Preparation and evaluation of 400 μm I.D. polymer-based hydrophilic interaction chromatography monolithic columns with high column efficiency

- Two kinds of 400 μm i.d. HILIC monolithic columns were prepared.
- High column efficiencies (up to 144,900 plates/m) were obtained on these columns.
- A clearly better separation performance was also observed for various polar analytes on these monoliths compared to the ZIC-HILIC column.
- These monolithic columns can be used for fast separation of nucleotides.

The quest for higher column efficiency is one of the major research areas in polymer-based monolithic column fabrication. In this research, two novel polymer-based HILIC monolithic columns with 400 μm I.D. × 800 μm O.D. were prepared based on the thermally initiated co-polymerization of N,N-dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl) ammonium betaine (SPP) and ethylene glycol dimethacrylate (EDMA) or N,N'-methylenebisacrylamide (MBA). In order to obtain a satisfactory performance in terms of column permeability, mechanical stability, efficiency and selectivity, the polymerization parameters were systematically optimized. Column efficiencies as high as 142, 000 plates/m and 120, 000 plates/m were observed for the analysis of neutral compounds at 0.6 mm/s on the poly(SPP-co-MBA) and poly(SPP-co-EDMA) monoliths, respectively. Furthermore, the Van Deemter plots for thiourea on the two monoliths were compared with that on a commercial silica based ZIC-HILIC column (3.5 μm, 200 Å, 150 mm × 300 μm I.D.) using ACN/H2O (90/10, v/v) as the mobile phase at room temperature. It was noticeable that the Van Deemter curves for both monoliths, particularly the poly(SPP-co-MBA) monolith, are significantly flatter than that obtained for the ZIC-HILIC column, which indicates that in spite of their larger internal diameters, they yield better overall efficiency, with less peak dispersion, across a much wider range of usable linear velocities. A clearly better separation performance was also observed for nucleobases, nucleosides, nucleotides and small peptides on the poly(SPP-co-MBA) monolith compared to the ZIC-HILIC column. It is particularly worth mentioning that these 400 μm I.D. polymer-based HILIC monolithic columns exhibit enhanced mechanical strength owing to the thicker capillary wall of the fused-silica capillaries.