Chromatography Forum

Hi
I`m developing a rel subs method and have a problem with the shape of one of my peaks. One of the impuities is an acid and shows severe tailing, at low levels it is almost flat. All my other impurity peaks are nice and sharp. After I wash my column out with water I can get a good peak shape but over a few injections the shape gets worse again.
Running on a waters UPLC system with a Zorbax XDB-C18 1.8um 3x100mm column. I`m using a gradient flow with ACN and 15mM ammonium acetate buffer pH 5.5
Appreciate anyones comments
jimmy

First you clear, After some injections, all peaks get worst or only problemetic (acid impurity) peak get worst ?

If only peak of acid impurity gets worst, its may be due to solution stability.
Regards,
Ha resh B. Patel

I would investigate using a lower pH mobile phase.

Hi
its just one peak that is the problem. I have 8 other impurities in my chromatogram and their peak shape is fine.
The problem impurity is an acid.
If I wash my column over night with 80:20 water:ACN the peak shape is fine for the first 3 or 4 injections then the peak starts to tail badly.
If I use a new column the peak shape is fine then it gets worse after a few injections.
All the other 8 peaks are sharp and consistent.

I`ve tried reduced the pH to pH 3 increasing my buffer conc to 25mM from 15mM and adding 0.1% acetic acid. When I tried this I couldn`t see the peak at all.

Perhaps there is a cationic impurity in your ammonium acetate or acetic acid? Try new bottles with different lot#'s or a different salt/acid (ammonium formate/formic acid) buffer.

If the peak gets worse as a function of the number of injections ( as opposed to, say, total exposure time of the column to the mobile phase), that suggests that something from your sample is accumulating on the column. Without knowing anything about your sample, it's hard to be more specific. A general-purpose fix would be the use of a guard cartridge (changed frequently! ) or perhaps 2 cartridges with a switching valve to allow alternate injection and wash.

Jimmy,

Please clarify if performance degrades only when you inject samples. In other words, when using a new or freshly washed column and you make one injection of sample followed by 4 injections of initial mobile phase then inject another sample does the second sample injection look OK?

Deterioration of just one peak among 8 peaks is interesting. May accumulation be a problem but the other 7 peaks -as you say- have good peak shapes. If you tried everything, there is not much thing to say. However, may be the pKa of the acidic compound about 5-6 so, it may tail. pH 3 is not appropriate for you because acidic compounds becomes more non-ionized and more retain in the column.
I am not an expert, but I suggest you to add some octylamine or triethylamine (%1 or less) to mobile phase and adjust pH to your previous pH (5.5). You will see sharpened peaks, including the acidic one...

Oversimplied hypothesis is that 1) there was a secondary interaction; 2) which is between the acid anion and the stationary phase, 3) the functioning component in stationary phase must be some cations; 4) if there are multiple interaction sites in this molecule(chelating) then it does not take a lot cation to cause this tailing.

A solution may be adding some chelating agent to the mobile phase such as EDTA.

Good Luck and please keep us posted with your results if you ever try.

Definitely try playing with the pH before you add any ion-pairing agents.

Let us know what happens. This is an interesting situation.

Kerri