We currently work on two very polar drugs (both contain -SH group) in human plasma. During MD, we used 25 mM phosphate/methanol (95 : 5). The resolution was about 1.3. At one time, we accidentally prepared the buffer at a concentration of 50 mM. To our surprise, the resolution became 3.4. In our case, the column (Zorbax SB-Ag), flow rate, and the HPLC system did NOT change. Therefore, the enhanced resolution must have come from higher buffer concentrations. We were surprised. Usually higher buffer concentrations result in better peak shape. We always got symmetrical peaks for both analytes. Any explanation is highly appreciated.

This is not a surprise.The salt-in/salt-out effect of a buffer is not the same for different molecules and depends on the buffer concentration. The buffer concentration works differently for different moleculs. Thanks god for all differenses involved in the chromatography.

Buffer strength as well as changes in ph will affect retention. As I.D. says, different molecules are affected differently. It can also cause changes in elution order, so if both of your peaks are of similar size, you may want to verify the elution order with standards.