Big issue with shlouldering peaks

[adrien16]

Hi everyone,

I need your help about this method : I use a gradient ACN/buffer pH 9.0

I observe sometimes shoulder peaks with pseudoephedrine HCL and triprolidine HCl. I know that the pH of the buffer is around the pKa of these compounds but with organic modifier (such as ACN at 10%) the pka is lower than this value.
But do you know if these conditions could involve a shouldering peak or not ?

My second interrogation is about the preparation of the buffer.
I prepare a 10mM aceta buffer adjusted with NH4OH. Is someone knows some issues about that ? Instability for exemple ?

Thank you

Adrien

[HW Mueller]

A first guess could be that you sometimes have a pH mismatch between sample solvent and mobile phase. 2nd guess: An organic modifyer mismatch.
There have been many extensive discussions on buffers here. My preference: Use a web pH calculator if possible. If not I would prepare an ammonium acetate solution which is 10mM in ammonia (since the pH buffering is here due to NH3/NH4) and which has a pH below 9, then another ammonium acetate solution with a pH above 9. The two solutions can than be mixed to final pH (9) without changing the molarity of the ammonia.

I am presently not in the lab, so I am guessing: Isn´t a HPLC grade NH4OAc available from Fisher? High grades of acetic are also available.

Furthermore, I would probably play with other pH and buffer concentrations.

[syx]

the peak shouldering problem was related to all of the peaks or just some of them? when all peaks are distorted, the main problem may be occured at the column inlet (frit blockage, column voids).

[Bruce Hamilton]

As noted above by Hans, shoulders are often the result of sample solvent being different to initial mobile phase, or having inadequate pH buffer ability.

One major issue can be the sample requires a higher concentration of buffer to achieve a sharp peak. This can be solubility or buffer strength, so try using a more dilute sample concentration to see if the shoulder disappears. If it does, keep the solvent composition but increase the buffer concentration in the sample solvent.

Another possible cause is junk in the sample, which is usually obvipous because the stds are OK.

Please keep having fun,

Bruce Hamilton