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Purification of fatty acids
before analysis



The techniques described below are useful to clean the sample before chromatographic analysis but mainly to fractionate a complex mixture into groups studied later by different techniques. Some of them may also be used as preparative procedures.


Purification by TLC

Separation of normal and hydroxy fatty acids

Separation according to unsaturation

Separation according to chain length

Separation of chlorinated fatty acids

Separation by counter-current





Purification by TLC



Spurious peaks can appear on chromatograms which are generated by various possible reactions, functional groups, contaminants present in reagents or in glassware and other lipid molecules (sterols, antioxidants, phytol, various non-saponifiable materials). Thus, sometimes it becomes essential to purify methyl esters prior to GLC analysis and sometimes to separate normal from hydroxy fatty acids.

Materials and Reagents:

Washed silica gel plates (migration in chloroform/methanol 1/1, v/v)
Hexane, diethyl ether


The FAME are purified by migration in hexane/diethyl ether (1/1, v/v) as developing solvent.
After a rapid drying, the spots are detected with UV light after primuline spray. Compounds are identified with the help of standard chemicals.
In a typical experiment, the Rf are: FAME, 0.58; hydroxy fatty acid methyl esters,0.32; free fatty acids, 0.27; hydroxy fatty acids, 0.09. Dimethyl acetals which are produced from aldehydes or plasmalogens in the same conditions as FAME migrate slightly slower than FAME (Rf: 0.52). All components are recovered from the scraped spots by two elutions with 2 ml hexane/diethyl ether (1/1, v/v).




Separation of normal and hydroxy fatty acids:


TLC separation

Fatty acids (as FAME) are separated by TLC on silica gel plates with hexane/diethyl ether (85/15, v/v) as eluent. Detected after primuline spray under UV light, spots corresponding to the two fatty acid classes are scraped and fatty acids are eluted with dichloromethane washings (2 times 2 ml). Standard solutions (one normal and one hydroxy fatty acid) are prepared by methylation with BF3/methanol of commercial compounds and chromatographed in parallel.
Normal fatty acids can be analyzed directly by GLC but hydroxy fatty acids must be previously derivatized by reacting with a reagent such as SIL-A from Sigma (100 µl, 15 min at 30°C) or BSTFA-TMCS from Alltech (100 µl, 4-5 h at 20°C). After these derivatizations, reagents are evaporated under nitrogen and residues dissolved in hexane before injection. The determination of 2- and 3-hydroxy fatty acids in food samples by gas chromatography and mass spectrometry has been described (Jenske R et al., J Agric Food Chem 2008, 56, 11578).

Column separation

An alternative method is proposed below to separate rapidly and quantitatively normal and hydroxy fatty acid methyl esters on a small Florisil column (Bouhours JF J Chromatogr 1979, 169, 462).

Materials and reagents:

Pasteur pipette (15 cm) with a sheet of fiberglass paper at the bottom of the large tube.
Florisil powder (60-100 mesh).
Hexane, diethyl ether, ethyl acetate.


Fill the pipette with 0.5 g of Florisil suspended in hexane. Wash the column with 5 ml of hexane/diethyl ether (95/5, v/v).
Load the fatty acids (in 1 ml hexane/ether) and elute the normal fatty acid with 6 ml of the same mixture, then elute the hydroxy fatty acids with 6 ml of ethyl acetate.
The first fraction is evaporated, dissolved in a small volume of hexane and analyzed by GLC.
The second fraction is evaporated, silylated, dried and re-dissolved in hexane before GLC analysis.

Comments: a similar separation may be obtained using a micro-column filled with silica gel (3 cm) suspended in hexane (fatty acids being dissolved in the same solvent). Normal fatty acids are eluted by 4 ml of hexane/diethyl ether (93/7, v/v) and hydroxy fatty acids by 4 ml of hexane/diethyl ether (50/50, v/v).
Another elution system using hexane and ethyl acetate has been used to determine the concentrations of medium-chain 2- and 3-hydroxy fatty acids in foodstuffs (milk, brain, suet, oils) (Jenske R et al., Food Chem 2009, 114, 1122). The analysis was done using gas chromatography with electron-capture negative ion mass spectrometry.

Solid-phase microextraction (SPME) was proposed to allow a rapid, precise and accurate methodology for the quantification of short-chain free fatty acids (C4-C10) in milk (Gonzalez-Cordova AF et al., J Agric Food Chem 2001, 49, 4603). The SPME device was from Supelco Co (Bellafonte, PA). A polyacrylate phase was shown to allow efficient recoveries when first conditioned in a gas chromatography injection port at 300°C for 2 h. The fiber was exposed to the sample headspace for 60 min and desorbed for 5 min into the gas chromatograph. The limits of quantification were shown to be about 0.1 ppm for C4, C6, and C8 and 1.6 ppm for C10.





Separation according to unsaturation

When complex fatty acid mixtures are analyzed or when information on the unsaturation degree are needed, a simple fractionation of the crude fatty extract can be achieved by urea adduct formation but a chromatography on silica gel impregnated with silver nitrate is required to separate individual components. These separations may be achieved either by TLC or column chromatography.


Urea fractionation


Upon crystallization, urea forms inclusion complexes with some long-chain aliphatic compounds. Saturated fatty acids (as FAME) form complexes readily (as trans fatty acids), their formation being less efficient with increasing number of double bonds or in the presence of branched chains. This procedure cannot be used as an analytical technique but is frequently applied to obtain a concentrate of polyunsaturated or branched-chain fatty acids.


Urea, methanol, pentane.


To a dry extract containing up to 100 mg FAME, add 10 ml methanol.
Warm at about 60°C and by mixing dissolve 3.5 g urea.
Cool the mixture at 20°C, wait 5-6 h and centrifuge to collect the methanol phase.
Extract the polyunsaturated fatty acids by two washings of pentane (5 ml). After pentane evaporation, add 2 ml water and 1 ml pentane. Fatty acids are recovered from the pentane phase.
Saturated fatty acids are recovered from crystallized urea after its solubilization in water by pentane or hexane washings.

As an application, this procedure was useful to prepare a lipid extract enriched in γ-linolenic acid from seed oil of Boraginaceae species (Campra-Madrid P et al., Chromatographia 2002, 56, 673). After direct saponification of ground seeds with KOH-ethanol at 60°C, acidification and hexane partition, urea fractionation was done with a ratio of urea to fatty acids of 4/1 (w/w) and a methanol ratio of 1/3 (w/v) at 0°C. In one step, the γ-linolenic acid concentration in the urea concentrate from Anchusa azurea was increased from 7% to 67% with a yield of about 91%. The production of γ-linolenic acid from borage oil has been studied in optimizing the formation of urea complexation (Spurvey SA et al., J Food Lipids 2000, 7, 163).  
Urea was also used to purify γ
-linolenic acid from a microalgae extract (Spirulina platensis), enabling a final purity of its ester of about 84% (Sajilata MG et al., Food Chem 2008, 109, 580).

To obtain high and quantitative yields (80-100%) of urea complex with gram amounts of lipids, it is recommended that the ratio of organic compounds / urea / methanol should be 1 / 5 / 7-20.

A simple, rapid and cost-effective preparative-scale separation using urea crystallization was developed for the isolation of gram-scale amounts of highly pure n-3 fatty acids from fish oil sources (Hidajat K et al., J Chromatogr A 1995, 702, 215). The individual n-3 fatty acids were fractionated by HPLC on a phenyl silica column and with an isocratic ternary mobile phase.
A simple procedure used to enrich fish oil in EPA and DHA was reported for squid visceral oil ethyl esters (Hwang LS et al., JAOCS 2001, 78, 473). A single complexation reaction raised the EPA content by 2.2 fold and DHA content by 2.4 fold, thus allowing further purification by molecular distillation. It must be noticed that cholesterol was also enriched by about 2.5 fold. EPA and DHA were optimally concentrated from chemically hydrolyzed sardine oil by urea fractionation using methanol at 4°C and urea/fatty acid ratio of 4/1 (w/w) (Chakraborty K et al., J Agric Food Chem 2007, 55, 7586). A further enrichment in EPA was obtained using argentation chromatography.
Concentration of DHA from algal oil has been done using complexation of free fatty acids with 2% urea in ethanol (95% in water). After crystallization at room temperature, the mixture was maintained at 4°C for 24h before fliltration. Saturated and monoene fatty acids were eliminated almost completely while DHA was enriched from 47.4 to 97.1% (Senayake SPJ et al., J Food Lipids 2000, 7, 51).


Silver ion chromatography


Since the introduction of silver ion chromatography in 1962 (Morris LJ, Chem Ind, London, 1962, 1238-1240), this separation had an increasing importance in the efforts to elucidate the structure of various lipids. It was mostly used as an efficient step to resolve a complex lipid mixture into simple molecular species. Silver ion is used either in TLC or in column chromatography (normal pressure, solid phase extraction or HPLC). This use is based on the ability of silver ion to complex with unsaturated compounds. The stability of the complex increases with an increasing number of double bonds but decreases with the increasing chain-length. The cis-isomers are more stable than the trans-isomers and conjugated polyenes form less stable complexes than do those containing methylene-interrupted double bonds.
Fatty acids are resolved practically on the basis of the number and the configuration of their double bonds. Silver ion TLC is also efficient in separating geometrical isomers, the trans isomers migrating ahead of the corresponding cis molecules. The migration order is tt > ct > cc for dienes and ttt > ctt > cct > ccc for the trienes. The resolution of fatty acids in the form of FAME with zero to six double bonds is feasible using two sets of developing solvents. Thus, argentation column chromatography was used with success for the separation of fish oil fatty acids in seven fractions using mixtures of ether in petroleum ether (Hoque M et al., JAOCS 1973, 50, 29).

The incipient wetness impregnation method was used to prepare a AgNO3/SiO2 adsorbent for the extraction of linolenic acid methyl ester from hexane solutions prepared from vegetal oils (McWilliams KM et al., Eur J Lipid Sci Technol 2016, 118, 252). This batchwise process makes some vegetal oils used as biodiesels a source of the omega-3 fatty acid and gives a product that is less sensitive to oxidative deterioration.

The followings references are highly recommended to anyone who wants to have a more complete picture of the role of silver ion chromatography in lipid analysis (Christie WW, High performance liquid chromatography and lipids. A practical guide, Pergamon Press 1987 – Nikolova-Damyanova B, in Advances in lipid methodology-One, by Christie WW, The oily Press 1992, 181-237).


Silver ion TLC



Silver nitrate, sodium thiosulphate,
hexane, diethyl ether, methanol, ethyl acetate, toluene
Silica gel plates, room with dim light, migration tank protected from light


Silica gel plates are impregnated with silver nitrate by dipping rapidly (1 min) in a 4% solution of silver nitrate in methanol/water (9/1, v/v). Plates are then drained and dried 2 min in dim light in a ventilated fume hood and for 20 min at 100°C. They are kept in a tightly closed box in the dark.
Plates are developed in:

– hexane/diethyl ether (90/10, v/v) to separate saturated, monoenes and dienes,
– toluene/ethyl acetate (90/10, v/v) to separate all types.

Fatty acid spots are eluted by two washings with 2 ml of hexane/diethyl ether (1/1, v/v) and may be analyzed immediately by GLC.

The solvent proportions may be altered slightly to improve the separations according to local experimental conditions (plates, humidity, temperature, geometry of the tank…). The addition of 1% acetic acid (v/v) to the mobile phase enables the resolution of free fatty acids.
Plates are dried in a ventilated hood, immersed about 1 min in a saturated solution of sodium thiosulphate in water and rinsed in running water for 1 min. Plates are dried in a ventilated hood and sprayed with primuline to detect the fluorescent fatty acid spots under UV light.

FAME mixtures obtained from a vegetal oil or a marine oil (cod liver) help in detecting the position of the respective fatty acids in combination with GLC.
The first eluent (A) enables the separation of FAME up to dienes, the more unsaturated staying at the origin region. The second eluent (B) enables the separation of all FAME except the saturated and monoenes which co-migrate near the solvent front.



Lane A: hexane/diethyl ether (90/10, v/v) as eluent; 1, saturated; 2, monoenes; 3, dienes; 4, other polyunsaturated.
Lane B: toluene/ethyl acetate (90/10, v/v) as eluent; 1, saturated and monoenes; 2, 20:2; 3, 18:2; 4, 20:3; 5, 18:3; 6, tetraenes; 7, pentaenes; 8, hexaenes.


It must be noticed that, with eluent A, trans-dienes and conjugated dienes are not resolved from cis-monoenes and that trans-monoenes migrate ahead of cis-monoenes fatty acids (between saturated and monoenes). These peculiarities allow to collect these isomeric fatty acids before a further analysis by HPLC.


Silver ion column chromatography


We describe below a simple procedure adapted from Christie (J Lipid Res 1989, 30, 1471) in which a commercial solid phase extraction column is converted to the silver ion form and fatty acid methyl esters are separated by stepwise elution.

Materials and Reagents:

Bond Elut SCX solid phase extraction columns (0.5 g of propylbenzene sulphonic acid)
Solution of 40 mg AgNO3 in 1 ml acetonitrile/water (10/1, v/v)
1 M HCl, 1 M NaOH
Acetonitrile, dichloromethane, hexane, acetone


Columns are regenerated by successive washes of 5 ml 1 M NaOH, 10 ml water, 5 ml 4 M HCl, water until neutral pH and acetonitrile/water (10/1, v/v) . Wrap the column in an aluminum foil to protect against light.
Column are converted to the silver ion form by allowing to percolate slowly 1 ml of AgNO3 solution. The column is then washed by 5 ml acetonitrile, 5 ml acetone and 10 ml dichloromethane.

Load 0.1 ml dichloromethane containing no more than 1 mg FAME.

– Saturated fatty acids are eluted by 5 ml dichloromethane
– Monoenes are eluted by 5 ml dichloromethane/methanol (90/10, v/v)
– Dienes are eluted by 5 ml acetone
– Trienes are eluted by 10 ml acetone/acetonitrile (98/2, v/v)
– Tetraenes are eluted by 10 ml acetone/acetonitrile (94/6, v/v)
– Pentaenes are eluted by 10 ml acetone/acetonitrile (90/10, v/v)
– Hexaenes are eluted with acetone/acetonitrile (60/40, v/v)

The elution is made at atmospheric pressure (flow rate of about 0.5 ml).

Notice that trans-monoenes and conjugated dienes are eluted with the monoene fraction (trans dienes migrate ahead of cis dienes and with cis monoenes).

The fractionation of polyunsaturated fatty acids from edible oils (vegetable or marine oils) using an open column with partially argentated silica gel as stationary phase has been described (Guil-Guerrero JL et al., Grasas Aceites 2003, 54, 116). Methyl esters of linoleic, a-linolenic, g-linolenic, stearidonic, eicosapentaenoic, and docosahexaenoic acids where prepared with a purity of 83 up to 100%. Technical details on the preparation of the argentated column and the elution steps are found in the cited paper.
The selective removal of polyunsaturated fatty acids from mixtures of fatty acid methyl esters prepared from vegetal oils has been described (Ghebreyessus KY et al., JAOCS 2006, 83, 645). It was shown that the 7% of linolenic acid in soybean oil could be reduced to 0.1% according a simple method involving a column filtration on silica gel impregnated with 20% AgNO



Separation according to chain length


To identify the very long-chain fatty acids present in some biological samples (bacteria, fungi, soil…), it is often necessary to enrich the samples in fatty acids having more than 22 carbon atoms.
Several efficient enrichment methods were described such as those involving either separation by TLC of fatty acid derivatives (Quereshi N et al., J Biol Chem 1980, 255, 182), reversed phase used in TLC or in HPLC (Rezanka T et al., J Chromatogr 1989, 472, 290). An efficient and reliable enrichment method was described for microorganisms using a simple solid-phase extraction cartridge packed with an octadecyl-bonded silica (Rezanka T et al., J Chromatogr 1993, 636, 249). A total amount of 1-2 mg of fatty acid methyl esters was applied to the SPE cartridge in a small volume of dichloromethane. Methanol/isopropanol (98/2, v/v) was allowed to flow and very long-chain fatty acids were eluted after an elution volume of about 2 ml.



Separation of chlorinated fatty acids


As chlorinated fatty acids are present in low concentrations in animal tissues as well as in aqueous biota, it is necessary to highly concentrate the lipid extracts and to selectively fractionate these fatty acid derivatives.
A simple isolation procedure was proposed using a small aminopropyl column (Akesson-Nilsson G, J Chromatogr A 2003, 996, 173). The lipid extract was methylated with BF3 in methanol and toluene.
A 500-mg aminopropyl column (Supelco, No. 57014) was connected to a vacuum manifold and conditioned with 2 ml of n-hexane. Fatty acids were applied on the column in 0.2 ml of hexane. The column was washed with 6 ml of hexane and the chlorinated derivatives were eluted with 4 ml of a solvent mixture made of hexane/ethyl ether/dichloromethane (89/1/10, v/v). After evaporation and dissolution in a small volume of hexane, chlorinated fatty acids may be analyzed by GLC using a halogen specific detector (XSD detector from Oico).



Separation by counter-current


The preparation of highly unsaturated fatty acids on a semi-preparative scale has been described using a counter-current chromatographic device based on a multi-layer coil separator extractor (Bousquet O et al., J Chromatogr A 1995, 704, 211). The fatty acids obtained by saponification are injected in the column with heptane as the stationary phase and acetonitrile/water as the mobile phase. Baseline resolution was obtained with four important n-3 fatty acids from marine algae (16:3, 18:4, 20:5, and 22:6) after optimization of the chromatographic parameters (mobile phase, flow rate, and coil rotational speed). Gram-level separations are possible using only a bench-top apparatus.


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