Can one sonicate an entire column?

[maisymouser]

Hi, this is my first time using Chromforum and I'm very grateful for such a community of chromatographers. I'm going to preface by saying that I'm not well-versed in the use of LC but am learning a lot during my graduate studies as a biological student!

I got a bit of protein stuck on my Agilent SB-C18 column. I have found in the past that flushing with 6M urea takes care of the problem really well, but this time when I did it, the method stopped while I was out of the lab. I'm a bit worried that there may be caked urea on the inside of the column. I stored the entire column last night in water (the LC was not operational and I couldn't immediately try to flush with water). Is it possible to sonicate the entire column while in water? Or should I remove the frit and just sonicate that? Or can I do them both while disconnected?

Thanks for your input!

[tom jupille]

Are you seeing any symptoms or problems? If not, a good policy to follow if "if it ain't broke, don't fix it". Run a test sample and see how the column is performing before you panic (if it has a problem, *then* you can panic!)

As far as sonicating the entire column, I don't think it will hurt the column, but I doubt that it will help anything either.

As far as removing the frit, DON'T DO IT except as a very last resort on an otherwise dead column; it has a very high chance of damaging the column bed. The only situation in which I would consider it is if all of these conditions apply:
- the column back pressure is unusably high or all peaks show the same kind of shape problem, and
- I have already back-flushed the column without success, and
- I do not have a replacement column available, and
- I have samples that must be run before a replacement column can arrive.
Even then, I would replace the frit rather than trying to sonicate.

[M Farooq]

Columns should never be sonicated, because the junk is not going anywhere. Imagine sonicating 3g of tightly packed of silica in a 2-3 mL water. Nothing will happen. The junk is going to stay right there. The typical volume of mobile phase inside the column is around 2-3 mL. The beds are so tightly packed that if you try to extrude packed silica, you will see nothing but rock-hard cylindrical pieces of the bed. Secondly silica columns should never be stored in water because silica likes to dissolve in water, albeit slowly.

Here is a column care manual and follow their instructions for cleaning, it is not a big deal to clean columns using a range of solvents. Simply follow the instructions in the Column Care section.
https://www.chem.agilent.com/Library/da ... 4-002f.pdf

[lmh]

Tom's right. Try putting some flow through the column and see what happens to back-pressure. If it is vaguely normal, try running a few test standards, and only panic if the results are consistently rubbish.

If the back-pressure is too high, yes, by all means follow Agilent's wash procedure, but if you've put a really strong solution of something through the column you might want to try a nice slow flow of nearly 100% water first, and gradually build up the flow rate as the back-pressure (hopefully!) comes down.

But the bottom line is that if you've killed the column, really really don't worry. Based in the UK I note that the columns I use generally cost somewhere between £400 and £1000, so if I've got 500 injections out of a column, the worst-case scenario is that killing it has cost me £2 per injection (and most columns do a lot more than 500). This is probably less than I spent on plastic-ware and vials during the preparation of the sample, and it's certainly irrelevant compared to my time. I also notice that some non-specialist groups buy a column, and it becomes a treasured lab asset, passed lovingly from Father to Son, from Mother to Daughter, and there is an expectation that it will carry on for ever. Columns are consumables. They deteriorate. Sometimes accidentally destroying one can be a good thing, if it's actually so old that resolution has already slumped.

[maisymouser]

Thanks all. I think I was getting too panicky, a simple wash with water did the trick. I wonder if I had left the column out overnight with 6M urea, if it would have been more difficult to flush with water. At any rate, all is well! Thanks for the quick replies. I'll be sure to visit ChromForum again soon.