Hydrophilic interaction chromatography of small metal species in plants using sulfobetaine- and phosphorylcholine-type zwitterionic stationary phases

Hydrophilic interaction chromatography (HILIC) was applied for the separation of small, noncovalent metal species, and free ligands in plants, using two different zwitterionic stationary phases at pH 7.3. Ligands ranged from amino acids and phytosiderophores to organic acids and synthetic chelators like EDTA. Our results confirmed the suitability of zwitterionic stationary phases for the separation of such ligands and their metal complexes. In particular, the chromatographic behavior of phytosiderophore complexes of copper, nickel, and zinc on a sulfobetaine-type material was compared to that on a phosphorylcholine-type material under otherwise identical conditions. A compression of the usable retention range for phytosiderophore species on the latter phase was found, which can be traced back to the reversed charge orientation of the zwitterionic functionalities. Differences were also found for the integrity of more labile metal chelates during separation on the two columns. In particular, Ni-malate could only be analyzed on the sulfobetaine phase, and Cu-glutathione, Ni-aspartate, and Ni-citrate complexes were only stable on the phosphorylcholine stationary phase at pH 7.3. Ni-histidine species were only found after lowering the pH to 4-5. The identification of separated species is possible by online ESI-MS in the negative ionization mode.