Chromatography Forum

Hi,

I am trying to separate the Vitamin B1 from hemolysed whole blood using a C18 collumn in a reversed phase HPLC system.

I am using a Phospahte buffer mobile phase in a gradient from 5 to 60% Methanol.
Sample hemolysed by temperature schock at -70 °C and then proteins are precipitated with methanol.
Derivatization with Potassium ferricyanide.

TDP (thiamine diphosphate) Calibrators prepared in water.

The problem that I have is that from time to time I get a second peak proportional to the concentration used that comes right after my peak (like a peak-split), even though they are pretty well separated.

Can anybody help me please?

Thanks a lot

Do replicate injections get the peak split from time to time? Or is it certain standards that do it and others don't? I would do 5 replicate injections from the same vial to make sure you get consistency. Split peaks usually come from blocked inlet frits in my experience.

I will try and let you know if it is the case.

Thanks

If it is possibly, please provide us two chromatograms (a good chromatogram and one with double-peak) where comparable conditions were used.