Column packings surface

Volumetric liquid rate per unit cross-sectional area of column Packing surface area per unit volume of column L uL... 

Let us first consider the situation when macromolecules do not exhibit any attractive or repulsive interaction with the porous column packing surface except for the effects caused by the imperviousness of the pore walls. This corresponds to AH=0 in Equation (17) and the sample retention volume is controlled exclusively by the entropy of process. The fictitious retention volume of eluent molecules corresponds to total volume of liquid within column, becanse the small molecules... 

The same problems bring about the system-peaks, which are common to mixed mobile phases. The latter result from the preferential solvation of dissolved macromolecules (see section 11.2.4), from the preferential evaporation of one eluent component from the sample solution, and also from the displacement of the mobile phase component, which is preferentially sorbed on the column packing surface. To stress again, the enthalpic interactions between macromolecules and colunm packings are to be carefully suppressed (compare section 11.5.2.3). Unfortunately, this is often not the case in the routine laboratory practice. 

Silica can be used as is for normal phase HPLC in which the mobile phase is nonpolar in nature, such as hex-ane/chloroform and the column-packing surface is polar. For reversed-phase HPLC in which the mobile phase is polar, such as water or methanol, and the column-packing surface is nonpolar, the silica must be chemically altered. Silanization reactions are carried out to covalently attach long chain hydrocarbon groups to the silica surface. Further details of the importance of silica supports are provided in Section III, Separation Techniques. ... 

If the packing surface is discontinuous in nature, a phase inversion occurs, and gas oubbles through the liquid. The column is not unstable and can be brought back to gas-phase continuous operation by merely reducing the gas rate. Analogously to the flooding condition, the pressure drop rises rapidly as phase inversion occurs. 

The analysis demonstrates the elegant use of a very specific type of column packing. As a result, there is no sample preparation, so after the serum has been filtered or centrifuged, which is a precautionary measure to protect the apparatus, 10 p.1 of serum is injected directly on to the column. The separation obtained is shown in figure 13. The stationary phase, as described by Supelco, was a silica based material with a polymeric surface containing dispersive areas surrounded by a polar network. Small molecules can penetrate the polar network and interact with the dispersive areas and be retained, whereas the larger molecules, such as proteins, cannot reach the interactive surface and are thus rapidly eluted from the column. The chemical nature of the material is not clear, but it can be assumed that the dispersive surface where interaction with the small molecules can take place probably contains hydrocarbon chains like a reversed phase. 

Water-soluble polymeric dyes have been prepared from water-insoluble chromophores, viz., anthraquinone derivatives. Unreacted chromophore and its simple derivatives, which are all water-insoluble, remain in solution due to solubilization by the polymeric dye. A method has been developed to separate and quantitate the polymeric dye and these hydrophobic impurities using Sephadex column packing. The solvent developed has the property of debinding the impiirities from the polymer, and further allows a separation of the imp irities into discrete species. This latter separation is based on the functional groups on the impurity molecules, having a different interaction with the Sephadex surface in the presence of this solvent. The polymer elutes at the void volume... 

Cleanup of sample extract. Pipet 2.5 mL of the solution derived from Module GPC into a long-necked round-bottom flask or a pear-shaped flask and add 10 mL of isooctane. By rotating the flask slowly, carefully evaporate the solution to ca 1 mL in a rotary evaporator (water-bath temperature set at 30 0 °C). If an odor of ethyl acetate is still present, add isooctane again and repeat the evaporation. Repeat, if necessary, until no odor of ethyl acetate is present the ethyl acetate must be completely removed. Allow the solution to drain to the upper surface of the column packing and then place a graduated test-tube under the column. 

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