Pre-Concentration of Minor Components

Determination of structures of fatty acids is not always straightforward. Derivatized fatty acids are not always sufficiently well resolved by gas chromatography, and minor components especially may be obscured. Analysis of the latter is greatly assisted if they can be concentrated by some means. Urea fractionation is a useful technique for concentration of polyunsaturated, branched-chain or cyclic fatty acids. However, chromatographic techniques, such as high-performance liquid chromatography (HPLC) in the reversed-phase mode or silver ion chromatography (either HPLC or thin-layer chromatography (TLC)) afford greater versatility and selectivity. Counter-current distribution equipment is also being used successfully for the purpose. Several published review articles on the topic are available [1-3]

The choice of the most appropriate method will depend on the nature of the sample, the amount of material available and the equipment available to the analyst.


1.  Urea Fractionation

When urea is permitted to crystallize in the presence of fatty acid methyl esters, it forms hexagonal crystals with a channel, into which some fatty acids may be able to fit and so be removed from solution. Saturated straight-chain acids (as the methyl ester derivatives) form such urea inclusion complexes readily, but the double bonds of unsaturated fatty acids or methyl branches increase their bulk so that they form complexes less readily or not at all. Monoenoic fatty acids are complexed to a limited extent but polyenes are not. When the crystallization process is complete, the saturated and a proportion of the monoenoic fatty acids are locked up in the crystals, while polyunsaturated, branched-chain and cyclic fatty acids remain in the mother liquor. Although the procedure is sometimes carried out on large amounts of material, the following simplified method is of more value to analysts [4].

Laboratory protocol:
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.

With care, there is little opportunity for harm to come to polyunsaturated esters. The author has also used this procedure after hydrogenation to isolate branched-chain and cyclic fatty acids to confirm their structures.


2.  Solvent Partition in the Form of Silver Ion Complexes

Although silver ion complexation is usually used in conjunction with chromatography to separate unsaturated compounds (see below), a simple solvent partition procedure has described that permits the isolation of a concentrate of polyunsaturated fatty acids [5].

Laboratory protocol:
The fatty acid methyl 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 x 10 mL). The hexane extract is dried over anhydrous sodium sulfate and evaporated to yield the polyunsaturated esters.

Note: Take great care to prevent spillages, which can leave an indelible stain on laboratory benches and equipment, and especially do not allow the silver ion solution to come in contact with the skin.


3.  Solid-Phase Extraction Methods

Fatty acid methyl esters from fish oils (5 mg scale) have been divided into two fractions, i.e. saturated plus monoenes, and polyunsaturated, on a solid-phase extraction column with bonded aminopropyl groups [6], and similar methodology is used for hydroxy/nonhydroxy acids [7]. Solid-phase extraction columns of the reversed-phase type have been employed for enrichment of methyl eicosapentaenoate from algal fatty acids [8]. We have no experience of these methods so can offer no further guidance.

Bond Elut™ columns packed with a silica-based benzenesulfonic acid medium can be converted to the silver ion form and used to achieve useful separations of methyl ester derivatives of fatty acids on a small scale [9]. No silver salts are eluted with the fractions.

Laboratory Protocol

Satisfactory resolution of components with zero to six double bonds is obtained. As substantial changes in solvent composition are utilized at each step, there is little cross-contamination, especially with the early fractions. It is important that the columns should not be overloaded or that the flow-rate be increased artificially otherwise resolution is lost. Some modification to these conditions may be required for different commercial brands of column. This methodology is described in greater detail on this website here...


4.  Reversed-Phase HPLC

Although HPLC in the reversed-phase mode is sometimes used for analytical separations of fatty acids, it is of much less practical value than gas chromatography, which affords much more accurate quantification and beter resolution. It is more useful for micro-preparative purposes, i.e. to isolate components for further analysis by GC-MS. The technique has been reviewed by Nikolova-Damyanova [10].

Octadecylsilyl (ODS)-bonded phases have become the standard for the purpose, and those that have been especially stabilized against attack by bases are useful for separation both of methyl esters and of the nitrogen-containing derivatives used in mass spectrometry. We use a column of Hichrom RPB™ for the purpose, but comparable types from other suppliers should also be suitable. Conventional ODS columns can also be used provided that amines are incorporated into the mobile phase [11].

In reversed-phase HPLC, unsaturated fatty acids are eluted substantially ahead of the saturated fatty acid with the same chain-length, each double bond reducing the retention time by the equivalent of approximately two carbon atoms. Thus oleic acid derivatives tend to elute in a similar region to the corresponding palmitic acid derivatives, though they are usually well resolved. Indeed, the resolving power of columns packed with modern micro-particulate phases is such that there need be little overlap of the main components of interest in natural samples, if the mobile phase is selected with care. On the other hand because of the nature of the separation, different fatty acids are easily confused, and it is necessary exercise care to ensure correct identification. Model mixtures of pure standards are invaluable in developing separations and as an aid to the identification of unknowns.

A separation of 3-pyridylcarbinol ('picolinyl') esters prepared from the fatty acids of borage oil, on a column of Hichrom RPB™ using acetonitrile only as the mobile phase is illustrated [2].

Reversed-phase HPLC separation of 3-pyridylcarbinol esters

In this example, the more highly unsaturated C18 esters elute first, followed by 16:0, then the C20 monoenoic fatty acids and 18:0.

Laboratory protocol:
To reproduce this separation, a column of Hichrom RPB™ (250 x 4.6 mm; Hichrom Ltd, Reading, UK) or equivalent is used with acetonitrile as mobile phase, with the flow rate programmed from 0.5 to 1.5 mL/min over 30 min, and held at this for a further 5 min. The temperature of the column is maintained at 20°C. The sample (5 μL) is injected in a solution of acetone-acetonitrile (1:9,v/v). UV detection at 254 nm, or an evaporative light-scattering detector with stream-splitter can be used.

If the column temperature is kept constant, the elution times are highly reproducible, but this is not essential. Here, evaporative light-scattering detection was used for convenience, but UV detection should also be possible by using the specific absorbance of the pyridine ring. Methyl esters and DMOX derivatives can be chromatographed under the same conditions, except that corresponding components elute a little earlier than for 3-pyridylcarbinol esters, presumably because the last are a little more hydrophobic. Again, UV detection should be possible for methyl esters, but at 206 nm. It should be noted that base-deactivated columns of this type are rendered unusable by exposure to acids.


5.  Silver Ion TLC and HPLC

Silver ion chromatography is an invaluable technique for separating fatty acids according to the number and configuration of the double bonds. However, a major new section of the AOCS Lipid Library on silver-ion chromatography is now this website, so some brief details only are sketched out here. The figure below illustrates GC analyses of fractions with zero to six double bonds from methyl esters of fatty acids from sheep liver obtained by silver ion TLC. It is easy to see that the simple fractions thus obtained are much easier to analyse by GC linked to mass spectrometry. Two substantial reviews by Nikolova-Damyanova have been published on the topic [12,13].


In brief, silver ion TLC is simple to undertake using silica gel layers into which silver nitrate is incorporated. Adaptations to simple partition and solid-phase extraction methodologies are described above. Silver ion HPLC uses columns containing ion-exchange materials with bonded phenylsulfonic acid groups to which silver can be linked. It is a simple task to prepare suitable columns in the laboratory [14], or they can be purchased ready made from Varian Inc. (ChromSpher Lipids™ columns). There is a specific protocol here...


6. Counter-Current Distribution

Simple bench-top counter-current distribution equipment is now available that permits separation of fatty acid derivatives under mild conditions on a much larger scale than is possible with conventional HPLC equipment, although the resolution is probably inferior. Separation is on the basis of chain-length and degree of unsaturation. We have no experience of the technique so cannot comment further, but recent publications from W. Vetter’s laboratory show the possibilities [15,16].



  1. Christie, W.W. Structural analysis of fatty acids. In: Advances in Lipid Methodology - Four, pp. 119-169 (1997) (edited by W.W. Christie, Oily Press, Dundee).
  2. Christie, W.W. Gas chromatography-mass spectrometry methods for structural analysis of fatty acids. Lipids, 33, 343-353 (1998) (DOI: 10.1007/s11745-998-0214-x).
  3. Christie, W.W. and Han, X. Lipid Analysis - Isolation, Separation, Identification and Lipidomic Analysis (4th edition), 446 pages (Oily Press, Woodhead Publishing and now Elsevier) (2010) - see Science Direct.
  4. Linstead, R.P. and Whalley, M. The formation of crystalline complexes between urea and esters, and their application to the separation of mixtures of esters. J. Chem. Soc., 2987-2989 (1950).
  5. Peers, K.E. and Coxon, D.T. Simple enrichment procedure for the estimation of minor polyunsaturated fatty acids in food fats. J. Food Sci. Technol., 21, 463-469 (1986).
  6. Wilson, R., Henderson, R.J., Burkow, I.C. and Sargent, J.R. The enrichment of n-3 polyunsaturated fatty acids using aminopropyl solid phase extraction columns. Lipids, 28, 51-54 (1993) (DOI: 10.1007/BF02536360).
  7. Zelles, L. Phospholipid fatty acid profiles in selected members of soil microbial communities. Chemosphere, 35, 275-294 (1997) (DOI: 10.1016/S0045-6535(97)00155-0).
  8. Cohen, Z. and Cohen, S. Preparation of eicosapentaenoic acid (EPA) concentrate from Porphyridium cruentum. J. Am. Oil Chem. Soc., 68, 16-19 (1991) (DOI: 10.1007/BF02660301).
  9. Christie, W.W. Silver ion chromatography using solid-phase extraction columns packed with a bonded-sulfonic acid phase. J. Lipid Res., 30, 1471-1473 (1989) (available here).
  10. Nikolova-Damyanova, B. Reversed-phase HPLC: general principles and application to the analysis of fatty acids and triacylglycerols. In: Advances in Lipid Methodology - Four, pp. 193-251 (ed. W.W. Christie, Oily Press, Dundee) (1997).
  11. Christie, W.W. and Stefanov, K. Separation of picolinyl ester derivatives of fatty acids by high-performance liquid chromatography for identification by mass spectrometry. J. Chromatogr. A, 392, 259-265 (1987) (DOI: 10.1016/S0021-9673(01)94271-2).
  12. Nikolova-Damyanova, B. Silver ion chromatography and lipids. In: Advances in Lipid Methodology - One, pp. 181-237 (ed. W.W. Christie, Oily Press, Dundee) (1992). Now available on this website here...
  13. Nikolova-Damyanova, B. Lipid analysis by silver ion chromatography. In: Advances in Lipid Methodology - Five, pp. 43-123 (ed. R.O. Adlof, Oily Press, Bridgwater) (2003).
  14. Christie, W.W. A stable silver-loaded column for the separation of lipids by high-performance liquid chromatography. J. High Resolut. Chromatogr., Chromatogr. Commun., 10, 148-150 (1987) (DOI: 10.1002/jhrc.1240100309).
  15. Hammann, S., Tillmann, U., Schroder, M. and Vetter, W. Profiling the fatty acids from a strain of the microalgae Alexandrium tamarense by means of high-speed counter-current chromatography and gas chromatography coupled with mass spectrometry. J. Chromatogr. A, 1312, 93-103 (2013) (DOI: 10.1016/j.chroma.2013.08.090).
  16. Schroder, M. and Vetter, W. Detection of 430 fatty acid methyl esters from a transesterified butter sample. J. Am. Oil Chem. Soc., 90, 771-790 (2013) (DOI: 10.1007/s11746-013-2218-z).

Updated May 5, 2014