Chromatography Forum

A mixture of pharmaceutical intermediates were run on a range of Primesep columns (A, 100, 200) using 5-90% ACN. A range of additives were tried, ie TFA, formic acid, acetic acid and ammonium acetate, (pHs of 2.0, 2.6, 3.4 and 6.9) .

Performance with TFA and ammonium acetate was good, but the basic intermediates were very strongly retained when using the formic and acetic acids (25mM), which we attribute to low ionic strength of the acid. We are investigating further, but has anybody else seen this behaviour and if so do you have an explanation? [/b]

You have two reasons for strong interaction when you use acetic acid and formic acid. When you do method development on Primesep columns you need to consider amount of ions you have in the mobile phase. In addition to that there is pH effect on a stationary phase. Primesep A column has pKa close to 0, Primesep 100 has pKa of 1 and Primesep 200 has pKa of 2. With 0.1% TFA you have pH around 2 and TFA is fully dissociated. At such low pH you start suppressing carboxylic acid on the surface of stationary phase (Primesep 100 and Primesep 200) and ion -interaction between stationary phase and analytes decreases. When you have 0.1% formic or 0.1% acetic acid, first of all you have less ions to facilitate ion-exchange interaction and second of all you pH is much higher (3-4 vs. 2), so there is no suppression of stationary phase.
When you use ammonium formate or acetate you might have same pH (3-5) but amount of ions in the mobile phase is much higher (full dissociation of ammonium formate producing both ions-formate and ammonium)
When eluting basic analytes you need to consider ionization state of stationary phase (controlled by pH) and amount of ions in the mobile phase. You can use various salts to create desired ion-strength and various acids to adjust pH to the desired level. We recommend that for buffers you use the same acid (formic acid for ammonium formate, phosphoric acid for phosphate, etc.). In case of basic analytes you would not see a change in ionization state when you operate within operating range of Primesep columns, but if you have pyridine based molecules or amino acids, by going to pH above 4 you will change ionization state of such molecules.
Also very often when people do gradient of CAN they forget that concentration of acid might change too (as well as dissociation of such acids at higher concentration of ACN).

You need to have appropriate column for you application: Primesep A might be too strong.

Regards,

Vlad