Chromatography Forum

Hi,

Does anybody know if the appplication of Stainless Steel detrimental to HPLC analysis of biomolecules?

Therefore is the use of Titanium or PEEK tubing systems more compatible?

Thank You

Some people claim that stainless does all kinds iof things to proteins, I havn´t seen this. Now, one should also consider that the TSK Gel SW 3000 column, (see another current chain on that) is in a stainless tube, though mainly considered for proteins. This is true of a lot of other protein specialty columns. Now if I were to work with hemoglobin I would be careful, at least initially, until Fe++, Fe+++ is mopped up. Other members might have an example of iron ion complexing with their protein, or whatever? A documented example would interest me considerably, also.

Well, when it comes to "bio-compatibilty" issues, one of the more sensitive separations must be RP-IP chromatography of dsDNA. MP A: 0.1mol/L triethylammonium acetate (TEAA, pH=7), MP B: 0.1 mol/L TEAA, 25% acetonitrile.
As long as all stainless steel parts are in good shape and passivated there is no problem. Stainless steel frits (high surface area) are especially critical. Very bad are those older style stainless steel solvent inlet filters (10-20µm porosity) that hang inside the mobile phase.

Somewhere I have an old Hewlett Packard application note in which they determined metal concentrations in mobile phase on a HP1090 (must have been late 1980s). It was important to degass the MP. The amounts of metals found were significantly higher in the not degassed MP than in the degassed MP (makes sense to remove as much of the oxygen as possible in order to prevent corrosion)

best regards,

It depends a lot on exactly which molecules you care about. Some proteins, especially phosphoyrlated ones, and some antibodies are known to be highly sensitive to iron contamination. Phosphopeptides and phosphosugars can vanish entirely due to metal contamination. (This is an underappreciated problem with electrospray interfaces.) There are other proteins that simply ignore metal contamination.

Years ago, when I worked for Pickering Laboratories, iron contamination was killing their glyphosate columns. I had to develop a special cleaning solution. Switching to non-metallic frits prevented most of the problem.

Passivation and deoxygenation are good practices to minimize corrosion and contamination, but may not always be sufficient. One of our customers validated an important protein assay using stainless steel HPLC equipment. After years of use, his equipment is shedding iron at a furious rate and poisoning the columns.

Titanium is less susceptible to acid corrosion than stainless steel, but there are several alloys of titanium used for HPLC construction, and they are not equally inert. Phosphorylated compounds do stick to titanium dioxide under acidic conditions (there is even a TiO2 column sold for that purpose).

PEEK systems have the lowest metal leaching. Dionex would be happy if you bought some.

In some applications, for instance dsDNA, it is usual practice to include EDTA in the mobile phase to complex divalent ions that interfere. I have demonstrated that pyrophosphate can mask or remove iron contamination in phosphopeptide analysis. These additives, unfortunately, are bad news for LC/MS.