Chromatography Forum

Dear Sir
I developed the HPLC _UV method for the estimation of two drugs . The drugs used for the present study was Glimepiride and Pioglitazone. There are number of methods published on simultaneous estimation of these two drugs but none of the author gave the mechanism why pH4.5 is requires for good separation of these drugs on C18 column.
I tried with 47% of buffer in different pH such 3.5, 4.5, 6.0 and 7.0. In all cases at pH 3.5 the Rs(resolution) was 21.698 where as at pH4.5 it was 12.675 and pH 6.0 it was 3.789. At pH 7.0 both drugs where not at all separated but eluted fast on the same rt. The pKa value for Glimepiride is 6.2 and Pioglitazone pKa 5.2 and 6.8.The both drugs structures are linked below given link.
Pioglitazone [http://www.chemblink.com/structures/112529-15-4.gif]
Glimepiride [http://www.drugs.com/pro/images/897e946d-075f-4048-bc32-ec5d6da9c388/chem-structure.jpg]
I will be very happy if any one will give mechanism of separation of both these drugs at different pH.
Thanking you in advanced
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The retention of glimepiride will decrease with increasing pH due to the ionization of the sulfonamide function.
Pioglitazone has a pyridine function that will ionize at low pH, i.e. the pKa of 5.2 is probably the pyridine. The CO-NH-CO function is the acid function and will ionize at high pH, with the pKa of 6.8, in a similar way as the sulfonamide.

Some of the pKa's shift with the solvent composition. The pyridine pKa will go to a lower pH and the other pKa will go to a higher pH.