Chromatography Forum

Hi,

I will start to try 100% aqueous isocratic liquid affinity chromatography on an Agilent 1200-6130 LC-MS (ESI+) (single quadrupole) system.

I have a protein coupled to the column (2.1 mm ID) and I would like to use approximately physiologic pH, about 7. I have planned to use 20-40 mM ammonium acetate. No organic will be used (not physiological). To obtain also a physiologic ion strength it would be nice to use eg 150 mM ammonium acetate... but I think that this would not work, because of both ion suppression and that salts may contaminate the MS, because they will not evaporate totally, due to the high concentration.

I will analyze different small molecules to investigate if hey have affinity to the target protein that is coupled to the column.

I am very grateful if someone has suggestions about instrument parameters and what (non-organic) mobile phase that can be used, including literature refs.

Thank you.

Regards,

Goran Karlsson, PhD
Linnaeus University
Kalmar
Sweden

What is physisiological about an immobilized potein and NH4OAc?
If you want to do affinity chromatogaphy you want separation, etc., etc., who cares about physiology? If you want to study affinity to the protein under physiological conditions than its immobilization introduces a huge unknown.

You are right, 150mM is too high. And also one thing to consider is the follow rate or the amount of water introduced to the ESI source, because water has a very high surface tension that greatly effect the ionization efficiency.