Polymeric stationary phase surface polymerization

The chromatographic behavior of C2-C12 alkane sulfonates was studied on a polymeric column (PRP-1) or a C18 siUca column with a metal cation (M, or as the only counterion in the mobile phase [4]. Retention of the sulfonates was beUeved to be accompanied by double layer formation at the stationary phase surface, and diffuse secondary layer is made up of counter cations. 

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. 

Chemical surface modifications The first surface modification for the purpose of eliminating EOF and protein adsorption was recommended by Hjerten.28 The attachment of vinyl silanes allowed the polymerization of a variety of molecules to the surface. Most of the chemical modifications used for preparing capillaries for electrophoresis originated from the experience acquired over the years preparing GC and LC stationary phases. Chemical modification should conform to certain requirements, including the prevention of adsorption, the provision of stable and constant EOF over a wide pH range, chemical stability, ease of preparation, and reproduciblity of preparation. The effects of silanization of the inner surface of capillaries on electrophoretic separations have been extensively studied.26-29... 

Reverse-phase HPLC (RP-HPLC) separates proteins on the basis of differences in their surface hydophobicity. The stationary phase in the HPLC column normally consists of silica or a polymeric support to which hydrophobic arms (usually alkyl chains, such as butyl, octyl or octadecyl groups) have been attached. Reverse-phase systems have proven themselves to be a particularly powerful analytical technique, capable of separating very similar molecules displaying only minor differences in hydrophobicity. In some instances a single amino acid substitution or the removal of a single amino acid from the end of a polypeptide chain can be detected by RP-HPLC. In most instances, modifications such as deamidation will also cause peak shifts. Such systems, therefore, may be used to detect impurities, be they related or unrelated to the protein product. RP-HPLC finds extensive application in, for example, the analysis of insulin preparations. Modified forms, or insulin polymers, are easily distinguishable from native insulin on reverse-phase columns. 

Another way to improve the performance of open-tubular columns was suggested by Sawada and Jinno [83]. They first vinylized the inner surface of a 25 pm i.d. capillary and then performed in situ copolymerization of f-butylacryl-amide and 2-acrylamido-2-methyl-l-propanesulfonic acid (AMPS) to create a layer of polymeric stationary phase. This process does not currently allow good control over the homogeneity of the layer and the column efficiencies achieved in CEC separations of hydrocarbons were relatively low. These authors also recently thoroughly reviewed all the aspects of the open tubular CEC technologies [84]. 

Reversed-phase liquid chromatography shape-recognition processes are distinctly limited to describe the enhanced separation of geometric isomers or structurally related compounds that result primarily from the differences between molecular shapes rather than from additional interactions within the stationary-phase and/or silica support. For example, residual silanol activity of the base silica on nonend-capped polymeric Cis phases was found to enhance the separation of the polar carotenoids lutein and zeaxanthin [29]. In contrast, the separations of both the nonpolar carotenoid probes (a- and P-carotene and lycopene) and the SRM 869 column test mixture on endcapped and nonendcapped polymeric Cig phases exhibited no appreciable difference in retention. The nonpolar probes are subject to shape-selective interactions with the alkyl component of the stationary-phase (irrespective of endcapping), whereas the polar carotenoids containing hydroxyl moieties are subject to an additional level of retentive interactions via H-bonding with the surface silanols. Therefore, a direct comparison between the retention behavior of nonpolar and polar carotenoid solutes of similar shape and size that vary by the addition of polar substituents (e.g., dl-trans P-carotene vs. dll-trans P-cryptoxanthin) may not always be appropriate in the context of shape selectivity. 

FIGURE 5.5 Synthesis schemes for chromatographic stationary phases (a) monomeric synthesis where X represents reactive (e.g., chloro or alkoxy) or nonreactive (methyl) substituents, (b) solution polymerization, in which water is added to the slurry and (c) surface polymerization, in which water is added to the silica surface. 

Sander et al. [63] investigated the effect of microparticulate silica pore size on the properties of solution-polymerized Cig stationary phases and observed both an increase in bonding density and shape recognition for wider pore (>120 A) silica. A size-exclusion mechanism was proposed, in which the reaction of the silane polymer on the surface is enhanced for wide pores and reduced for narrow pores. Polymeric Ci8 phases prepared on substrates with narrow pores exhibited monomeric-like chromatographic properties. This effect may be the result of an increase in competitive surface linkage with the less sterically hindered monomers that coexist with the bulkier oligomers that have polymerized in the reaction solution (Figure 5.13). 

Analytical shape computation techniques were applied for the detection of cavities and the calculation of molecular surface properties of isolated cavity features and other ordered formations within these resultant alkyl stationary-phase simulation models [227]. Deep cavities (8-10 A wide) within the alkyl chains were identified for Cig polymeric models representing shape selective stationary phases (Figure 5.23). Similar-structure cavities with significant alkyl-chain ordered regions (>11 A) were isolated from two independent Cig models (differing in temperature,... 

FIGURE 5.26 (See color insert following page 280.) A representation of the slot model illustrating potential constrained-shape solute (BaP) interactions with the conformational ordered cavities of a polymeric Cjg stationary-phase simulation model. Also included on the chromatographic surface is an identical-scale molecular structure of 1,2 3,4 5,6 7,8-tetrabenzonaphthalene (TBN). 

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