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.

Characterization of binding affinities in a chromatographic system by suspended state HR/MAS NMR spectroscopy.

In the current work a racemate of (R)- and (S)-benzylmandelate was separated with a stereoselective polysaccharide-based chiral stationary phase by HPLC. To elucidate the occurring chiral molecular recognition processes in the heterogeneous system used, NMR spectroscopy was chosen under high resolution/magic angle spinning (HR/MAS) NMR conditions in the suspended state. Therefore, and as a proof of concept, a combination of several NMR methods such as spin-lattice relaxation time (T(1)) measurements (T(1)), the saturation transfer difference, and the 2D experiment of the transferred nuclear overhauser enhancement spectroscopy technique were applied. With HR/MAS NMR it is feasible to combine NMR and chromatography to achieve further insights into the separation process.

Architecture and dynamics of C18 bonded interphases with small molecule spacers.

The relationship between alkyl phase structure and chromatographic performance is investigated for a series of octadecyl (C(18))-modified silica surfaces with defined spacing of the alkyl surface by a "pre-end-capping" technique. Stationary phases were prepared by a two step process with (1) reaction with less than stochiometric amounts of a small monofunctional silane, followed by (2) solution or surface polymerization with octadecyltrichlorosilane. The results of solid-state and suspension nuclear magnetic resonance (NMR) spectroscopy are correlated with the chromatographic behavior regarding shape selective separations. Two sets of six different stationary phases were prepared by solution and surface polymerization approaches, yielding materials with surface coverages from 2.7 to 5.6 micromol/m(2). (13)C cross-polarization magic angle spinning (CP/MAS) NMR spectra show a predominance of trans conformations for the set of surface polymerized phases with a C(18) coverage greater than 4.5 micromol/m(2). For the solution polymerized phases, no predominance for the trans conformation was observed, even for surface coverages greater than 5.1 micromol/m(2). Proton spectra in suspension indicate the trend that a higher coverage for the surface polymerized materials correlates with a more rigid alkyl chain conformation. The set of solution polymerized stationary phases confirms this tendency but minor deviations are observed for high coverages. These structural abnormalities are confirmed by differences in the (29)Si CP/MAS spectra. Furthermore, the (29)Si CP/MAS spectra indicate a lower amount of cross-linking for the materials with the highest amount of placeholder (spacer). The use of the different spectroscopic and chromatographic methods provides a wealth of information on the surface morphology of the systematically prepared C(18) materials and extends the understanding of surface morphology of alkyl modified silica and its influences of the molecular recognition process in liquid chromatography.

1H HR/MAS NMR in the suspended state: molecular recognition processes in liquid chromatography between steroids and a silica hydride-based cholesterol phase.

A mixture of eight steroids was separated by HPLC on a silica hydride-based stationary phase modified with a moiety of cholesteryl 10-undecanoate. Within the steroid mixture progesterone showed the longest retention time suggesting the strongest interaction with the stationary phase material. To obtain more detailed information upon the effective separation process, interaction studies were performed by high resolution/magic angle spinning (HR/MAS) NMR spectroscopy recorded in the suspended state. (1)H saturation transfer difference (STD) NMR experiments with T(1rho) suppression showed significant hydrophobic binding affinities of progesterone toward the cholesterol column material. These NMR results can be correlated directly to the chromatographic data obtained by HPLC.

Synthesis, characterisation and chromatographic evaluation of polyether dendrimer stationary phases.

Supercritical carbon dioxide has attracted attention as a potential replacement for traditional organic solvents due to its simplified workup procedures and reduced environmental impact--providing a green chemistry approach for organic solvent-free functionalisation. In addition to the environmental benefits, the enhanced diffusivity observed in supercritical solvents can often enhance reaction rates. We have applied these valuable features to the preparation of silica-bonded stationary phases and examined their potential in liquid chromatography. We report the successful preparation and characterisation of polyether silica based on Frechet dendrimers--this significantly enhances the range of stationary-phase chemistries that can be prepared in supercritical fluids. First- and second-generation polyether silicas were prepared, characterised, end-capped and evaluated for use as stationary phases for liquid chromatography.

Synthesis, characterization, and high-performance liquid chromatographic evaluation of C14 stationary phases containing branched and unbranched alkyl groups.

Reversed-phase materials with branched and unbranched alkyl groups were prepared by modifying porous, spherical silica gel in a two-step reaction-immobilization of a trifunctional alkoxysilane (3-glycidoxypropyltrimethoxysilane) on the silica surface followed by reaction with a branched and an unbranched octanoic acid. The chromatographic sorbents were characterized by solid-state (29)Si and (13)C NMR spectroscopy. The chromatographic behaviour of the stationary phases was evaluated by use of a test mixture according to the Standard Reference Material 870 set from the US National Institute of Standards and Technology, in order to study the effect of branched and unbranched alkyl chains.

Monitoring the interactions of tocopherol homologues with reversed-phase stationary HPLC phases by 1H suspended-state saturation transfer difference high-resolution/magic angle spinning NMR spectroscopy.

The separation process in reversed-phase high-performance liquid chromatography employing C18 phases is mainly due to hydrophobic interactions. The separation of tocopherol isomers, exhibited by the C30 phases, however, is additionally driven by shape selectivity. This phenomenon is investigated by suspended-state nuclear magnetic resonance spectroscopy using the saturation transfer difference technique, which was originally introduced to study protein-ligand interactions. The interaction strength between beta-/gamma-tocopherol and three different stationary phases was estimated qualitatively. The nuclear magnetic resonance data are compared to chromatographic data, and a similar mode of interaction between the analytes and the stationary phases is elucidated.