Chromatography Forum

Hi everybody,

I have a gradient HPLC method for the assay of related and degradation products. The MF consists of:
- MF A 950%:50%=Ammonim acetate buffer pH=4:Acetonitrile
- MF B 750%:250%=Acetonitrile: Ammonium acetate buffer pH=4
pH of the buffer is adjusted befor mixing with the organic solvent.
Everything was flawless for a while until few weeks ago when my peak started to show an increased retention. Instead appearing at 19-20min, it is now appearing cca.23-24min?!

I have eliminated the system and the column as a possible problem, so now my only concern is the MF. Any suggestions?
Has anybody experienced some problems when using ammonium acetate buffer?

Thank you all in advance,
for your help and for your attention

If you have a low pressure gradient system please check the magnetic valves and check flow of each channel. And try to rinse your column with water:acetonitrile at elevated temperature. And than see if you get back the starting retention time for your compound.

Have you any problem with ohter analysis?

If you've a pump then HPLC system problems, there problems are in all analysis that you start.

The peak shape is varied?

Any changes in the way you are making the mobile fase? Do you make stock for buffer? Changes in the pHmeter? Calibration solutions? Any possibility for acetronitrile is being evaporeted?

I have eliminated the system and the column as a possible problem,

How did you eliminate the system and column?

I performed the analysis on 3 different HPLC systems and different columns. The problem persists... That's why I think there's something going on with the mobile phase.

Any changes in the way you are making the mobile fase? Do you make stock for buffer? Changes in the pHmeter? Calibration solutions? Any possibility for acetronitrile is being evaporeted?

I had a colleague make and mix the mobile phases instead of me and still the problem persists. No, I don't make a stock solution, the buffer is always made extempore. I did notice shifts in the buffer pH, but I doubt that 0,05-0,1 pH units can make such large RT jump cause during method validation the robustness was performed with buffer change of +/-0,2 pH units and the peak retention was still 22min...
At first I thought too that pH (more precisely lower pH) must be the problem, so I adjusted the buffer's pH cca 4.05 but the retention didn't decreased significantly. We have 3 pH meters in our laboratory, they are all in use daily and nobody else seems to be having problems with pH...
I also doubt that the ACN can evaporate so fast, cause I get an increased retention right on the first run after the mobile phase has been on the system for not longer than 1 hour; Rt doesn't shift during the hole analysis, it's constant...

Have you any problem with ohter analysis?

If you've a pump then HPLC system problems, there problems are in all analysis that you start.

The peak shape is varied?

Absolutely no other problems with other analysis, and until recently this method was also functioning flawless. The peak shape and the hole chromatography is intact only shifted right for 3-4 minutes. It's not the HPLC pump cause I've tried 3 different systems and the problem persists.

Natasha:
Are you using the same ammonium acetate? Changes in the suplier? Old flasks?

Compare the old method file with the new one. Maybe someone* change flow rates in one of gradient steps ?
I know its sounds funny but strange things can happen in laboratory.

The sample preparation, dilution no changes ?

Good luck.

When you tried 3 different HPLCs and different columns you still consistently got the same wrong RT?? That's weird to begin with. Is this a gradient run?
Are the HPLCS running one method over a network?? If that's the case I'd say method.


If not, sounds like you narrowed it down to reagents so far

Are the different columns you tried from different batches of sorbent? Have any of them shown a "normal" separation before ? If not, this could be a column/sorbent batch related problem. In my experience this is rarely the case but if all columns that have thIs problem are from a new batch and all previous columns behaved as expected this could be the problem.

We really need more information about your conditions and the nature of your analytes (im assuming basic, but very weak base, i.e. pKa ~4?) to offer anything more useful.

A change in your pH value of 0,1 can have drastic impact on the RT of your compound.
Please remember pH is also linked to temperature.
GLP is to adjust and to messure pH with a calibrated pH meter.

It may be caused by microbial growing in the mobile phase A. you might want to try re-run it with new column and fresh prepared mobile phases to eliminate it. just another thought.

naythewave, do you control the column temperature and if so, at which temperature. Also how is the temperature controlled if it is?

Best Regards