Why decreases signal in ESI-negative increasing % of water?

[crlar]

For the HPLC-MS/MS analysis of molecules with carboxilic groups, we are using ESI-negative.
We have better signal at higher pH (I suppose due to the increasing quantitiy of anions) and higher content of ACN.
But for getting any retention in the chromatographic column, I have to increase the acqueous content of the mobile phase...and then the signal decreases dramatically (ten times with a 20% of water or buffer)... Anyone knows why? (The instrument I use is an API-365)

Thanks!

[Uwe Neue]

It appears that you are running this isocratically (i.e. no gradient). Under these conditions the peak volume increases and the sample concentration decreases. Since ESI is for the most part a concentration-sensitive detector your signal will decrease.

[crlar]

Thanks for your answer Uwe.

But I have confirmed this fenomenon also injecting without column, directly from the injector to the MS...

[MG]

You might need to adjust your source temperature and gas flows for the higher aqueous mobile phases. The temperature is the big one. For that source, optimum can be 250-300 for mostly organic, and 400-450 for mostly aqueous.

[Victor]

Crlar,

I think your observation is a common one and applies to both positive and negative ESI. I think it may be due to the surface tension of the droplets which increases as the concentration of water increases. High surface tension affects droplet formation, and I guess also desolvation of the droplets. I think this is why in some cases alternatives to RP-HPLC are chosen, even if sacrifices must be made on the chromatgraphy side.

[JNF]

Hi,

I agree with Victor. The more water you add the less volatile your mobile phase is, thus the less efficient the spray is at producing the required ions (at given conditions).

The solution is to increase the spray efficiency by increasing temperature of the neb. gas, increase the gas flow or (maybe) the diclustering potential.


Regards
JNF

[crlar]

Thank you all for your comments.
I'll try to test the effect of increasing the DP or the nitrogen flow. Related to the temperature, I am working at 400ºC that is the maximum recomended...