Preparation and evaluation of a bacitracin-bonded silica stationary phase for hydrophilic interaction liquid chromatography

• A novel bacitracin-bonded silica stationary phase was successfully prepared. • Polar analytes can be well retained and separated in hydrophilic mode. • The developed stationary phase possessed good repeatability and stability. New stationary-phase materials are of great interest for use in hydrophilic interaction liquid chromatography (HILIC), a relatively new technique capable of separating highly polar compounds. In this study, a new strategy was established to prepare a novel Sil-Bac stationary phase by bonding bacitracin to silanised silica particles using hexamethylene diisocyanate as a spacer arm. The obtained material was characterised by Fourier transform infrared spectroscopy, Brunauer–Emmett–Teller analysis, transmission electron microscopy (TEM), and elemental analysis. The network layers observed through TEM and change in specific surface area from 315.0820 to 169.6758 m 2 ·g −1 after modification reflected the addition of bacitracin. The chromatographic behaviour of the Sil-Bac column showed that it can be used for HILIC. The influence of the chromatographic conditions on substance retention was explored by varying the acetonitrile content, column temperature, buffer concentration, and pH of the mobile phase. Using cytosine, uracil, nicotinamide, and isonicotinic acid as probes, the Sil-Bac column was demonstrated to have acceptable repeatability and stability, with a relative standard deviation of the retention time in the ranges of 0.05–0.18% for intra-day precision ( n = 3) and 0.16–3.28% for inter-day precision ( n = 3). Finally, the stationary phase was applied in separating basic nucleosides, water-soluble vitamins, and amino acids. A variety of bases and nucleosides could be separated within 7 min in a mobile phase of ACN/ammonium acetate (pH 3.0) = 90/10 (v/v), T = 30 °C. The Sil-Bac column was also compared with three commercial columns, and the results indicated that the analytes retained on the Sil-Bac column showed better retention and selectivity from an overall view. This novel stationary phase has a good separation ability and selectivity in HILIC mode and provides new ideas for the separation of polar compounds.