates of other polyunsaturated fatty acids.

(iii) Low-temperature crystallisation:  Fractions enriched in polyunsaturated fatty acids are readily obtained by low-temperature crystallisation, a technique that demands little by way of expensive equipment. Most methods employ acetone for crystallisation of samples in the form of the methyl ester derivatives, or acetone, diethyl ether or hexane for the free acids, which are generally preferred for the purpose, at temperatures down to −70°C (attainable with solid carbon dioxide as refrigerant).

Three fractions enriched in saturated, monoenoic and polyenoic fatty acids, respectively, can be prepared [137]. As an example, the free acids are taken down to the lowest working temperature (about −50°C generally), at a concentration of 1 g per 10 mL of acetone, and are held there for up to 5 hours with gentle swirling until equilibrium is reached. The solution is then filtered through a Büchner funnel, cooled to just below the solution temperature, and the crystals are washed with a small amount of cold solvent. The material in solution consists mainly of polyunsaturated fatty acids, although it is always contaminated by small amounts of saturated and monoenoic components. If the volume of solvent is reduced until a 10% solution is again attained, the process can be repeated and improved separations obtained. The crystals may be redissolved in fresh solvent and further fractionated at slightly higher temperatures into components enriched in monoenoic and then in saturated fatty acids. In the method's favour, large quantities of fatty acids can be processed in a single operation, and very little harm can come to polyunsaturated acids at the low temperatures employed.


2.  Silver ion chromatography

Since its introduction by Morris and others in 1962, TLC on silica gel impregnated with silver nitrate has been of enormous value to the lipid analyst (see our specialist pages on this topic for more information here..). It is sometimes termed "argentation" chromatography. The basis of the separation is the facility with which the double bonds in the alkyl chains of fatty acids form polar complexes reversibly with silver compounds. Fatty acids can be separated according to both the number and the configuration of their double bonds and sometimes, with care, according to the position of the double bonds in the alkyl chain. HPLC has been slow to make a mark in this area, because of problems in preparing stable columns, but many of the major difficulties now appear to have been resolved. However, most of the data on the elution characteristics of silver complexes of unsaturated fatty acids have been obtained by TLC.

Usually, 3 to 5% by weight of silver nitrate relative to the weight of silica gel is incorporated into the slurry used to make the plates, which are then activated in the normal way. They must be stored in the dark and are stable thus for a month or so. Plates with better keeping properties have been prepared by incorporating 30% ammonia into the slurry, but a well-ventilated area is needed for activation [998]. Excellent results have recently been claimed for pre-coated alumina plates, impregnated by immersion in a 10% solution of silver nitrate, followed by drying and re-activating [127]. On exposure to light, silver-impregnated plates darken rapidly, and it is important that they be handled and developed in a darkened room or cupboard whenever possible. Fatty acids (as methyl esters) on the plate can be visualised under UV light after spraying with 2',7'-dichlorofluorescein in 95% methanol (0.1% w/v), when they appear as yellow spots on a red-purple background.

It is reportedly possible to separate fatty acid methyl esters with zero to six double bonds into distinct fractions on a single plate with a double development in hexane-diethyl ether-acetic acid (94:4:2 by volume), provided that the atmospheric relative humidity is below 50% [231]. It is more usual, however, to attempt to separate methyl esters of fatty acids with zero to two double bonds on one plate, and those with three to six double bonds on another as illustrated in Figure 6.2, although complete separation of components with four or more double bonds is never easy. Hexane-diethyl ether (9:1, v/v) will separate components with up to two double bonds, and the same solvents in the ratio 2:3 will separate polyunsaturated esters [163]. After visualising with the spray reagent, components with zero to two double bonds are eluted from the adsorbent with diethyl ether or chloroform; chloroform-methanol (9:1, v/v) may be necessary for complete recovery of polyunsaturated compounds. Unwanted silver ions contaminating fractions can be eliminated from the extracts by washing them with dilute ammonia (about pH 9). Up to 5 mg of esters can be separated on a 20 × 20 cm plate coated with a layer 0.5 mm thick of silica gel containing 5% (w/w) silver nitrate.

Figure 6.2. Separation of methyl ester derivatives of unsaturated fatty acids by TLC on silica gel G impregnated with 10% (w/w) silver nitrate. Plate A: mobile phase hexane-diethyl ether (9:1, v/v). Plate B: as A but solvents in the ratio 2:3. Silver ion TLC chromatogram of unsaturated fatty acid esters

The elution characteristics of a wide variety of unsaturated esters have been studied. For example, the complete series of methyl cis- and trans-octadecenoates [323] and methylene-interrupted cis,cis-octadecadienoates [157] have been subjected to silver nitrate TLC. When run in order on a single TLC plate, each series migrates in the form of a sinusoidal curve similar to that observed with isomeric polar esters on silica gel alone (see below), with a minimum at the 6-18:1 isomer (or at 6,9-18:2 in the case of the dienes) and a maximum at the 13-18:1 isomer. trans-Isomers migrate consistently ahead of the corresponding cis-isomers, with the exception of the cis-2 component, which not only migrates ahead of its trans-analogue but also ahead of methyl stearate. With natural samples, monoenoic fatty acids containing trans-double bonds can be estimated by separating them from the cis-compounds by means of silver nitrate TLC with hexane-diethyl ether (9:1, v/v) as developing solvent and eluting them, together with the band containing the saturated components, from the adsorbent. If the samples are analysed by GC before and after the separation, the amount of the trans-acids in the mixture can be determined [181]. Alternatively, the cis- and trans-components can be eluted individually and quantified by GC with an internal standard.

With care, it is possible to separate positional isomers of unsaturated fatty acids by silver nitrate TLC. The most consistent and successful separations of this kind were achieved by Morris et al. [622], who utilised silica gel impregnated with up to 30% silver nitrate, developing the plates several times in the same direction if necessary, with toluene as developing solvent at temperatures as low as −5 to −25°C (complex formation is stronger at low temperatures). With this system, the methyl 6-, 9- and 11-cis-octadecenoates can be cleanly separated from each other. Layers containing such high proportions of silver nitrate are very friable, but are not required for more routine separations.

An acetylenic group is less polar than a cis-double bond on silver nitrate TLC, so methyl stearolate migrates just ahead of methyl oleate [620] and methyl crepenynate ahead of methyl linoleate [325]. The allenic ester, methyl labellenate, also migrates ahead of methyl oleate [326]. Esters with conjugated double bonds are less strongly retained than similar compounds with isolated double bonds; for example, methyl 9-cis,11-trans-octadecadienoate migrates with methyl oleate when hexane-diethyl ether (9:1, v/v) is the developing solvent, and just ahead of it when toluene is used [161]. It is worth noting that compounds which are not resolved with hexane-diethyl ether solvent systems are frequently separable with aromatic solvents such as toluene and vice versa. Cyclopropane and saturated branched-chain esters co-chromatograph with normal saturated straight-chain compounds.

It appears likely that silver ion HPLC will soon begin to supplant TLC procedures. As the author has reviewed the technique in some detail elsewhere [168], separate web pages on this site now deal with the topic in some detail (here..). Several groups have reported successful separations of unsaturated fatty acid derivatives on microparticulate silica gel impregnated with silver nitrate [88,372,378,379,685,814]. It appears that the most suitable grade of silica gel for the purpose is one with a large pore size. Unfortunately, silver nitrate leaches rapidly from such columns, contaminating samples and reducing the working life of the column.

A better approach consists in binding the silver ions to a cation-exchange resin, as the silver ions are thus retained more strongly on the column. Some of the first attempts showed promise, but did not make full use of the benefits of HPLC technology [33-36,246]. Better results were obtained with eicosanoids on columns containing sulfonic acid-bonded phases loaded with silver ions [736], and the author has had considerable success with a column of this type in the separation of the more conventional fatty acids. A pre-packed column (250 × 4.6 mm i.d.) of Nucleosil™ 5SA was loaded with silver ions simply by injecting small aliquots of silver nitrate, via a Rheodyne™ injector, into a mobile phase of water [169]; when the excess silver ions began to emerge from the end of the column, the aqueous phase was flushed out with organic solvents of decreasing polarity (there is further information here..).

Silver ion HPLC chromatogram of fatty acid methyl esters

Figure 6.3. Separation of the methyl ester derivatives of the fatty acids of Rapana thomasiana by silver ion HPLC. A column of Nucleosil 5SA™ (250 × 4.6 mm), loaded with silver ions, was eluted with a gradient of methanol to methanol-acetonitrile (9:1, v/v) over 40 min at a flow-rate of 0.75 mL/min. An evaporative light-scattering detector was used with a stream-splitter at the end of the column. Further details are given elsewhere [175].


To illustrate the power of this column, an application to the separation of the fatty acids of the sea snail Rapana thomasiana is illustrated in Figure 6.3 [175]. The column was eluted with a gradient of acetonitrile into methanol, and an evaporative light-scattering detector was used (UV detection at low wavelengths would also have been possible). The major part of the eluent was diverted by a stream splitter at the end of the column for manual collection of fractions as they were seen to emerge. Fractions with zero to six double bonds were clearly resolved, with no cross-contamination and no contamination with silver ions, in less than 25 minutes (and on about the 0.5 to 1 mg scale). After evaporating the solvents, the fractions were examined by GC on a WCOT column coated with Silar 5CP™ as shown in Figures 6.4 and 6.5. The GC trace of the unfractionated methyl esters was so complex that even the more common fatty acids were not easily distinguished (published elsewhere [175]).

GC chromatograms of fractions obtained by silver ion TLC

Figure 6.4. GC separations of the fractions separated by silver ion chromatography (illustrated in Fig. 6.3) [175]. (A) Saturated; (B) monoenes; (C) dienes. A WCOT column (25 m × 0.22 mm i.d.) coated with Silar 5CP™ was used with hydrogen as carrier gas at a flow-rate of 1 mL/min; the column was maintained at 155°C for 3 minutes, then the temperature was raised at 4°C/min to 195°C, and was maintained at this for a further 17 min. Peak designations: 1, 4,8,12-trimethyl-13:0; 2, 14:0; 3, 14:1(n-5); 4, 15:0; 5, iso-methyl-15:0; 6, 16:0; 7, 16:1(n-7); 8, 16:1(n-5); 10, C17-cyclic (unidentified); 11, iso-methyl-16:0; 12, anteiso-methyl-16:0; 13, 17:0; 14, 17:1(n-8); 15, 14-methyl-17:0; 16, 18:0; 17, 18:1(n-13); 18, 18:1(n-11); 19, 18:1(n-9); 20, 18:1(n-7); 21, 18:1(n-5); 22, 18:2(n-6); 26, 19:1; 27, 19:1(n-8); 28, 20:0; 29, 20:1(n-13); 30, 20:1(n-9); 31, 20:1(n-7); 32, 5,11-20:2; 33, 5,13-20:2; 39, 21:1(n-14); 40, 22:1(n-15); 41, 7,13-22:2; 42, 7,15-22:2.

GC chromatograms of fractions obtained by silver ion TLC

Figure 6.5.  As Figure 6.4 except: (A) trienes; (B) tetraenes; (C) pentaenes with hexaene (dotted line) superimposed. Peak designations: 9, 16:3(n-4); 23, 18:3(n-3); 24, 18:4(n-3); 25, 18:5(n-3); 34, 20:3(n-9); 35, 20:3(n-6); 36, 20:3(n-3); 37, 20:4(n-6); 38, 20:5(n-3); 43, 22:3(n-6) (?); 44, 22:4(n-6); 45, 22:5(n-3); 46, 22:6(n-3).


After fractionation, some relationships between various components are immediately apparent. Both the chain length and the number of double bonds in each fatty acid derivative in the chromatograms are ascertained, while relative retention times can be correlated with those of constituents of the unfractionated material. By careful measurement of ECL values, it is then frequently possible to assign double bond positions. In this sample, the identities of nearly fifty different fatty acids in the fractions were confirmed by GC-mass spectrometry following conversion to the picolinyl ester derivatives (see Chapter 7).

The saturated fraction contained the usual range of straight-chain even- and odd-numbered fatty acids, and many branched-chain constituents including the isoprenoid 4,8,12-trimethyl-13:0. In the monoenes, there were a number of positional isomers of the C16 (2), C18 (5), C20 (4) and C22 fatty acids, not to mention the odd-chain constituents, with 7-20:1 (20:1(n-13)) surprisingly being the single most abundant component. The diene fraction presented real problems of identification, since it contained trace amounts only of the conventional methylene-interrupted compounds, with a number of fatty acids with several methylene groups between the double bonds, i.e. 5,11- and 5,13-20:2 and 7,13- and 7,15-22:2. After this the polyene fractions may appear somewhat of an anticlimax, but components from the (n-3) and (n-6) families were identified (a minor component identified initially as 10,13,16-22:3 [175] may in fact be 7,13,16-22:3).

Fractionation of the sample in this way compensated to a considerable degree for any loss of resolution in the GC-MS system and permitted the identification of many more fatty acids than was possible with the unfractionated sample. Although the example chosen here for illustrative purposes is rather unusual, the technique has proved equally valuable with more conventional animal, algal and plant fatty acids.

With such a rapid elution scheme, separation of configurational isomers would not be expected. Better resolution is possible by employing a gradient from 1,2-dichloroethane-dichloromethane (1:1, v/v) to this solvent mixture containing 10% of methanol-acetonitrile (1:1, v/v) as the mobile phase, as shown in Figure 6.6. Here the fatty acid esters of human plasma lipids are separated, and cis- and trans-isomers of monoenoic fatty acids are resolved as are some positional isomers of the polyunsaturated constituents. Excellent separations of positional and configurational isomers of derivatives of monoenoic fatty acids from hydrogenated oils have been accomplished by utilising dichloroethane-dichloromethane as the isocratic mobile phase (Christie, W.W. and Breckenridge, G.H.M. J. Chromatogr., 469, 261-269 (1989)).

Silver ion HPLC of methyl ester derivatives of fatty acids 

Figure 6.6.  Separation of methyl ester derivatives of fatty acids by HPLC on a silver ion column (as in Fig. 6.3) with evaporative light-scattering detection. A gradient of 1,2-dichloroethane-dichloromethane (1:1) was changed to 1,2-dichloroethane-dichloromethane-methanol-acetonitrile (45:45:5:5 by volume) over 40 min at a flow-rate of 1.5 mL/min.


It is not hard to predict that separations of this type will prove invaluable to lipid analysts in future when identifying unknown fatty acids. One problem with the column appears to be that it is unsuited for certain applications, as residual sulfonic acid groups on the stationary phase bring about transesterification in some circumstances, although this can be avoided with care. A buildup in operating pressure tends to occur in prolonged use that appears to be due to impurities accumulating on the column; these can be removed by elution with acetonitrile-methanol (1:1, v/v) into which a few milligrams of silver nitrate in acetonitrile is injected. In general, it is probably advisable to avoid ether-containing solvents as silver nitrate can catalyse a reaction between traces of hydroperoxides and double bonds to form epoxides [154]. Recently, a column consisting of silver-loaded mercaptopropyl silica gel was described that shows promise [234]; further results are awaited with interest. In addition, the author (Christie, W.W. J. Lipid Res., 30, 1471-1473 (1989)) has obtained good results with Bond Elut™ cartridges, packed with a bonded sulfonic acid medium, in the silver ion form. Further details here....


3.  Chromatography of mercury adducts

Before silver nitrate chromatography was developed, some similar separations of unsaturated compounds were achieved by TLC of mercuric acetate derivatives and some workers continue to find it of value. The procedure is rather tedious as the derivatives must be prepared prior to the analysis (see Chapter 4), then decomposed when the fractionation is over, before components can be analysed further by other procedures. Also, the resolutions that can be obtained are not as good as those accomplished with silver nitrate chromatography. Usually the methoxy-bromomercuri-derivatives are prepared, as these are less polar than the acetoxy-mercuri-compounds and can be fractionated according to the number of double bonds by TLC [614,827,830,972,973] or column chromatography [198] procedures. Sébédio et al. [827,830] utilise hexane-dioxane (3:2, v/v) as the solvent for development in TLC, and detect the bands by spraying with diphenylcarbazone in ethanol (0.2%, w/v). One advantage of the method in some circumstances is that there is no separation of the cis- and trans-isomers, which can be collected as a single band and re-chromatographed by other procedures later.


4.  Reversed-phase HPLC

Before gas chromatography was developed, liquid-liquid partition chromatography was the most useful technique for separating individual (or critical pairs of) fatty acids from natural mixtures. After a period in abeyance, the instrumentation developed for HPLC has been applied to utilise this principle to effect excellent separations of fatty acid derivatives on an analytical or semi-micro-preparative scale. Again, as the procedure has been reviewed rather comprehensively by the author [168], there is no need to repeat this here. However, a brief summary of the principles and of some selected applications that complement GC analysis is worthy of discussion.

The technique involves partition of a solute between a stationary and a mobile phase as in GC, except that in the former both phases are liquids; the term "reversed-phase" implies that the mobile phase is more polar than the stationary one. By far the most widely used stationary phase consists of octadecylsilyl ("C18" or "ODS") groups, linked to a silanol surface by covalent bonds, although C8 phases are increasingly being found to have some utility. Invariably, unsaturated fatty acids are eluted appreciably ahead of the saturated fatty acids of the same chain length, each double bond reducing the retention time (or volume) by the equivalent of about two carbon atoms. Thus, oleic acid derivatives tend to elute in the same region as palmitate; as these are always major components of plant and animal tissues, it is essential in assessing separation conditions for natural samples that these fatty acids should be adequately resolved. 14:0, 16:1, 18:2 and 20:4 fatty acids form a further troublesome group. Because of technical improvements in the production of the microparticulate phases, resolution has become less of a problem in recent years. On the other hand, due to the nature of the separation, the various fatty acids are easily confused, and it is necessary to be especially vigilant to ensure that components separated by reversed-phase HPLC are identified correctly.

As methyl ester derivatives are by far the most useful for chromatography in general and for GC in particular, it is perhaps most relevant to consider HPLC of fatty acids in this form here. The main difficulty lies in the choice of detection system, as lipids lack chromophores that facilitate spectrophotometric detection (see Chapter 2 also). Aveldano et al. [67] made a systematic study of the separation of methyl ester derivatives with real samples (as opposed to standard mixtures) of mouse brain fatty acids on a column (250 × 4.6 mm) of Zorbax™ ODS, maintained at 35°C; the mobile phase was acetonitrile-water (7:3, v/v) changed to acetonitrile alone, and with UV detection at 192 nm. The nature of the separation is illustrated in Figure 6.7. When the nature of the separation process is understood, the order of elution of different components is logical, but a newcomer to the technique would certainly find it puzzling. Nonetheless, a number of different components are sufficiently well resolved, for structure determination, say. As isolated double bonds contribute most to absorption at low wavelengths, the response of the detector is strongly dependent on the degree of unsaturation of each constituent fatty acid.

Reversed-phase HPLC of the methyl ester derivatives of fatty acids

Figure 6.7.  Separation of the methyl ester derivatives of fatty acids from the phospholipids of mouse brain by reversed-phase HPLC with spectrophotometric detection at 192 nm [67]. In essence, the column of Zorbax™ ODS phase, maintained at 35°C, was eluted stepwise with acetonitrile-water (7:3, v/v) then with acetonitrile alone. Methyl elaidate was added as an internal standard. (Reproduced by kind permission of the authors and of the Journal of Lipid Research, and redrawn from the original paper).


It is noteworthy that cis- and trans-isomers are well resolved, and reversed-phase HPLC has been suggested as a means of estimating such fatty acids (as the methyl esters) in hydrogenated fish oils [894]. In this instance, an isocratic mobile phase, consisting of methanol-water (89:11, v/v), was used with refractive index detection. Some positional isomers can also be resolved, and as an example various conjugated trienoic acids have been separated by the technique [900].

Alternative procedures for reversed-phase HPLC of fatty acids have been developed, in which fatty acid esters containing strongly UV-absorbing substituents in the alcohol moiety are prepared, so that components emerging from the columns can be detected by means of UV spectrophotometry. Borch [119] was one of the first to exploit this technique and obtained some remarkable separations of fatty acids, in the form of the phenacyl esters, by means of HPLC with a C18-bonded stationary phase and acetonitrile-water mixtures as the mobile phase, in conjunction with UV-detection. A wide range of components are separable, including polyunsaturated fatty acids and isomers in which the position or configuration of the double bond varies. Indeed, oleic and petroselinic acid are resolved in this system, a feat that is not readily achieved by other techniques. Innumerable separations of fatty acids in the form of derivatives such as the 2-naphthacyl, p-bromophenacyl (for example of C30 to C56 fatty acids [742]), or methoxyphenacyl esters have been recorded, generally with gradients of methanol-water or acetonitrile-water mixtures as the mobile phase [168]. Separations of geometrical and positional isomers of fatty acid derivatives of this kind have also been studied [986,994]. The author (unpublished) has observed that phenacyl esters are readily hydrolysed or transesterified by the procedures described in Chapter 4, if this is required.

One advantage of the method is that any impurities not converted to UV-absorbing derivatives are not detected, so cannot obscure the separations. On the other hand, it is likely again that the principal use of such methods will be for small-scale preparative separations of single components for structural analysis or for liquid scintillation counting. The technique may also prove invaluable for determination of the fatty acid composition of seed oils containing thermally labile fatty acids, such as those with cyclopropene moieties [987]. GC is likely to remain the method of choice for analytical separations of most natural fatty acids in the foreseeable future.

One further reversed-phase separation worth recounting is of picolinyl ester derivatives of fatty acids, since these are particularly valuable for structural identification by GC-mass spectrometry (see Chapter 7). Figure 6.8 illustrates a separation of the picolinyl ester derivatives of the fatty acids of cod liver oil on a column containing an octylsilyl bonded phase with stream-splitting for collection purposes and evaporative light-scattering detection [184]. The solvent reservoirs contained methanol (A) and water-pyridine-acetic acid (B) (98.5:1.5:0.025 by volume), and a gradient of the solvents in the ratio (A:B) from 80:20 to 92:8 was generated over 40 min at a flow-rate of 0.75 mL/min. Components which form critical pairs in this system are generally resolved well by GC. On subsequent examination of each of the fractions from the column in turn by GC-MS, 39 different constituents were positively identified.

Reversed-phase HPLC of the picolinyl ester derivatives of the fatty acids of cod liver oil

Figure 6.8.  Separation of the picolinyl ester derivatives of the fatty acids of cod liver oil by HPLC in the reversed-phase mode on a column (250 × 5 mm) of Spherisorb™ C8 (see text for elution conditions) [184]. The main components in each peak are as follows: 1, 14:1; 2, 18:4; 3, 16:2; 4-5, 14:0, 20:5, 18:3; 6, 16:1; 7, 22:6, 20:4; 8, 18:2; 9, 22:5; 10, 16:0; 11, 18:1; 12, 20:2; 13, 18:0; 14, 20:1; 15, phytanic acid; 16, 22:1. (Reproduced by kind permission of the Journal of Chromatography, and redrawn from the original paper).


5.  Adsorption chromatography

In general, fatty acids that differ only in chain length or degree of unsaturation cannot be separated by adsorption chromatography, although short-chain and polyunsaturated fatty acids migrate more slowly on silica gel and other adsorbents than do C16 to C20 saturated or monoenoic components, so that fractions enriched in these compounds can sometimes be obtained. If polar functional groups (especially oxygenated moieties) occur in the alkyl chain, however, some useful separations are possible. It is again more usual to separate fatty acids as the methyl ester derivatives, but unesterified fatty acids can also be chromatographed if 1% of acetic or formic acids is incorporated into the mobile phase when silicic acid is the adsorbent. Sufficient material for many structural analyses can be obtained by preparative TLC, and the elution characteristics of polar fatty acid esters on thin layers of silica gel have been thoroughly examined. Inevitably, most work in this area has been done with TLC systems, but HPLC will certainly replace these in due course with the TLC experience serving as a guide.

In particular, Morris et al. [623] have studied the chromatographic behaviour of the complete series of methyl hydroxy-, acetoxy- and keto-stearates. When each series is arranged in order as a line of spots on a single TLC plate, which is then developed in various hexane-diethyl ether mixtures, the compounds migrate in the form of a sinusoidal curve with a minimum at the 5-substituted isomer and a maximum at the 13- or l14-substituted isomer. Isomers with functional groups in positions 2 to 7 can often be separated from each other and from the remaining compounds. This sinusoidal effect is exhibited by other isomeric series of polar aliphatic compounds. As an example of a practical separation, methyl 6-, 14- and 2-hydroxy-stearates were resolved by TLC on silica gel layers, with hexane-diethyl ether (85:15, v/v) as mobile phase [955]. Figure 6.9 illustrates the elution characteristics of some hydroxy esters with this solvent system. Positional isomers of dihydroxy esters, i.e. methyl 6,7- and 9,10-dihydroxy-stearates have been separated by TLC, with diethyl ether-hexane (4:1, v/v) as developing solvent [621]. Up to 20 mg of esters can be separated on a 20 × 20 cm plate coated with a layer of silica gel 0.5 mm thick. As with silver ion TLC, bands are detected by means of a 2',7'-dichlorofluorescein spray, and they are recovered by elution from the adsorbent, after scraping it into a small column, with diethyl ether or a chloroform-methanol mixture (about 9:1, v/v).

Figure 6.9. Schematic TLC separation of methyl esters of oxygenated fatty acids on silica gel G, with a mobile phase of hexane-diethyl ether (85:15, v/v). Abbreviations: a, 16:0; b, methyl 9,10-epoxystearate; c, methyl 12-ketostearate; d, methyl 2-hydroxystearate; e, methyl 14-hydroxystearate; f, methyl 6-hydroxystearate; g, methyl 9,10-dihydroxystearate. TLC chromatogram of methyl esters of oxygenated fatty acids

Threo- and erythro-isomers of vicinal dihydroxy esters can be separated on thin layers of silica gel impregnated with boric acid (10%, w/w) as a complexing agent, with hexane-diethyl ether (60:40, v/v) as the developing solvent (the threo-isomer migrates more rapidly); the compounds cannot be separated on silica gel alone [618]. Sodium arsenite-impregnated layers give even more remarkable separations of isomeric polyhydroxy fatty acids.

A great deal of work has been done on the separation of hydroperoxides of unsaturated fatty acids by HPLC and columns of silica gel (reviewed by the author [168]). As the separation is carried out anaerobically at room temperature, little harm can come to the samples. Conjugated double bonds usually present in such fatty acids permit sensitive and specific detection by UV spectrophotometry at 235 nm. The technique has been used with other oxygenated fatty acids, and as an example, epoxy fatty acids were isolated from the total fatty acids of a seed oil by HPLC with a column of Partisil™ silica gel with hexane-diethyl ether (9:1, v/v) as the mobile phase [871].

Where the fatty acids contain double bonds in addition to more polar groups, silver ion chromatography may be of further assistance in achieving separations to complement those by GC.



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.

Go to Part 2 of this Chapter.

Updated July 5, 2011