Chromatography Forum

From my collegue:
I transferred my bile acids analysis method to our new Bruker LC-QTOF system from our older Waters LC-MS/MS. I made new solvents: 0,2 % HCOOH and 10 mM ammonium acetate in A) H2O and B) MeOH, all LC-MS grade. Solvents were fresh, ammonium acetate 2 years old. Column used: Thermo Accucore polar Premium, 2,6 um, 2,1 x 150 mm + Accucore polar premium guard column
I saw a huge contamination in the blank run. I thought that I had overloaded the column in the previous instrument and the peaks were caused by our compounds, since no one else had any problems with the instrument. I tried to wash the column with methanol, iso-propanol, 95 % methanol and with following procedures that manufacturer proposed:
1. protocol:
1. 90% water / 10% acetonitrile (for buffer removal if required)
2. 50% THF / 50% acetonitrile
3. acetonitrile
4. Re-equilibrate with mobile phase
2. protocol: (reverse flushing)
1. Flush with HPLC grade water, inject 4 aliquots of 200 µL DMSO during this flush.
2. Flush with methanol
3. Flush with chloroform
4. Flush with methanol
All washing steps 40-60 column volumes.
These did not remove the contamination.
I ordered a new, similar column and saw a huge contamination in a blank run. I tried with another column (Kinetex C18 2.6 um, 2.1x 100 mm) and saw the same contamination. I made a run without buffers and without column and the blank was pure. So the contamination is not stuck into instrument lines. I also ran blanks without buffers in these columns, but contamination is stuck there.
My guess is that ammonium acetate is contaminated somehow, possibly by some wax. The m/v value of the biggest contaminant peak is 604.383 (may be protonated form or ammonium adduct of some molecule). Any ideas?

jusuom wrote:
I also ran blanks without buffers in these columns, but contamination is stuck there.

Can you elaborate this part, since it does not seem to align with the rest of your observations? Do you see a contamination peak in this experiment or not?

I don't advice running LC-ESI-MS methods with plain water and plain MeOH as part of contamination troubleshooting, since you don't know whether your contamination can actually ionize in the absence of additives.

I suggest, no matter what, to trow away the acetate and start with a fresh bottle. Same story for the formic acid. Mobile phase additives are such a common source of contamination, especially if it's used by several people in the lab. We recently introduced a rule to not touch the LCMS formic acid with anything - we pour about 1mL in a (new) glass test tube, and pipet it from the test tube in our mobile phase bottle.

I'm slightly suspicious this is a new system. In my (admittedly limited) experience, new systems do sometimes have strange plastic-related ions that come out of tubing, and which manufacturers love to blame on the user's solvents and methods. They make you buy a specific grade of isopropanol and flush your system and after three weeks of fiddling around, everything's fine. Then a year or two later, one of the engineers lets slip that they've realised it was a piece of tubing that was releasing a contaminant for a bit, until it's washed out...
If you have moved your method from an old system to a new, can you take the column back to the old to check whether it has genuinely become contaminated with something, or whether this is a problem specific to the new instrument?
(of course replacing the mobile phase additives makes perfect sense as it's an extremely cheap thing to do, certainly can't do any harm, and may well solve the problem).