The Lipid Library

SILVER ION CHROMATOGRAPHY

Part 3.  Some Practical Separations

Summary:  There are two major types of application for silver ion high-performance liquid chromatography of lipids, i.e. for the separation of fatty acids and for molecular species of triacylglycerols. With the former, fatty acids can be separated into groups depending on the numbers of double bonds, or cis and trans isomers or positional isomers can be separated. Triacylglycerol fractions separated by silver ion chromatography complement those obtained by reversed-phase chromatography.

My previous article on the mechanism of high-performance liquid chromatography in the silver ion mode might be considered too academic for some tastes. Here, I describe some useful practical separations. To recapitulate briefly, silver-ion HPLC uses very little silver nitrate, which is linked by ionic bonds to the stationary phase and remains there during chromatography; clean fractions – no dyes or silver nitrate - are obtained in micro-preparative applications, with high resolution. The mobile phase can either be chlorinated solvents, such as dichloromethane-dichloroethane mixtures, or hexane with acetonitrile as a modifier. Hexane (or isohexane)-based mobile phases are obviously less toxic, but my impression is that greater sample loads and higher resolution is possible with chlorinated solvents. On the other hand, Richard Adlof (personal communication) believes that the columns may last longer if hexane-containing solvents form the basis of the separation.

Two main types of application to lipids of silver ion HPLC appear to have been established, i.e. to fatty acid derivatives and intact triacylglycerols, although the technique would seem to be applicable to a wide range of other aliphatic and alicyclic compounds, including phospholipids (and derivatives thereof), sterols, terpenoids, insect pheromones, etc.

Fatty Acid Analysis

With fatty acids per se, our main applications are to separation of cis/trans or positional isomers, or to the simplification of complex mixtures for analysis by GC-MS. The latter technique is discussed briefly in the mass spectrometry section of this website. Some natural samples have so many different fatty acid components, over a hundred in some sponges, for example, that even the best modern GC columns have insufficient resolution. The problem is compounded if several different derivatization techniques must be used for identification purposes. Then, analysis is made much easier if fractions are separated according to degree of unsaturation prior to derivatization and GC-MS.

It now seems to have been generally accepted that silver ion chromatography must be an essential component of any definitive method for complete and accurate analysis of trans fatty acids, especially monoenes. Many years ago in a paper that is now largely forgotten, I described a method for trans analysis involving separation of a saturated plus trans band (methyl ester derivatives) by silver ion TLC combined with GC analysis of the sample before and following the separation; the saturated fatty acid components of the sample served as an internal standard [1]. Later, my collaborators and I updated this to silver ion HPLC [2]. By using a short silver ion column, we could get complete separation of saturated, and trans- and cis-monoenoic derivatives in less than 10 minutes. The analysis was completed by collecting fractions for quantification by GC. By using modern highly polar phases in capillary columns, comprehensive analyses of geometrical and positional isomers are possible.

I believe silver-ion HPLC could be used much more by biochemists. For example, base-line separation of three natural isomers of octadecenoic acid, i.e. 6-, 9- and 11-18:1, is easily accomplished, as illustrated in Figure 1 [3]. No other procedure could hope to match this. In particular, studies of the biosynthesis of these or other isomeric fatty acids with isotopically labelled precursors could be facilitated by the use of this technique. It should be noted that methyl esters are not always best for the purpose, and phenacyl or p-methoxyphenacyl esters can offer much better resolution in many applications.

Figure 1. Silver-ion HPLC separation of p-methoxyphenacyl esters of cis-11-, 9- and 6-18:1 fatty acids. A Nucleosil 5SA™ column in the silver ion form was utilized with a mobile phase of dichloromethane-1,2-dichloroethane-acetonitrile (50:50:0.025 by volume) at a flow-rate of 1.5 mL/min [3] (redrawn with permission of the journal).

In recent years, the technique has been widely used to isolate, identify and quantify isomers of conjugated linoleic acid (CLA), either as free acids, methyl esters or phenacyl esters [4]. Similarly, the technique has proved invaluable for the isolation of isomers of the complex mixture of cyclic fatty acids formed as artefacts in heated frying oils for structure determination [5].

Triacylglycerol Analysis

In silver-ion HPLC of triacylglycerols, the degree of unsaturation of all three fatty acids in the molecule is important in the separation. The simplest elution scheme I could devise was a gradient of acetone into dichloroethane-dichloromethane, but this is suitable only for fats with a relatively small proportion of linoleic acid, such as ruminant adipose tissue or milk fat. The trisaturated species are eluted first followed by disaturated-monoenoic, saturated-dimonoenoic and so forth. Indeed, it was possible to separate not only the usual fractions with saturated and cis-monoenoic residues but also those with trans double bonds. The procedure may therefore have some potential for the analysis of partially hydrogenated fats such as those in margarines.

Most samples of potential interest contain a higher proportion of linoleic acid, and this was accommodated with a ternary gradient system simply by introducing acetonitrile into acetone after the first fractions eluted, as illustrated for palm oil in Figure 2 [6]. The retention time of one dienoic acyl residue is equivalent to about 2.5 monoenes. One triene (18:3(n-3)) is exactly equal to two dienes (18:2(n-6)), so there is some overlap of dienoic and trienoic fractions when alpha-linolenic acid is present in a sample. On the other hand, molecular species of triacylglycerols containing gamma-linolenic acid, as in evening primrose oil, are retained a little less strongly and the separation is quite distinctive.

Figure 2. Silver-ion HPLC separation of palm oil triacylglycerols. Column as above, and a ternary gradient elution scheme [6] (redrawn with permission of the journal). Abbreviations: S, saturated; M, monoenoic; D, dienoic; T, trienoic fatty acyl residues.

The order of elution of the triacylglycerol species with these systems is thus easy to understand, in contrast to HPLC in the reversed-phase mode. A similar 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 rapid separation of the important disaturated-monoenoic species. 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. By extending the gradient, we can get acceptable resolution of fish oils even.

Nowadays, most analysts prefer to use mobile phases based on heptane or hexane and acetonitrile because of their reduced toxicity [7]. Applications of mass spectrometry with atmospheric-pressure chemical ionization [8] or electrospray ionization [9] to the detection and identification of molecular species of triacylglycerols separated by silver ion HPLC are also worthy of note.

Reversed-Phase HPLC Complements These Separations

The alternative to silver ion chromatography in many applications is likely to be reversed-phase HPLC. With the latter technique, separation is both by chain-length and degree of unsaturation of fatty acyl-chains, each double bond reducing the retention time by the equivalent of about two methylene groups. I look on these as complementary techniques rather than as competitors. Reversed-phase HPLC is a robust technique that affords excellent resolution in many applications with isocratic elution or simple gradients, although positional isomers of polyenes especially tend to be less well-resolved than with silver-ion HPLC. The latter requires much more complex gradients, and does not give separation by chain-length. When the two techniques are used in tandem, much more information can be obtained about samples, and it is often easier to isolate single components in a high degree of purity in micro-preparative applications.

References

1. Christie, W.W. and Moore, J.H. Structures of triglycerides isolated from various sheep tissues. J. Sci. Food Agric., 22, 120-124 (1971).
2. Toschi, T.G., Capella, P., Holt, C. and Christie, W.W. A comparison of silver ion HPLC plus GC with Fourier-transform IR spectroscopy for the determination of trans double bonds in unsaturated fatty acids. J. Sci. Food Agric., 61, 261-266 (1993).
3. Nikolova-Damyanova, B., Christie, W.W. and Herslöf, B.G. Mechanistic aspects of fatty acid retention in silver ion chromatography. J. Chromatogr. A, 749, 47-54 (1996).
4. Adlof, R.O. Application of silver ion chromatography to the separation of conjugated linoleic acid isomers. In: Advances in Conjugated Linoleic Acid Research, Volume 2., pp. 37-55 (ed. J.L. Sebedio, W.W. Christie and R.O. Adlof, AOCS Press, Champaign, IL.) (2003).
5. Dobson, G., Christie, W.W., Brechany, E.Y., Sebedio, J.L. and Le Quéré, J.-L. Silver ion chromatography and gas chromatography-mass spectrometry in the structural analysis of cyclic dienoic fatty acids formed in frying oils. Chem. Phys. Lipids, 75, 171-182 (1995).
6. Christie, W.W. Separation of molecular species of triacylglycerols by HPLC with a silver ion column. J. Chromatogr. A, 454, 273-284 (1988).
7. Elfman-Borjesson, I., van den Hark, S. and Harrod, M. Gradients of n-heptane and acetonitrile in silver-ion high-performance liquid chromatography analyses of cis and trans bonds in lipids. J. Am. Oil Chem. Soc., 74, 1177-1180 (1997).
8. Neff, W.E. and Byrdwell, W.C. Triacylglycerol analysis by high-performance liquid chromatography atmospheric-pressure chemical-ionization mass spectrometry - Crepis alpina and Vernonia galamensis seed oils. J. Liqu. Chromatogr., 18, 4165-4181 (1995).
9. Schuyl, P.J.W., de Joode, T., Vasconcellos, M.A. and Duchateau, G.S.M.J.E. Silver-phase high-performance liquid chromatography-electrospray-mass spectrometry of triacylglycerols. J. Chromatogr. A, 810, 53-61 (1998).

This article has been updated appreciably from one by the author that first appeared in Lipid Technology, 10, 113-115 (1998).

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