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FAQ: I am observing broad, split peaks in Aqueous Normal Phase (ANP) when using acetonitrile/methanol diluent, What can I do to improve peak shape?

Reference Number: AA-02128 Created: 02/07/2014 03:30 PM Last Updated: 09/12/2020 02:48 PM

Question: 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?

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 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.


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