Chromatography Forum

Hi All,
I'm developing a method using 10mM AMA with pH adjusted to 3.0. I started seeing retention time slowly shifted to the left. Could it be due to the pH was out of range as recommended 3.8-5.8? Would pH 3.0 be okay if the pKA of acetic acid is 4.8?
Thanks in advance,

The column I am using is Zorbax Eclipse XDB C8, and the other mobile phase is ACN.

Keep the pH at ±0.5 of the pKa. For Acetate buffers it would mean pH=4.26 to 5.26 (pKa=4.76).
You might get away with ±1.0 of the pKa but for maximum buffer capacity try to stay close to the pKa.

best regards,

Robert Haefele

Would there be any impact if I maintain the pH of the 10mM AMA buffer at 3.0? Would it do any damage to the column, chromatogram,etc.? I am about to start validating the method and my "deadline" is at the end of next week.
thanks,

ntruong,

well, to keep the pH constant this is exactly what a buffer is for. If you operate too far from the optimum pH (±0.5 from pKa) the buffer becomes very quickly useless because it is no longer able to function as a buffer.
I don't know if pH 3 in your case has any negative impact on your column but you will lose robustness (which you mentioned in your 1st post: shifting retention times). Personally, I have never used AMA (ammonium actetate: correct?). I assume you cannot use pH=4.8 and your buffer has to be volatile?

cheers,

Robert Haefele

Is there any reason not to use ammonium formate? The pKa is 3.7 so you will need less acid. Your baseline will be less noisy.

Using acetic acid to adjust 10mM ammonium acetate to pH 3.0 will take so much acid that it becomes part of the solvent system.

Thanks!!!
I learned it the hard way for going against the conventional wisdom. The elution of the main component is dancing cha..cha . It eluted at 13 minutes on one mobile phase and 11 minutes the next day when another fresh mobile phase is prepared.