choice of ion-paring agent

[miro2009]

Hello,
I need some help figuring out how to choose the bestion-pairing agent for developing an RPHPLC method
Usually the most common setup is using 0.1% TFA in acetonitrile, which I found usually gives the best profiles with a number of proteins regarding peak shape, resolution,..
for one method I needed a mobile phase of pH in th range of 5-6 to avoid degradation of my sample, so tried to use TEA and djusted the pH with phosph acid, but obtained the worst profiles ever! Also tried out formic acid... TFA is always the best option regarding the profile

So when actually are other ion pairing agents used? and on what basis are they chosen, just comparing profiles or is there a scientific basis for choosing the best agent, for eg. v\certain properties of my sample?
And why is TFA the only agent that gives me acceptable profiles so far?!

[grzesiek]

"I needed a mobile phase of pH in th range of 5-6 to avoid degradation of my sample" - it degrades during the course of one chromatographic run?

"Also tried out formic acid... " - did you try formic with hfba for example?

"And why is TFA the only agent that gives me acceptable profiles so far?!" - how many did you try? probably due to low pH (better peak shape) and good ion-pairing (higher retention)

[miro2009]

Hello again, in reply to your questions:
Sample degradation: my sample is a protein that degrades in acidic pHs to alfa and beta chains, this was also validated by performing a native SDS-PAGE analysis of the sample at different pHs and observe the lower the pH, the significantly higher the band intensity of the separated chains. So by RPC, the sample actually shows 2 peaks (the 2 chains) in addition to a third minor peak (low % of remaining intact).. so, yes it degrades in 1 single run
As for my general trials of comparing ion pairing agents, I tried formic acid, TEA, phosphate buffer...

[grzesiek]

could you post your results of this bad profile experiment and write what column do you use?

[HW Mueller]

If you got sharp peaks with TFA it is probable that the protein disintegrated before injection or very early on, while at higher pH this degradation was slower and occured throughout its presence on the column, thus giving broad peaks. Maybe a good book on protein LC and or purification will give you some ideas on how to use organic modifyers, chaotropes, detergents, ionic strength.
Incidentally, what do you mean by "it ws also validated . . ."? Proven, shown, indicated?
Furthermore, where does the idea come from that formic acid, TEA, TFA, phosphoric acid are ion pairing agents here?