Chromatography Forum

Dear,

I am using the Acquity C18 column 1.7 µm, 2.1*50 mm from Waters 2 years succesfully on our LC-MS/MS system. Normally, after 800-1000 injections (depending on the cleanliness of the samples, which is not evident since I work in a forensic lab), I have to replace the column because of bad peak shape, increased pressure etc.

I recently replaced an old column, and did 10 injection succesfully on the new column. Then, the pressure increased. The mobile phase I use is 10 mM ammonium bicarbonate at pH 9 and methanol, flow rate 0.5 ml/min, oven is at 40°C. The method is fully described in two of my papers: Verplaetse R and Tytgat J, For Sci Int (epub ahead of print) and J Chrom B 2010, 878, 1987-1996.

I flushed the column already with water, methanol, acetonitrile and isopropanol without improvement.
We have two similar LC-MS/MS- systems. On both systems the problem is seen and another new unused new column has normal pressure. This excludes a problem with the hardware or mobile phase and point directly to the colmun. I have no idea what the problem could be, but considering the price of these columns, I hope I can still solve the problem, since it was only used 10 times...

Any suggestions...?

You might invest in a guard column or a pre-column filter. They are cheaper to replace.

You might try backflushing the column with various mobile phases. One method I read about starts off with high aqueous (acidic or not) then goes through methanol, acn, isopropanol, hexane, isopropanol, ACN, methanol/Aq.
other methods include DMSO.

Backflushing using higher conc. acetic or formic acid in the aqueous might help.

You could also try changing the frit on the column if all else fails.

I already backflushed the column with water, methanol, acetonitrile and isopropanol. I will try hexane and higher buffer concentrations. Normally I use 10 - 25 mM, which concentrations would you suggest?

I didn't mention it, but I use a precolumnfilter! The increased pressure is however caused by the column, as the precolumnfilter works fine on another column and has no increased pressure on its own!

Thanks for your help!

Dear,

I tried to further flush the column without any improvement.

Because of urgent analyses, I started with a new column. AFTER 18 INJECTIONS PRESSURE WAS AGAIN INCREASED ABOVE THE MAXIMUM OF OUR SYSTEM (which is 660 bar)!!!

I have no idea what the problem could be. Hopefully some of you forummembers do!

"INCREASED ABOVE THE MAXIMUM OF OUR SYSTEM (which is 660 bar)"

This information is essential. I have made a quick calculation about backpressure generated by your system. Using MeOH and an Acquity column 50x2.1mm, 1.7um at 40°C, the pressure generated by the column alone at 45% MeOH (maximal pressure) is about 630 bars. The system itself will generate a few bars as well, which brings you very close to the pressure limit, maybe slightly higher. Have a try with flow rate at 400 ul/min and tell us what happens.

Dear,

Thanks for your suggestion, but I have been using this method for 2 years succesfully, so normally the backpressure is fine! The method is also validated at a flow rate of 0.5 ml/min, so it will be hard to change the flow rate...

I believe there is something coming off the system (the tubing the pump...?) which causes a blockage only seen at the very high pressures... Has anaybody any experience what this could be?

Thanks!

I have experience with 6 Acquity systems in 2 different countries. Instruments are extremely robust as you probably experienced yourself last 2 years you mentioned.
To me it sounds like you have mechanical contamination from debris or from polymer. It may not be possible to remove it at all. Waters normally covers columns by insurance, check replacement conditions with them.
Use col. prefilters and keep an eye on filter brand for solvents, they should be strictly correct as you may end up with plastic in your mob. ph. Use good brands of filters. It is best to use LCMS grade solvents, LCMS grade buffers, LCMS grade acids. Question your water, vials, vial septum. Change mob ph at least 2 times/wk. Develop protocol for washing columns after batch and strictly follow it. We developed proprietary protocol supported by full scan LCMS examination of wash. For our chemistry it was ~2 hrs. Some of our samples are crude extracts, and we manage to do up to 5,000 injection from 100x2.1column. Glassware should be UPLC dedicated, read elsewhere how to treat and maintain glassware for MS and that should be strictly dedicated ONLY to UPLC work. Protect from dust.

I like the phrase that was written on a box from some company “Chemistry is good quality”. That is pretty much correct. Alternatively do the risk assessment, if all of above is more expensive than 1 lost column/year (~$600-800), then don’t bother.

Have you eliminated the samples as the cause of the problem ? - early on you mentioned that they might not be very clean.

Peter

I managed to get rid of the increased pressure by backflushing the column for several hours...

I will now use your suggestions: I willl replace all mobile phase and the bottles. Then I will run the gradient from the method, 25 times times but without injections. If this works, the system&mobile phase should be ok. Then I will inject some samples again 25 times to make sure the problem is not situated in the sample/vial/injector.

Hope I can find the cause this way, thank you very much for all your suggestiosn!

You asked me what concentration acid to backflush with.
You can check literature on column cleaning for various suggestions.

4 or 5% acetic acid wouldn't hurt your column at all, but it would need a good rinse afterwards.

That your column has the problem but not the precolumn filter would suggest that whatever it is, it is getting past the precolumn filter. What is the "pore" size on it? Is it possible you have some form of organism growing in your mobilephases?

Alp