Size-exclusion chromatography silica-based packings

Also, silica-based packings are available for aqueous size-exclusion chromatography. The commercially available ones are coated with a glycidoxypro-pylsilane bonded phase. While they do not swell or shrink, they are not compatible with basic pH values and contain residual silanols that may interact with the polymer. Also, only a limited pore-size range is available compared to the aqueous size-exclusion chromatography columns based on glycidyl methacrylate. 

Two classes of micron-sized stationary phases have been encountered in this section silica particles and cross-linked polymer resin beads. Both materials are porous, with pore sizes ranging from approximately 50 to 4000 A for silica particles and from 50 to 1,000,000 A for divinylbenzene cross-linked polystyrene resins. In size-exclusion chromatography, also called molecular-exclusion or gel-permeation chromatography, separation is based on the solute s ability to enter into the pores of the column packing. Smaller solutes spend proportionally more time within the pores and, consequently, take longer to elute from the column. 

For the classical form of size exclusion chromatography in organic solvents, packings based on highly cross-linked styrene-divinylbenzene are used. For SEC of polar polymers using polar or aqueous solvents, packings based on a polar methacrylate polymer are used. Diol-derivatized silica is used for the separation of proteins and other polar polymers. The different packings will be discussed in sections dedicated to their different application areas. 

Protein-Pak packings are designed for the size exclusion chromatography of proteins and related compounds. They are based on silica, which is deactivated with glycidylpropylsilane. The diol function prevents the interaction of the target analytes with the silica surface. However, because coverage of the silica surface is always incomplete, residual acidic silanols can interact with the analytes. For this reason, most applications are carried out with a salt concentration above 0.2 mol/liter, which eliminates the interaction of analytes with surface silanols. Protein-Pak packings are stable from pH 2 to pH 8. 

Neutral hydrophilic polymeric packings are also useful for HILIC (6). Examples are the hydrophilic methacrylate phases, which were originally designed for aqueous size-exclusion chromatography. The surface of these packings consists of polyol groups, which are very hydrophilic. These phases tend to be more retentive than silica-based bonded phases. 

Modified Silica-Based Packing Materials for Size Exclusion Chromatography... 

Unfortunately, exclusion chromatography has some inherent disadvantages that make its selection as the separation method of choice a little difficult. Although the separation is based on molecular size, which might be considered an ideal rationale, the total separation must be contained in the pore volume of the stationary phase. That is to say all the solutes must be eluted between the excluded volume and the dead volume, which is approximately half the column dead volume. In a 25 cm long, 4.6 mm i.d. column packed with silica gel, this means that all the solutes must be eluted in about 2 ml of mobile phase. It follows, that to achieve a reasonable separation of a multi-component mixture, the peaks must be very narrow and each occupy only a few microliters of mobile phase. Scott and Kucera (9) constructed a column 14 meters long and 1 mm i.d. packed with 5ja... 

The factors that control separation and dispersion are quite different. The relative separation of two solutes is solely dependent on the nature and magnitude of the Interactions between each solute and the two phases. Thus, the relative movement of each solute band would appear to be Independent of column dimensions or particle geometry and be determined only by the choice of the stationary phase and the mobile phase. However, there is a caveat to this statement. It assumes that any exclusion properties of the stationary phase are not included in the term particle geometry. The pore size of the packing material can control retention directly and exclusively, as in exclusion chromatography or, indirectly, by controlling the access of the solute to the stationary phase in normal and reverse phase chromatography. As all stationary phases based on silica gel exhibit some exclusion properties, the ideal situation where the selective retention of two solutes is solely controlled by phase interactions is rarely met in practice. If the molecular size of the solutes differ, then the exclusion properties of the silica gel will always play some part in solute retention. 

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