I am analyzing Pyrazinoic Acid by Aqueous Normal Phase HPLC (ANP) 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?

Suggestion: 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 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.

Pyrazinoic Acid