Chromatography Forum

Hi,

I am doing metabolomic analyses using an LTQ-Orbitrap Discovery. Unfortunately, stearic acid and palmitic acid seem to be continuously present at high levels in the spectra during these analyses. The first samples had the highest signals for these fatty acids and the signals gradually diminished over time. After 80 samples both ions were still present however.

I am using a Zorbax Eclipse Plus C18 1.8 µm 150x0.5 mm column with a flow rate of 15 µl/min. Mobile phase A=5 mM ammonkum acetate Mobile phase B= 97.5 % acetonitril, 2.5 % water 5 mM ammoium acetate. I run a gradient from 7% B to 40% B. Then I quickly go to 100% B and then equilibrate for 10 minutes at 7% B.
I am not using soap to clean the glasware. Anyone an idea where these fatty acids come from? Could this be due to column bleeding?

Koen

I guess it may come from several sources but if they are continuously present over your chromatographic run, I would incline towards an MS source contamination. Maybe someone cleaned the source without using gloves? If the contamination was located before the column (mobile phase, filters, sample etc.), I would expect the fatty acids to be retained on the C18 column and suddenly elute as a sharp or broad peak according to their concentration.

There are some simple tests you can do to determine where they come from. For example direct infusion of some clean mobile phase will tell you if it comes from the MS source.