Question: I am analyzing pyrazinoic acid (see structure below) by Aqueous Normal Phase ANP HPLC using the Cogent Diamond Hydride™ column. I observe two broad, split peaks for the analyte. I am using an acetonitrile / DI water / formic acid based mobile phase and my diluent is 50:50 methanol / acetonitrile. Retention is also low. What can I do to improve peak shape and / or retention?
Answer: The absence of water in your diluent may be contributing to the split broad peak shape. This has often been observed when using non-aqueous containing diluents with an aqueous/organic ANP mobile phase. Try a diluent of 50:50:0.1 acetonitrile / DI water / formic acid. If that does not solve the problem, try an ammonium acetate-based mobile phase and diluent (10mM).
You will probably be using negative ion mode LCMS in that case. With the carboxyl group ionized, you will probably be looking for the [M-H]– ion. You can expect stronger retention with the carboxyl group ionized.