Silver ion TLC with Quantification by a Gas Chromatography Method

The Author: William W. Christie, James Hutton Institute (and Mylnefield Lipid Analysis), Invergowrie, Dundee (DD2 5DA), Scotland.

As detailed in an earlier web page, silver nitrate TLC enables separation of triacylglycerols, containing a normal range of fatty acids with zero to three cis-double bonds, into simpler species with up to nine double bonds in the fatty acid moieties per mole of glycerol. To summarise, components tend to migrate in the order -


- where S, M, D, and T denote saturated, mono-, di- and trienoic acids, respectively (they do not indicate the positions of the fatty acids on the glycerol moiety), although there may be some changes in this order depending on the nature of the solvent mixtures used for development.

Densitometry or gravimetric methods are widely used for quantification, but a useful alternative is to scrape off fractions, add an internal standard such as the methyl ester of an odd-chain fatty acid (17:0 or 19:0) not present naturally in the sample, or a synthetic triacylglycerol containing only the selected acid. Fractions are then recovered from the plates and transesterified for analysis by gas chromatography. The fatty acid composition of each fraction is determined in this way, enabling identification, and the amount of the sample is found by relating the total area of the fatty acid peaks to that of the standard ester [1].

I only have experience of this technique with home-made plates, but I see no reason why it cannot be applied to commercial pre-coated plates. Up to 10 mg of triacylglycerols can be separated on a 20 × 20 cm plate (0.5 mm thick layer), and excellent separations of large numbers of components have been obtained with 20 × 40 cm plates by others. I have used plates with up to 10% silver nitrate incorporated into the layers, but Nikolova-Damyanova and Momchilova recommend much lower silver nitrate concentrations.

The solvent systems generally employed consist of hexane-diethyl ether, toluene-diethyl ether or chloroform (alcohol-free)-methanol mixtures. As all the fractions listed above cannot be separated on one plate, it is common practice to separate the least polar fractions first with hexane-diethyl ether (80:20, v/v) or chloroform-methanol (197:3, v/v), and then to separate the remaining fractions with more polar solvents such as diethyl ether alone or chloroform-methanol (96:4, v/v). Some representative separations are illustrated in many of the web pages in the Silver ion Chromatography section of this website.

Bands are detected under UV light as yellow bands on a dark background after spraying with a solution of 0.1% (w/v) 2',7'-dichlorofluorescein in 95% methanol, components are recovered from the adsorbent and they are identified and determined by gas chromatography of the fatty acid constituents with an added internal standard (the concentration depending on the size of the sample) as follows

Laboratory protocol:
Bands are scraped from the plate into test tubes and a solution of the internal standard, for example methyl heptadecanoate in methanol solution (1 mL), is added to each followed by hexane-diethyl ether (1:1, v/v; 3.5 mL) and 20% aqueous sodium chloride (1 mL). The contents are mixed thoroughly by shaking and using a vortex mixer, and centrifuged briefly to precipitate the solids. The top layer is pipetted off, and the aqueous layer is washed twice more with similar volumes of hexane-ether. The combined extracts are washed with 0.05 M Tris buffer (pH 9.0; 2 mL) before being dried over anhydrous sodium sulfate.

The washing step removes any silver nitrate and dye that co-elute with the triacylglycerol fraction. After evaporation of the solvent, the fractions are transesterified for analysis by GC, for example with sodium methoxide in anhydrous methanol. The fractions can then be identified from the relative proportions of the various fatty acid components and quantified in relation to the internal standard.

As a check, when all the fractions have been collated, it is advisable to use the quantitative data for each fatty acid in each fraction to calculate the composition of the original triacylglycerols for comparison with a separate analysis of the intact sample. Any errors/problems in the procedure can then be detected.

While this procedure is more time consuming than densitometric procedures, a disadvantage when large numbers of samples have to be analysed, it is capable of appreciable accuracy.

Analogous procedures can be used to separate methyl esters of fatty acids into fractions differing in degree of unsaturation as an aid to subsequent identification, for example by gas chromatography-mass spectrometry. Also, procedures of this kind have been used frequently to determine the content of fatty acids with trans double bonds in samples.