Column problems - help needed

[Tomaz]

I am analysing cytokinins with Waters Nova-Pak C18 columns and am having poor peak quality.

My system was not used by anyone for a while so I checked everything step by step. Finally it came down to the column and I changed it for a new one. Problem solved! Peaks were great for a few runs of standards, but again after a week (this one week the system was on hold) the resolution of my peaks got worse. Also, my prime injection is a complete chaos.

I am runnning a gradient where the polar phase is water, and the organic phase methanol/acetonitrile=50:50. I am also adding TEAA 1ml/liter solvent to have my peaks nice and sharp.

Any ideas??? Previously I was always storing the system in 95:5=water:organic which also stabilizes the column; and so was this time.

I ran out of ideas,
thanx for all your coments

[gbalock]

Tomaz,
Leaving your system with such low organic can be a breeding ground for bacteria. I woulld suggest that if you are going to leave your system for more than overnight you switch the systme to 100% acetonitrile. But, be careful switching back to you mobile phase if you have buffer salts dissolved in it as they will precipitate out.

[Sentenced]

I agree with gbalock, you should store your columns with a higher percentage of organic solvent (acetonitrile or methanol), to avoid bacterial or fungical growing. I preffer 50:50 AcN:H2O, because I use buffered mobile phases and it prevents an intracolumn precipitation.
Also, to recover your columns, you should follow a regeneration procedure, like this:

H2O (20 column volumes)
H2O:AcN 50:50 (20)
AcN (20)
Isopropanol (20)
Hexane (40)
Isopropanol (20)
AcN (20)
H2O:AcN (20)

Hope i helped you.

[Uwe Neue]

I also recommend to check the pH of your TEAA (since you did not mention it, it is probably not a buffer, but just a solution of the TEAA that you bought, and the pH could be alkaline).

[sksrinivas]

It's probably due to phase collapse. We have this problem all the time in our lab, since we use highly aqueous mobile phases with some of our C18 columns, and sometimes even 100% water.

Typically, peaks are more or less symmetrical to begin with, and tailing factors get progressively worse over time. Phase collapse can occur with virtually any reverse phase column, and it happened even with some "cross-linked" C18 columns that we tested.

You need to regenerate the column regularly. In our case, we regenerate the column every two days, since our systems run 24 x 7. We use Agilent 1200's and 1100's, so it's not difficult to program the pump to flush the column with 100% AcN for about 30 minutes, at regular intervals. This usually is enough to restore normal column performance.

Storing the column in water or buffer for more than a week is not a good idea, so avoid it if you can.

[Uwe Neue]

Dewetting (which is what Sksrinivas called "phase collapse") is rare in gradients, especially if your final composition reaches more than 50% organic. At over 50% organic, you drive the mobile phase back into the pores, even if dewetting has happened. If you run only limited gradients, say from 5% to 25% organic, dewetting can happen, but it is due to the initial mobile phase.
If dewetting does happen, a flush with 2 or 3 column volumes of organic solvent (e.g. acetonitrile) will do the rewetting. It is an instantaneous event. No need to wait.
Also, there was a publication that demonstrated that dewetting can be prevented by putting a stronger restriction behind the column.

[Tomaz]

First I would like to thank you for all your comments and suggestions.

I tried rewetting with acetonitrile and my results have improved, but at the same time I am starting to get tails and shoulders and that means column degradation...and just from leaving my column in the aqueous phase

[Kostas Petritis]

I think that the problem was not the storage of the column itself, phase collapse/dewetting or microbial growth but as Uwe indicated the pH of the mobile phase is probably alkaline which degrades the column especially during the storage period...

You may want either to buffer and/or reacidify before storage. Storage in higher organic is also a good idea...