Chromatography Forum

hi all,
I am trying to optimize the detection of heme (hemin) via LC-MS. I have been running C18 with H2O/ACN and 0.1%FA. I detect 616,1766mz on Thermo QE or 616.1->556.9-557.4 on Thermo TSQ.
My main issue is that I have a very broad peak for hemin. Iron free protoporphyrin IX also runs broad, Coproporphyrin and biliverdin are reasonable. Suggestions on how to improve on this?
Furthermore, a strange thing is that on QE I see two peaks for heme (only with lower dilutions of standard, like 0.1µM) but on TSQ just one broad peak. What could those different peaks be? Finally, porphyrins often leach into my blanks.

I welcome your suggestions. Thanks!

Bory

What you describe is common where two different forms of the same molecule can interchange during the separation. An example would be for an acid when the mobile phase pH is near the pKa of the acid.

There is always a possibility that broad peaks come from chelating properties of the molecule and that heme kicks out iron.

great suggestions, thank you! I do monitor the iron free porphyrin too - it has a very distinguishable mass and RT, separate from heme (behaves like a more nonpolar molecule). Would you think changing formic acid concentration and/or temperature of run make a difference?
Thanks, Bory

This article seems to be getting a nice peak with a C18 column and H2O/ACN/0.1%FA mobile phases.

I would compare your conditions to those. It might be a matter of changing the initial conditions to higher % aqueous, or indeed the column temperature. Make sure that your injection solvent matches the composition of the initial mobile phase - this can also cause split/broad peaks.

great suggestions, thank you! I do monitor the iron free porphyrin too - it has a very distinguishable mass and RT, separate from heme (behaves like a more nonpolar molecule). Would you think changing formic acid concentration and/or temperature of run make a difference?
Thanks, Bory

Start adding more formic acid gradually and see if peak shape improves

thank you so much everybody! I will give it a try with your suggestions.
B