Chromatography Forum

We are working developing an analytical RP-HPLC method for one protein.
Mobile phases are composed of 0.27%TFA in AcN and 0.3% TFA in water.

So far we did not have any problems. But this week we have seen that our protein became oxidized during the elution from the column. We have prepared fresh mobile phases and analyzed the same sample and it was all just fine, i.e. no oxidation.
Initially, we have thought the problem was that the first time we prepared the aqueous mobile phase we did not vacuum filter it and that our degasser was unable to remove the oxygen from the mobile phase.
But yesterday we have prepared the mobile phases again. And this time we filtered the aqueous mobile phase. We have analyzed the same sample again and the same problem with oxidation reappeared.
Up till now, i.e. for the last 3 months, we have been using purified water as defind in USP and Ph.Eur. except that the water has no specifications regarding nitrate and heavy metal contents. I was wandering whether it would be possible, as now the fertilizers are sprinkled heavily on the nearby fields, that the nitrogen compounds coming from these fertilizers causes the oxidation of our protein.

If this is not a possible explanation for our problem, please, do you have any idea what else might cause the oxidation?
Let me just say that the column is still the same and quite new (2 weeks old), TFA is from the same lot, and I think that also acetonitrile is of the same lot.
Thank you very much for your help.

How do you know that your protein oxidized? How do the fertilizer nitrates get into your HPLC water....do you use water straight from the tap? It is very difficult to remove traces of O2 from liquids, and it is especially difficult to be consistent. If I was sure that I had an oxidation problem I would go to a pH where this doeesn´t happen, if not posible, one can add a trace of antioxidant.

How do you know that your protein oxidized?

OK, I cannot be 100% sure, but the chromatograms looks exactly the same as the chromatograms of my protein when I exposed it to oxidation-inducing conditions, i.e. hydrogen peroxide (0.1 or 0.5% for 2h, 1% overnight). I also know, that my protein is quite susceptible to oxidation.

How do the fertilizer nitrates get into your HPLC water....do you use water straight from the tap?

We are using water purified by reverse osmosis, so water contamination by nitrates in principle should not occur. Unfortunately, this water is not checked for the presence of nitrates. During the weekend we have tried with MilliQ water, i.e. water that is initially purified by reverse osmosis passes also through MilliQ Gradient system and this water works just fine (so far).

I think you have gone a long way toward solving your problem by using a good water purifier. RO is not really good enough for HPLC applications, and should be used only as a first stage purification. Keep your Milli-Q system in good working order and you will prevent a great many mysterious troubles with your HPLC.

If you have nitrate or nitrite in your tap water, it is easily detectable with nitrate test strips; Merck for example makes them. If the level exceeds 40ppm, alert your health department. Actually, here in California, nitrate pollution is mostly caused by cattle farming rather than fertilizer.

Another possibility is iron or copper contamination; these are redox catalysts for air oxidation.

Mr. Tracy,

thank you very much for your answer. You reassured me a lot. Thank you also for explaining me what other chemicals besides nitrogen compounds may induce oxidation.