HPLC Separation of Aminopyridines Isomers in Hydrogen-Bonding mode on a Zodiac HST HB HPLC Column

Pyridines and aminopyridines are hydrophilic, basic compounds commonly analyzed by GC or HPLC. In HPLC, separation is often achieved using reversed-phase chromatography with ion-pairing reagents, or alternative modes like HILIC. However, isomeric forms of substituted pyridines can be challenging to resolve.

We have developed a novel separation approach using the Zodiac HST HB column, which employs hydrogen-bonding interactions as the primary retention mechanism. This method enables effective separation of aminopyridine isomers based on their differential hydrogen-bonding interactions with the stationary phase.

The mobile phase consists of a combination of acetonitrile and methanol with additives such as formic acid and ammonium formate. Retention times and selectivity are highly responsive to changes in mobile phase composition, including the ratio of solvents and the concentration of additives. Notably, the elution order of isomers can be tuned by adjusting these parameters.

This method is fully compatible with LC/MS and preparative chromatography and is also suitable for separating other pyridine-based compounds and their isomers.