Silver ion columns for HPLC, i.e. ChromSpher LipidsTM columns (4.6 mm i.d. × 250 mm stainless steel; 5 μm particle size; silver-ion impregnated), can be purchased from Varian-Chrompack International (Middelburg, Netherlands) and used just as they are received. Most analysts find them perfectly satisfactory. So why make your own? One obvious reason is cost – homemade columns can cost only a third as much as commercial columns (which are probably manufactured by a similar method) – remember that Scots have a reputation for thrift. This is not a false economy, as it appears that homemade columns are as efficient as those available commercially.
In our experiments, we use pre-packed columns with phenylsulfonic acid groups bonded chemically to silica, i.e. Nucleosil 5SATM. Such strong cation-exchange materials can be converted very readily to the silver ion form.
Note! Not all manufacturers use the same nomenclature to designate their columns. Thus, Spherisorb SATM, for example, is an anion-exchange material and is not suitable for our purpose.
The procedure for preparing the columns has been published, and the practical details follow . In essence, an aqueous solution of silver nitrate is introduced to the column via multiple injections through the Rheodyne valve of the HPLC system, followed by organic solvents to remove the bound water. The procedure is simple and takes about 4-5 hours to complete.
Protocol: A pre-packed column (4.6 × 250 mm) of Nucleosil 5SATM (or equivalent) is connected to an HPLC pump fitted with a 100 μL loop on the injection valve; the outlet is fed into a reservoir for disposal. The column is flushed with 1% aqueous ammonium acetate solution at a flow rate of 0.5 mL/min for 1 hour, then with distilled water at 1 mL/min for 1 hour. While water is pumped through the column, silver nitrate (0.2 g) in water (1 mL) is injected via the Rheodyne valve in 50 μL aliquots at 1-minute intervals. 20 Minutes after the last injection, the column is washed by elution with methanol for 1 hour (1 mL/min), then with dichloromethane-1,2-dichloroethane (1:1, v/v) for 1 hour, and finally with hexane for 1 hour. The column is then ready for use.
Note: I have recently learnt that Nucleosil 5SATM can be very variable from batch to batch, making it less suitable for silver ion chromatography ! When ordering from the manufacturer (Macherey-Nagel), it is advisable to specify the purpose and to request columns packed with stationary phases of a higher carbon content. This particular commercial product was selected because at the time it was the only 5-micron packing of this type that was available. Alternatives can surely be purchased now that have smaller particle sizes, higher capacity or different chemistry and might be worth evaluating for silver ion HPLC (another reason for making your own columns).
Care of columns: At the end of each working day, the column should be flushed out with hexane for storage. Silver ions can be converted irreversibly to silver oxide by hydroperoxides in solvents, so column life can be prolonged by using freshly distilled solvents whenever possible.
- Christie, W.W. A stable silver-loaded column for the separation of lipids by high-performance liquid chromatography. J. High Resolut. Chromatogr., Chromatogr. Commun., 10, 148-150 (1987) (DOI: 10.1002/jhrc.1240100309).
- Nikolova-Damyanova, B. Retention of lipids in silver ion high-performance liquid chromatography: Facts and assumptions. J. Chromatogr. A, 1216, 1815–1824 (2009) (DOI: 10.1016/j.chroma.2008.10.097).