High-efficiency hydrophilic interaction chromatography by coupling 25 cm x 4.6mm ID x 5 microm silica columns and...

Recently, hydrophilic interaction chromatography (HILIC) has emerged as a valuable orthogonal tool to reversed-phase liquid chromatography (RP-LC) as it allows for resolution of highly polar ionisable compounds. The relationships between separation efficiency, column length and speed of analysis for 4.6 mm ID x 5 microm silica particle columns in HILIC are demonstrated using kinetic plots. The kinetic plots constructed for conventional pressure systems operating at 350 bar and at 30 degrees C and 80 degrees C are confirmed using experimental data for different column lengths. Efficiencies of more than 130,000 theoretical plates could be achieved by connecting up to six columns of 25 cm. As expected, a significant gain in analysis speed without loss of efficiency could be obtained by operating at 80 degrees C compared to 30 degrees C. The advantages of using long columns in HILIC in combination with elevated column temperature for the pharmaceutical industry are illustrated using test mixtures comprised of commercially available ionisable compounds (including some containing functional groups with potential genotoxic typical structural alerts) as well as real polar ionisable pharmaceuticals.