We use the Agilent's ZORBAX Eclipse Plus C18 RRHD 2.1×50 mm 1.8u columns, now for several years.
I use K-phosphate buffer as A and methanol as B.
This particular column was taken new recently, I've run few analysis*, then washed with water and methanol thoroughly and stored. I think in the meantime nobody else used this one column. Last week I took it out and noticed the pressure with 0.05ml/min (our no-analysis mode) was about 30 MPa (usually it's something like 8-12MPa). During the analyses the pressure rose from 45 to 60MPa, but the samples were supposed to be clean (enzymes reactions I run always with purified enzyme). The peaks looked good, just the few first analyses had high background in UV (rise of baseline). Next I've run other samples, the pressure went slowly down to 55MPa and peaks still looked well (tailing f. 1.2 and width 0.08), the pressure after analyses was 20 MPa. During today's runs, the pressure was at 45MPa (since the beginning) and now is at 10 MPa, but the peaks were very broad in the beginning (0.115) with tailing f. 1.3, so almost normal. However, during the analyses the width went to 0.09 again.
I'm almost scared to run it next time, because the pressure will be probably zero.
What do you think the problem could be? Is the column bad? Or could I have done something wrong?
*I just checked and the samples were not even for me, so I cannot really check how it looked, but probably nothing suspicious.
Hi Tomás,
this is a very weird story. In the beginning I thought it could be a precipate in the column creating the huge backpressure (maybe the phosphate salt precipitating with too much MeOH). My suggestion would be then to alwas use pre-columns/exchangable pre-filters attached to the column or extensively wash with water to get all salts out of the column. But that's what you did actually.
Due to the fact that the uv-signal had a high background with time and the pressure is dropping dramatically even during analysis, I think it may be the stationary phase saying "bye bye" to the column, so released to the outlet and traveling through the DAD. It is not possible because the companies put a filter in the column outlet so that not staionary phase can ever leave. It is actually possible if the column is attached in the wrong direction. We had the problem once when cleaning a column backwards and the pressure went up too high so that the silica from the column blocked the DAD inlet, *great*. Your particles are so small so that they may leave to the waste without blocking anything. I do not want to offend you with the "wrong direction mounting" suggestion, but this came into my mind. You could attach the column to the system with collecting the outlet liquid into a small beaker. If the column material is coming out, you'll see it right away/white precipitate.
Did you checked the pH of your buffer? It may be too high destroying the silica gel/hydrolyzing it.
You could also ask Agilent for the properties of the column/batch number and if costumers had identical issues with that LOT#. Especially if it is possible that the company did a mistake and put the mounting direction sticker in the wrong direction...
Hi,
I'm sorry, I forgot to mention. When I saw the huge pressure, I changed the in-line filter, but I saw the pressure drop immediately after disconecting the column (with the in-line filter still inline), so that obviously that was not the problem.
And it is not recommended to back-flush this column.
I'll check the direction of the column and to be sure* also the pH of the buffer.
*but I don't think there was any pressure drop after disconnecting the detector
Did you tried to replace the column with a new one from the same type? How is the pressure then? Maybe you had bad luck with the column you use right now and there is something stuck in there which was released and now the packaging of the column is destroyed. Most probably the column is bad and I guess you could get full refund from Agilent if you explain the issue to the service people.
If they do not give refund, I would still try to backflush the column with very low flow rates keeping the pressure below 10 bar and leave over night with cleaning solvent (is there a cleaning procedure you could try?). Maybe this removes something from the column inlet and could recover it. Otherwise I don't know how to fix the problem. I mean, if the peak shape and separation is still fine and corresponds to the elugrams you had before, why not keep using it?
It seems like there is something in the sample you are injecting that is clogging the column in the long term. Have you tried a longer post-injection wash step to clean it out?
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