The influence of properties and structure of nitrogen-containing heterocycles on their retention on amide stationary phase in acetonitrile-rich eluent

The retention behavior of 57 aromatic nitrogen-containing five- and six-membered heterocycles were studied. The conditions included amide stationary phase BEH Amide (Waters), characterized by reduced acidity of silanol groups, and acetonitrile-rich eluents containing ammonium formate. It was found that imidazole, pyridine, 1,2,4-triazole and triazine derivatives with log P from –1 to 1 and significantly different p K a (BH + ) were retained well. The factors promoting retention on the amide stationary phase were identified: protonation of bases with a decrease in the eluent pH; introduction of electron-donating sub-stituents (alkyl and amino groups) to the molecule, leading to an increase of the bases’ strength; the arrangement of heteroatoms in the cycle, providing a higher dipole moment of the molecule. Chromatographic separation of diazole, triazole, diazine, dimethylimidazole, alkyl-1,2,4-triazole, and aminopyridine isomers was demonstrated. The observed retention trends provide insights into the retention mechanism of nitrogen-containing heterocycles and can be used to optimize liquid chromatography-mass spectrometry conditions.