I am looking to do some comparative testing on a inert HPLC system versus a metalic system. Does anyone know of a good sample to test with? A protein or peptide that has a high affinity to stainless steel.

Thanks!

Though there is not a great experience with proteins, here, there is some doubt creeping in on a practical role for affinity to metals. Different proteins have a different affinity for all kinds of different things. One generally searches for mobile phase modifiers which give an acceptable chromatography.
If anybody knows a reference on a systematic study I would also greatly appreciate to be informed.
Complexation of metal ions with proteins is a different matter, but there also seems to be a lack of believable systematic studies on the importance of this.

The problem with using a stainless-steel instrument and doing protein chromatography is much more basic. You don't really need to demonstrate the effect of metal on protein chromatography. The real issue is that you can't safely use chloride containing eluents without a great deal of precautionary effort on a stainless-steel instrument. We have several columns which make use of chloride containing eluents and we often see columns returned after use on stainless steel instruments because the "column went bad". Invariably, we find a huge amount of iron deposited on the top of the column as the cause of column failure with symptoms including: loss of capacity, loss of the efficiency and high pressure. You can generally remove a lot of the deposited iron oxide which has contaminated the column with oxalic acid but you can rarely conclude the restore performance of a column which has been bestowed with a healthy load of iron oxide.

Chris, that sounds very interesting. What concentrations of chloride and what pH where involved? Are the "iron oxide" deposits on the column black? Is it really iron oxide?
An example of our experience: A 0.15M solution of Cl- at a ph=7.2 (part of PBS) does not change the appearance of stainless steel for several month, while conc. HCl (6M or 12M...donīt remember) immediatly blackens stainless.
There were some articles in LC.GC on this, but I donīt remember details....have to check.
Hans

Hans,

Actually, typically the iron oxide deposits are "rust brown". As I'm sure you know, iron oxide has several different characteristic colors depending upon the form but the most commonly occurring corrosion byproduct which accumulates on columns is a brown color. We know that this is iron oxide from several different characterizations but the most simple and direct is to treat the deposit with oxalic acid. Oxalic acid is fairly efficient at dissolving iron oxide but the process is relatively slow. By soaking a contaminated column inlet frit in 0.2 molar oxalic acid overnight, you can typically completely solublize the iron oxide. If the brown contamination is some sort of organic material derived from samples injected onto the column, treatment with oxalic acid will have no effect. We recently received columns from a customer using up to 0.1 molar sodium chloride eluent under mildly alkaline conditions and they were reporting loss in capacity and high pressure. Because the columns has been operated on a stainless-steel HPLC system, we immediately suspected iron oxide contamination as a source and indeed we demonstrated partial restoration of operating pressure and increase in retention time after treatment with oxalic acid. However, even after treatment over the weekend we were not able to fully restore the column capacity or return column pressure to normal. Presumably this is because iron is not the only metal in stainless steel and oxalic acid is not as efficient at removing other oxides.

Chris

Strange, 0.1molar, alkaline? They must have had stainful steel in their system!?
Normally, Fe+++ oxides are read, the Fe++ counterparts are green, but the only certain remembrance is that the Fe oxide chemistry is very complicated. The black in ancient greek vases is also an iron oxide... any inorganic chemists around? Why does HCl not dissolve the oxides, why does it turn stainless black?
Is there anybody out there who has used PBS and other Cl- solutions extensively without any problms (like we have)?