Hydrophilic interaction liquid chromatography of hydroxy aromatic carboxylic acid positional isomers

Hydrophilic interaction liquid chromatography (HILIC) has become increasingly popular as an alternative to reversed phase LC due to its ease of separating complex polar compound mixtures and the compatibility of the mobile phase with mass spectrometry (MS). Using a plain silica column (150  mm × 4.6  mm), we have shown a mixture containing three hydroxybenzoic acid isomers plus syringic and vanillic acid and three hydroxycinnamic acid isomers plus ferulic and sinapic acid can be separated using a mobile phase comprised of 90% acetonitrile (MeCN) and 10% 20 mM ammonium acetate at pH 6 in under 45 min. This method is appropriate when using UV detection at 275 nm. However, for improved MS compatibility, a buffer concentration of 10 mM is recommended but this greatly decreases the analyte retention factors. A second more nonpolar organic solvent component in the mobile phase (particularly pentane which has not been previously considered for HILIC) is found to offset this loss in retention. The optimum mobile phase is found to be 90% MeCN, 5% 10 mM ammonium acetate pH 6, and 5% pentane with resolution of eight of the ten compounds with a separation time of 30 min. Using UV detection, we have shown that detection limits range from 36 to 133 pmole and quantitation limits are spread from 94 to 376 pmole for six of the analytes. Upon testing this method on two other silica columns from different manufacturers, it is found that while resolution is similar, further optimization of the mobile phase is recommended.