Time-dependent column performance of cholesterol-based stationary phases for HPLC by LC characterization and solid-state NMR spectroscopy

Three different cholesterol‐based stationary phases were investigated with respect to their time‐dependent separation behavior. The examined stationary phases differ in the used spacer molecule and the synthesis route and were used under routine laboratory conditions over a period of two years. The chromatographic behavior of the three phases was determined by using a standard reference material in addition to a separation of a steroid mixture. The surface chemistry and the modification of these with the chemically bonded moiety were investigated with nuclear magnetic resonance (NMR) spectroscopy and elemental analysis. Through applying different techniques we determined changes in retention and selectivity; solid‐state NMR spectra showed changes in the surface chemistry dependent on the synthesis route. Superior long‐term stability was observed for the undecanoate‐cholesterol (UDC‐Chol) column in terms of hydrophobic retentiveness and selectivity.