Why the changes in pH of buffer?

[Sazabi]

Hi guys,

Just had random thought pop up on top of mind, which is about the glucosamine pka and buffer pH used in Mobile phase.

To my understading, glucosamine HCl has a pka at 6.5, where it would ionised in acidic condition. however, when my team was compare USP 32 and USP 33 monograph for glucosamine HCl, we found USP 32 use buffer pH 3.0 and USP 33 use buffer pH 7.5. can anyone kindly explain why there are two different pH being used and how it related to the separation for glucosamine?

USP32 - HPLC condition: mobile phase - Buffer:ACN (3:2) stationary phase - C8, 250 x 4.6mm

USP33 - HPLC condition: mobile phase - Buffer:ACN (1:3) stationary phase - Amino(NH2), 150 x 4.6mm

Many thanks

[sepscientologist]

Generally when you are analyzing and acid or base you want it to be in either it's acid or base form, ie, you want the buffer pH to be
far away from the pKA of the analyte. If you are near the pK of the analyte then small changes in pH of the buffer will change
the ratio of the acid/base form of the analyte and usually the retention time also.

I would be surprised if the C8 column worked under any conditions.

The second HILIC method on amino sounds pretty good.