Chromatography Forum

Hey All,

I am running HILIC mode LCMS (metabolomics) using 20mM ammonium acetate pH 9.6 and ACN gradient. The problem I am running into is that the pH of ammonium acetate buffer decreases by about 0.1 units per day and quite a few metabolites shift greatly in both intensity and retention time. Labs running similar methods say they prep the buffer fresh daily, but I would like to find a longer term solution. I initially tried using a gas tight bottle cap with a one-way air valve, which helped a little, but pH still drops fairly rapidly and so I had a few questions:

1) Would chilling the buffer in a fridge help while it was connected to the LC? I have in-line heaters I can add to the LC system to bring the temp back up before it hits the column.
2) I have a N2 generator in lab and so would it be helpful to add a stream of N2 into the bottle (to help with CO2 absorbtion)? If so is there a general how-to on how to set this up (i.e. what pressures to use)?

Thanks for any help in advance!

Aqueous eluents at alkaline pH tend to absorb CO2 from the air. This yields ina decreasing pH.

Avoid exposition of the eluent to air. You may use a CO2 adsorber or just purge the eluent with a CO2-free gas.

How did you adjust the pH to 9.6? At this pH, ~ 50% ammonium acetate might be existing as free aqueous ammonia. No wonder the pH will drift with time.

Regards,

Farooq

keeping the buffer in the freezer is not going to help either. In low temp solubility of gases is higher than in normal temp so the decreasing in pH value will be progressing more rapidly by absorption with CO2 like Markus said.

Why not prepare a 10x stock of your buffer, sparge with helium (re-adjust pH if necessary), stopper, and dilute to 1x daily?