层析填料的选择及其装柱技术介绍幻灯片.ppt

Speaker′s notes In 90% of all RPC applications the stationary phase may be made up of silica (SiOH). The silica particles are formed into beads, which, in laboratory scale work, normally range between 3 μm - 15 μm in diameter. The smaller bead size, 3 μm, will give a very high resolution and the larger, 10 or 15 μm, is used for more preparative scale work. The bead surface is covered with ethers and residual silanol groups. The beads also have pores which may either be in the range of 50-100 ? (?ngstr?m) or in the range of 300 ?. The more narrow pore sizes will give a more restricted mass transfer and are on stationary phases used for peptides. The larger pore sizes give a more improved mass transfer and are used for separation of proteins, larger peptides and DNA. Speaker′s notes The beads are conjugated with alkylsilanes forming the C2, C8, C18 ligands. Also C1 and C4 ligands are used. The C2 (and C1) are often used for so called end capping, i.e. blockage of the residual silanol groups. These silanol groups will otherwise give rise to nonspecific adsorption of the solute and tailing peaks, which will be more asymmetrical as indicated by a large asymmetry factor. So capping these silanol groups with the C2 group is frequently used and very effective. The remaining silanol groups are conjugated with either C8 or C18 carbon chains and these are the ligands that provide the hydrophobic layer of the beads. The C18 is the longer and the more hydrophobic ligand and C8 is the less hydrophobic ligand. Stationary phases with the two kinds of ligands are to some extent used for the separation of different molecules. It is normally considered that the C8 is most suitable for separation of large peptides while C18 is most suitable for less hydrophobic substances like smaller peptides or organic molecules. One stationary phase which has proved very efficient for peptide mapping and separation of peptides in a protein digest is the μRPC C2/C18 3 μm. The small bead size guarant