The Lipid Library

SILVER ION CHROMATOGRAPHY

Part 1. Introduction

Summary:  The principles and practical aspects of the technique of silver ion chromatography are introduced and described.

Most lipid analysts will come across the technique of silver ion (or "argentation") chromatography at some stage in their career. The principle is very simple and is dependent on the fact that the pi electrons of double bonds in the fatty acyl residues of lipids react reversibly with silver ions to form polar complexes, the greater the number of double bonds the stronger the complexation effect. The technique has been adapted to liquid-liquid partition, gas-liquid, column and thin-layer chromatography, but most older readers will associate it with the last of these and with the name of Lindsay Morris of Unilever Ltd. He described some of the first practical separations and published a definitive review which is still required reading for anyone who really wishes to understand the technique more than forty years later [1]. Of course, the technique has moved on since then and there are some more recent and substantial reviews available [2-4]. Much more information on silver ion chromatography is available on this site here...

Silver Ion Thin Layer Chromatography

In thin-layer chromatography (TLC), the silver nitrate is simply incorporated into the aqueous slurry used to suspend the silica gel and the plates are spread and activated in the usual way, though some care is necessary to minimise exposure to light. Alternative methods are available for commercial pre-coated plates. Lipid samples are applied to a plate and this is developed with a suitable mobile phase. Fully saturated lipids do not form complexes and migrate to the top of the plate, those containing one monoenoic fatty acyl residue come next and components of increasing unsaturation then follow; one dienoic fatty acid is retained more strongly than two monoenes. Many different classes of lipid have been analysed in this way, including methyl esters of fatty acids, sterol esters, and phospholipids and derivatives, but most industrial analysts will associate the technique with the fractionation of molecular species of triacylglycerols. In particular, it was for many years the method of choice for the determination of cocoa butter equivalents in confectionery fats. With the latter, it is even possible to distinguish between monoenoic species in which the unsaturated fatty acid is in either the central or the outer positions of the glycerol moiety.

There is a number of drawbacks to silver ion TLC procedures, however, and one these is the "purple finger syndrome"; silver nitrate on the hands rapidly darkens leaving a stain that takes weeks to fade away. In addition and more seriously from a scientific standpoint, silver ions are eluted from TLC plates and contaminate fractions in preparative applications. Silica gel and dyes used for detection purposes are further contaminants. On the other hand, the equipment required is simple, inexpensive and available in many laboratories, so the technique will no doubt continue to be widely used.

Because of the disadvantages of silver ion TLC, analysts have looked to high-performance liquid chromatography (HPLC) for the separation of molecular species of lipids, especially triacylglycerols, in recent years. The technique has been employed in the reversed-phase mode in the main with bonded octadecylsilyl groups (ODS) as the stationary phase with mobile phases containing acetonitrile as the major component (reviewed elsewhere [5]). Separations are then based on both the combined chain-lengths and the number of double bonds in the fatty acid residues, one double bond reducing the effective chain-length of an acyl moiety by the equivalent of about two methylene groups. By using long columns and small particle sizes (3 micron), some remarkable separations have been achieved of molecular fractions from relatively simple commercial fats such as palm oil and cocoa butter. Isocratic elution is often possible so that a variety of detection and quantification procedures can be used. Clean fractions are obtained in semi-preparative applications. The equipment and running costs may be high, but labour costs can be lower.

Nonetheless, there are still some problems and the chief of these is the identification of components. Because of the dual nature of the factors involved in the separation, intuitive identification of peaks on the recorder trace is generally not possible. This is not an insuperable problem with relatively simple seed oils, but can lead to great difficulties with such complex triglycerides as those from fish oils or milk fat.

Silver Ion High-Performance Liquid Chromatography

There have been many attempts to adapt silver ion chromatography to HPLC and these at first met with limited success only. Not surprisingly perhaps, Unilever laboratories have been at the forefront of the development, and Hammond and co-workers have published a number of contributions on the topic. Their approach was to impregnate an HPLC grade of silica gel with silver nitrate and only then to pack it into columns (reviewed elsewhere [6]). Both the grade of silica gel and the method of impregnation were found to be critical. Isocratic and gradient elution procedures were developed with transport-flame ionization detection to obtain excellent resolution and direct quantification of confectionery fats, for example. The chromatograms are relatively easy to interpret, in contrast to those from reversed-phase HPLC. Unfortunately, applications to more highly unsaturated fats have not been published, and as by now must appear inevitable there is a major problem, i.e. silver ions elute continuously in the mobile phase. A pre-column of silver nitrate extends the life of the column and protects the resolution, but samples and the detector are contaminated.

The approach, which I was among the first to adopt, was to load a silica-based ion-exchange medium (chemically-bonded sulfonic acid groups) with silver ions [7]. Preparation of the column could not be simpler - it involves taking a standard pre-packed column with the appropriate stationary phase (Nucleosil™ 5SA) and introducing the silver ions via a Rheodyne™ injector while pumping water through the column. Finally, the aqueous phase is replaced with organic solvents. Probably only 50 mg to 80 mg of silver ions are bound to the stationary phase, but this is quite sufficient for very many useful separations. Silver ion columns are now manufactured commercially and have been widely used by lipid analysts (Chromspher Lipids^TM, Varian-Chrompack International, Middelburg, Netherlands).

For all of my work with this silver ion column, I have used evaporative light-scattering detectors from various manufacturers (discussed in more detail on this website here...). By introducing a stream-splitter between the end of the column and the detector, fractions can be collected for analysis by other means, for example, for determination of fatty acid compositions.

Some of the early experiments with a phase of the ion-exchange type linked to silver ions were disappointing, largely because methanol was incorporated into the mobile phase and a small proportion of residual free sulfonic acid moieties catalysed transesterification when lipids were on the column. However, if aprotic solvents are used, there are no problems of this kind. The simplest elution scheme, which I could devise, was a gradient of acetone into dichloroethane-dichloromethane, suitable only for fats with a relatively small proportion of linoleic acid, such as sheep adipose tissue or milk fat, from which we can separate not only the usual fractions with saturated and cis-monoenoic residues but also those with trans double bonds [8]. Subsequently, Richard Adlof described a mobile phase consisting of hexane containing a small proportion of acetonitrile, which is now being widely used [9]. It is my impression that excellent resolution is possible with both types of mobile phase, but that those based on chlorinated solvents permit higher loads in preparative applications.

Most samples of potential interest in the oils and fats industry contain a higher proportion of linoleic acid, but this can be accommodated with the ternary gradient system simply by introducing acetonitrile into acetone after the first fractions are eluted, as demonstrated with safflower oil here (Figure 1). There is excellent resolution of most fractions as can be seen. The retention time of one dienoic acyl residue appears to be equivalent to about 2.5 monoenes. One triene is exactly equal to two dienes, so there is some overlap of dienoic and trienoic fractions when alpha-linolenic acid is present in a sample. The same elution scheme was employed for palm oil and cocoa butter, and this type of analysis is perhaps well suited to confectionery fats where we get a separation of the important SSM species rapidly. Indeed the resolution is such that there is ample scope for speeding up the separation by using shorter columns, faster flow-rates or a steeper gradient, if this is advantageous for quality control say.

Fig. 1. Separation of safflower oil triacylglycerols by silver ion HPLC (Redrawn from [8] with permission). Abbreviations: S, saturated; M, cis-monoenes; D, dienes.

While species of the type SMS were not separated from SSM, i.e. according to the position of the acyl residue on the triglyceride molecule, by this solvent system, such separations were subsequently achieved with solvents based on hexane-acetonitrile [10].

In nearly all work of this kind, quantification has been accomplished by collecting fractions, adding an internal standard and transesterifying for analysis of the methyl ester derivatives by gas chromatography. After careful calibration with appropriate standards, there is no reason why the response of the mass detector should not be used directly.

The methodology has been applied to samples as unsaturated as linseed oil and fish oils with considerable success. Many more applications will no doubt be devised.

The next article in this series deals with the mechanism of silver ion chromatography.

References

1. Morris, L.J. Separation of lipids by silver ion chromatography. J. Lipid Res., 7, 717-732 (1966).
2. 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).
3. Dobson, G., Christie, W.W. and Nikolova-Damyanova, B. Silver ion chromatography of lipids and fatty acids. J. Chromatogr. B., 671, 197-222 (1995).
4. 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).
5. Nikolova-Damyanova, B. Reversed-phase high-performance liquid chromatography: general principles and application to the analysis of fatty acids and triacylglycerols. In Advances in Lipid Methodology - Four, pp. 193-251 (edited by W.W. Christie, Oily Press, Dundee) (1997).
6. Hammond, E.W. and Irwin, J.W. Determination of lipids. In: HPLC in Food Analysis, pp. 95-132 (edited by R. Macrae, Academic Press, London) (1988).
7. Christie, W.W. A stable silver-loaded column for the separation of lipids by high-performance liquid chromatography. J. High Res. Chromatogr. Chromatogr. Commun., 10, 148-150 (1987).
8. Christie, W.W. Separation of molecular species of triacylglycerols by HPLC with a silver ion column. J. Chromatogr., 454, 273-284 (1988).
9. Adlof, R.O., Copes, L.C. and Emken, E.A. Analysis of the monoenoic fatty acid distribution in hydrogenated vegetable oils by silver-ion high-performance liquid chromatography. J. Am. Oil Chem. Soc., 72, 571-574 (1995).
10. Adlof, R.O. Analysis of triacylglycerol positional isomers by silver ion HPLC. J. High Resolut. Chromatogr., 18, 105-107 (1995).

This article has been updated appreciably from one by the author that first appeared in Lipid Technology, 1, 50-52 (1989).

Updated: 28/6/2007 Credits/disclaimer	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland	Right click on the icon to open in a new window