Chromatography Forum

Question.
I have 2 ways of preparing my mobile phases A and B. The 2 mobile phases preparations gave me a different baseline profile for the same elution gradient, though the retention time of my analytes were not affected at all.
Mobile Phase A= 80:20 10mM NH4OAc, pH 5: ACN
Mobile Phase B= 20:80 10mM NH4OAc, pH 5: ACN

Preparation 1 for 1L each of mobile phase
Mobile phase A = 100mL 100mM NH4OAc, pH5 + 700mL water + 200ml ACN
Mobile phase B = 100mL 100mM NH4OAc, pH5 + 100mL water + 800ml ACN
This gave me a upward sloping baseline at 225nm

Preparation 2 for 1L each of mobile phase
Mobile phase A = 800mL 10mM NH4OAc, pH5 + 200ml ACN
Mobile phase B = 200mL 10mM NH4OAc, pH5 + 800ml ACN
This gave me a downward sloping baseline at 225nm

I am surprised to see the big difference in the baseline profile at 225nm with different mobile phase preparations.
Does anyone have the explanation for such an observation?

If I did the mathematics right then the buffer concentrations for both preparations are actually different.
Preparation 1 gives you 10mM Acetate in both A and B
Preparation 2 gives you 8mM in A and 2mM in B.

Acetate shows considerable absorption at 225nm. Therefore, these different concentrations will lead to different baseline drifts. With preparation 1 the absorption of A and B due to acetate are the same, therefore you'll see the absorption differences of water and acetonitrile which usually leads to an upward slope. With preparation 2, you mainly see the drift due to acetate, going from higher to lower acetate concentration yields a downward drift.

I see.

Thank you for the clear explanation. I understand now.