SILVER ION CHROMATOGRAPHY
Part 2. The Mechanism
Summary: The increasingly widespread use of high-performance liquid chromatography in the silver ion mode has enabled the mechanism of the technique to be studied in much greater detail than has been possible hitherto. It is now apparent that one silver ion can interact with two unsaturated sites in an aliphatic chain simultaneously.
Silver Ion HPLC versus TLC
In the first article in this series the principles of silver ion chromatography are discussed, and the relative merits of using the technique in conjunction with thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) are described. References to major review articles are listed.
While TLC gives acceptable results for most purpose and is simple to use, it is messy. For example, when the technique is used for micro-preparative purposes, silica gel and silver nitrate contaminate fractions and have to be removed by additional washing steps. Purple stains appear on benches, floors and fingers! More importantly, if we wish to learn more of the mechanism of the technique, TLC in the silver ion mode is influenced by such factors as temperature, humidity, the concentration of silver ions in the silica gel, storage conditions, and the time and temperature of activation prior to use. Some of these factors are not at all easy to control. In addition, we know nothing of the physical arrangement of the silver ions within the silica gel. Is it crystalline or amorphous, for example?
In contrast, silver ion HPLC uses very little silver nitrate, which is linked by ionic bonds to the stationary phase, and remains there during chromatography so that clean fractions are obtained. More importantly, much better resolution is possible, and because so many of the chromatographic parameters are fixed, analytes have reproducible retention characteristics under given sets of conditions. Again, the technique for preparing such columns was described in the first article. Two types of mobile phase have been described – those using chlorinated solvents and those with based upon hexane-acetonitrile. Both are capable of giving excellent separations under practical conditions, but in mechanistic studies the former are best. For example, there may be some residual silanol groups on the surface of the silica matrix, but these should not influence separations greatly when relatively polar chlorinated solvents are used in the mobile phase.
In silver ion HPLC, we have a reasonable understanding of how the silver ions are bound to the stationary phase via the phenylsulfonic acid groups. We can control both the composition and flow rate of the mobile phase with a high degree of accuracy. Finally, we can control the temperature of the column, an important factor in the complexation reaction between silver ions and double bonds. Accurate chromatographic retention data can thus be obtained for a variety of lipid analytes of known structure.
Mechanisms
Silver ion HPLC has helped us greatly to unravel the mechanism of silver ion chromatography, and this is not merely an academic exercise. It is important in that it may lead us to further improvements to the methodology or in suggesting new applications. It is well known that silver ion chromatography is based on the distinctive property of unsaturated organic compounds to complex with transition metals in general and silver in particular. The complexes are of the charge-transfer type, so the unsaturated compound acts as an electron donor and the silver ion as an electron acceptor. To be pedantic, the preferred model assumes formation of a sigma type bond between the occupied 2p orbitals of an olefinic double bond and the free 5s and 5p orbitals of the silver ion, and a (probably weaker) pi acceptor backbond between the occupied 4d orbitals of the silver ion and the free antibonding 2p pi* orbitals of the olefinic bond.
When we did a literature search that took us outside the normal realms of lipid chemistry, we found that silver ions had been shown to form stable complexes with two molecules of ethylene, Ag+(C2H4)2. X-Ray studies of crystalline complexes of silver ion with some diolefins had shown again that coordination of one silver ion with two double bonds could occur simultaneously. This helped us enormously in interpreting our own experimental data.
Computational probes involving theoretical calculations of the molecular orbital interactions between silver ions and double bonds or various other ligands in fatty acyl chains have now shown that the stability of such complexes depends on the positions of C=C double bonds in molecules, while a significant part of the complexation energy is contributed by the interaction of Ag(I) and alkoxy-(or aryloxy-) carbonyl groups of fatty acid esters [1].
Interaction with Fatty Acids
It was our experiments with silver ion HPLC and unsaturated fatty acid esters of various kinds that first lead us to believe that if silver ions interact with two double bonds at the same time, they could also react in a similar manner with one double bond and the unpaired electrons on the carboxyl moiety as shown schematically in Figure 1.
Figure 1. Schematic representation of the interaction of a silver ion with the phenacyl ester derivative of petroselinic acid.
This explains how different positional isomers of fatty acids can be separated by this technique. For example, electron-rich esters, such as the phenacyl derivatives illustrated, are held much more strongly than are methyl esters when the double bond is within about 8 carbons of the carboxyl group, and the elution patterns of series of isomers are very different [2]. From 9-18:1 onwards, when the possibility of such a simultaneous interaction would seem to be less likely, there is no significant difference between methyl and phenacyl esters. Experiments with esters with a variety of different electron-donating and electron-withdrawing substituents provided firm evidence for this hypothesis [3].
Such experiments have also allowed us to develop greatly improved silver ion TLC procedures. Nearly 30 years ago, Lindsay Morris and colleagues were able to separate methyl esters of 6-, 9- and 11-18:1 by using TLC with a very high proportion of silver nitrate at −20°C. Now we know that this can be accomplished with phenacyl esters at room temperature on TLC plates with low silver nitrate levels [4].
An interaction between one silver ion and two double bonds at the same time also explains the chromatographic behaviour of fatty derivatives with two double bonds in the acyl chain on silver ion HPLC. When the distance between the double bonds is optimum, i.e. with a 1,5-cis,cis-diene system, fatty acids are very strongly retained, and the effect diminishes as the number of methylene groups between the double of bonds is varied. If the double bonds interacted singly with silver ions, it might have been anticipated that the kinetics of the system would be such that retention would be comparable in magnitude to the sum of the individual parts, but this is clearly not so. The computational study [1] subsequently gave considerable support to these suggestions.
This theory of complexation between silver ions and bis-double bond systems could potentially be applied to polyenoic fatty acid derivatives. It would predict that a triene would be held twice as strongly as a diene, a tetraene three times as strongly and so forth. Such a simple relationship is not in fact found (the degree of the complex formation is even greater than anticipated), probably because interactions with the ester moiety have to be taken into consideration and because the conformations of polyenes may permit some interactions between silver ions and double bonds that are remote from each other, via the formation of pseudo-cyclic structures.
Interaction with Triacylglycerols
Analogous physicochemical studies of the behaviour of triacylglycerols on silver ion chromatography suggest that a dual interaction is important in this instance also. For example, highly unsaturated triacylglycerols are retained especially strongly; a species with nine double bonds is held 10,000 times as strongly as one with only a single double bond [5]. It is the strength of this interaction rather than the efficiency of the column per se that is responsible for the quality of the separations.
Many analysts switch off mentally when chromatographic theory is mentioned. However, it is not simply an esoteric subject to be left to academics. If it helps us to better practical results, we should welcome it.
The third article in this series deals with some practical separations.
References
- Damyanova, B., Momtchilova, S., Bakalova, S., Zuilhof, H., Christie, W.W. and Kaneti, J. Computational probes into the conceptual basis of silver ion chromatography: I. Silver(I) ion complexes of unsaturated fatty acids and esters. J. Mol. Struct. - THEOCHEM., 589-590, 239-249 (2002).
- Nikolova-Damyanova, B., Herslöf, B.G. and Christie, W.W. Silver ion high-performance liquid chromatography of derivatives of isomeric fatty acids. J. Chromatogr., 609, 133-140 (1992).
- 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).
- Nikolova-Damyanova, B., Christie, W.W. and Herslöf, B.G. Improved separation of some positional isomers of monounsaturated fatty acids, as their phenacyl derivatives, by silver-ion thin-layer chromatography. J. Planar Chromatogr., 7, 382-385 (1994).
- Nikolova-Damyanova, B., Christie, W.W. and Herslöf, B.G. Retention properties of triacylglycerols on silver ion HPLC. J. Chromatogr. A, 694, 375-380 (1995).
This article has been updated appreciably from one by the author that first appeared in Lipid Technology, 10, 17-19 (1998).
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Updated: 26/6/2007 |
Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland
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