Contamination on UPLC

[BMol]

Hi, I'm having trouble getting clean blank injections on Waters H-Class UPLC systems. I'm running a gradient method using 20mM ammonium acetate as mobile A and ACN as mobile B. I've previously used these methods on these same systems without any issue. We have had trouble in the lab before with contaminated water from our Elga purification system but I'm currently using bottled water. I've done the test for enrichment by running a number of blank injections with increasing equilibration times and there does seem to be an increase in the contaminant peaks with longer equilibration. As a result of that I've changed mobile phase a number of times, including another batch of bottled HPLC grade water, freshly opened ammonium acetate and new ACN. I'm rinsing all glassware with organic solvent (ACN) then water prior to making up the mobile phase and I'm using ACN directly from the bottle. I've changed the column and changed system but the problem seems to be following me! I'd be grateful for any ideas!!

[shaun78]

You likely have contaminent built up on the head of your column. Change the column and I bet the problem goes away.

[BMol]

I've changed the column and no difference. I've flushed both columns that we're using with ACN as well overnight and still no improvement. When I replace mobile A with just water the peaks disappear however the pH of the buffer is 4.6 so I'm not sure how representative the water is as A.

[danko]

Your ammonium acetate must be the source of the polution.
Try another lot.
If you can't get purer ammonium acetate, you could filter the buffer through a guard column or a discarded analytical column prior to use.
Another possible polution source could be the pH electrode, if you're adjusting the pH in the eluent/buffer.

Finally, the pH adjusting reagent. What are you using for adjusting the pH?

Best Regards

[BMol]

I've used 2 lots of ammonium acetate with no change in the chromatography. Just had a conversation with a very helpful waters engineer who has suggested a couple of things to try. The mobile phase is pH'd with acetic acid. Thats next on my hitlist to check out!

[BMol]

Turns out it was the water. We used 3 batches of water from 2 different suppliers and when we tried our in house purified water (after a long sanitation of the purification system) the peaks disappeared.

[HW Mueller]

Did you use this "long sanitation" water in the experiment described in your reply from Aug 3, 4:46 am? You changed two things, but told us only about a change of one? Danko could have saved his time . . . .

[BMol]

The lab water came from the same source as the original water I used and the sanitisation on those treatment units is done monthly. The first mobile I used was made using this water and we had no reason to think there was anything wrong with it particularly when the bottled water showed the same contamination!