Chromatography Forum

Hello friends,
This is my first post in this forum. I am struggling with ghost peaks in revere phase chromatography gradient blank. I am using 10mM Ammonium Acetate as mobile phase A and Acetonitrile as Mobile phase B. My initial composition starts from 95% A and goes upt o 95% B. I am getting the blank peaks most of the time in the region where gradient composition corsses from 55% A to 65% B. I've almost tried anything (Changed the solvent grades, salt, pH and columns) but unable to resolve the problem. Surprisingly some day this problem disappears without doing anything, but comes again with the same mobile phase and column the very next day. Please suggest.

There have been many posts on the subject. Please check! There can be contaminants that leak from pipes, tubing, solvent reservoirs etc. If the description is correct that the same mobile phase does not show the ghost peaks wehn freshly made, but does a day later, this would make the solvent reservoirs, solvent lines and anything in it (stirrers etc) suspect.

The first thing to do in order to see if the peaks are mobile phase/LC related is to allow higher equilibration times and see if these peaks increase or not. Also some information on the detection that you are using would be helpful...

Dear Friend,
Thanks for your suggestions. But the peaks I am getting are more likely to be of gradient only because when I run the same mobile phase on the same column on the same day with gradient starting from 65% buffer and ends with 95% organic I do not get any peak.
But I've to start my gradient atleast from 90% buffer to get the desired resolution.
I've also read the earlier posts but of no use in this case, please come again with something which I could try to resolve the problem.

...when I run the same mobile phase on the same column on the same day with gradient starting from 65% buffer and ends with 95% organic I do not get any peak.
But I've to start my gradient atleast from 90% buffer to get the desired resolution...

This is what I would expect from a water based contaminant. Search my posts for the word Empore - you will find several threads detailing waht's going on and how to fix it with an Empore Extraction disk.

As DR says it is a water or additive contamination. The reason that you do not see it at 65% is that it is not retained in the column at such % of ACN and thus you do not have any accumulation of the contaminant.

A simple confirmation is to filter the contaminant out by passing 200 mL or so of your mobile phase A through a C18 column and then using it for your under the same experimental conditions you use for your analysis.

Dear Ajay...

I had same problem before...specially when % of ACN is high.

I fixed it by degassed the mobile phase thoroughly.

most likely the ghost peaks are micro air bubles that comes from the solvent mixer if you use gradient elution.

you can try helium sparging method...buble the mobile phase with helium gas vigorously for 10 mins. that should take care the problem...

hope it helps....

Ernest Basarah