Retention mechanism of column materials

Chromatography finds applications in many scientific fields for analyses and separations of chemical and biological mixtures. Bonded-stationary-liquid phases are made of siloxane coatings on porous silica, or silica-based substances, which are formed as uniform, porous, and mechanically sturdy particles. It is a constant problem to understand why these surfaces retain solute molecules in fabricating stationary phase materials and designing methods for separations.

We are applying xenon NMR for studying the retention mechanisms. The appreciable polarizability and its relative inertness render xenon a sensitive probe to study local environments including molecular interaction, structures and dynamics. In addition, Xenon NMR signal can be increased for 4-5 orders of magnitude through optical pumping method, which makes xenon more powerful for exploring surface molecular structure and interaction.

SEM Image of a particle of Zorbax SB-C18 HPLC Column Material. Particle Size: 5 micrometers, Pore size: 80 Å.

Diagram of the HPLC packing materials and the solvated xenon atoms where the particles, the pores and the spaces between the particles are indicated. The hair in the pores and on the surfaces of the particles represents the hydrocarbon chains of the surface coating, and the dots represent xenon atoms.

VT 129Xe NMR spectra of xenon in the SB-C18 column material. The experimental temperatures are labeled with their corresponding spectra. At 22 °C, the sharp peak at 3.0 ppm shows the gas-phase xenon inside the silica pores and the spaces between the silica particles, and the broad peak at 54.5 ppm shows the xenon solvated in the stationary phase. At -70 °C, the sharp peak at 227.4 ppm arises from the liquid xenon inside the silica pores and the spaces between the silica particles, and the broader peak at 211.9 ppm is due to the solvated xenon in the stationary phase. At -150 °C, the peaks at 302.9, 297.4, and 276.5 ppm arise from the solid xenon possibly outside the column material, in the spaces between the silica particles, and in the pores of the silica particles, respectively, and the peak at 219.7 ppm is from the xenon trapped in the stationary phase of the column material.

129Xe 2D Exchange NMR spectra at -140 0C with 500 ms exchange time. It shows the diffusion of Xe atoms between the solid phases inside the pores and outside the pores. No diffusion between the adsorbed phase and the solid phases was observed. The broadening of the adsorbed Xe phase shows the slower motion of the C18 chains.

129Xe VT NMR spectra of the sample of SB-C18/cyclohexane/xenon.

129Xe NMR is sensitive to probe the interfacial interactions of the solvents with the stationary phase of the column packing material. It was observed that the highly polar ethylene glycol molecules do not mix with the alkyl chains of the SB-C18 stationary phase. Because the solvent does not wet the stationary surface resulting in capillary depression, the solvent is not able to enter the pores and the spaces between the particles. Three phases in the sample are clearly defined as stationary/xenon phase, xenon gas phase (in the pores and the spaces between the particles) and ethylene glycol/xenon phase (the solvent droplets coated the packing particles). In contrast to ethylene glycol, the nonpolar solvent cyclohexane was observed to be well mixed with the SB-C18 stationary phase. The capillary rise effect allows the solvent to enter the pores and the spaces between the particles. This actually resulted in a stationary/solvent phase. Two phases in this sample are defined as stationary/cyclohexane/xenon phase and cyclohexane/xenon phases. The properties of ethyl acetate are between those of ethylene glycol and cyclohexane, but closer to cyclohexane. This sample also formed two phases as stationary/ethyl acetate/xenon phase and ethyl acetate/xenon phase. However, the solubility of ethyl acetate in the stationary phase is lower than that of cyclohexane. We have chosen three solvents with typical polarity indices (0.04 for cyclohexane, 4.4 for Ethyl acetate and 6.9 for Ethylene glycol) for the study of polarity effect on the miscibility of solvents with the stationary phase. We think that the more popularly used solvents, acetonitrile (polarity index 5.8) and methanol (polarity index 5.1) would have close interactions to similar interactions with the stationary phase as that of ethyl acetate.

The 129Xe NMR results show that a gas could be trapped in the pores and small spaces between the packing particles when a solvent does not wet the stationary phase completely. This can decrease the contact interface between the mobile phase and the stationary phase; thus, decrease the rate of matter exchange between the mobile phase and the stationary phase. This observation shows the rational that reversed-phase HPLC columns should be conditioned from highly solvating to more polar solvents. Partition chromatography considers the equilibrium distribution of a solute in the stationary phase and the mobile phase. The 129Xe NMR results also show that pure stationary phase exists only when a highly polar solvent is used in reversed-phase chromatography. For a solvent with lower polarity, a stationary/solvent phase actually forms. This, together with the mobile phase, determines the selective factor for separating mixtures.

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