Silica-based Reversed-phase Materials

Some materials are specially made for low-pH applications (pH 1-2), while others are made for use up to pH 9-11. The polymeric materials provide better protection of the silica surface and will generally he better for use at high or low pH. However, even the polymeric silica-based materials should never he stored at 

The surface silanization will never be complete and all materials contain a number of residual silanol groups that may participate in secondary interactions. The extent of such interactions depends on the protective properties of the alkyl groups in the silane reactants. 

Depending on the extent of surface coverage by protecting groups, different Cl8 materials may behave very differently especially toward solutes with amino groups. 

The hydrophobic character of the stationary phase increases with the chain length of the alkyl chain in the silica-based packings. Chains with up to 30 carbon atoms (C30) and down to Cl are commercially available, but the C18 (octadecylsilane, ODS) packings dominate for most purposes. 

The C30 materials have been used mainly for separation of configuration isomers. 

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For silica-based reversed-phase materials without any special modifications, the following rules apply ... 

McKeown, Euerby, and Lomax have made a detailed assessment of silica-based reversed-phase materials for the analysis of basic compounds. Walhagen et al. have... 

Recent advances in column stationary phases are remarkable. High performance silica-based reversed-phase 3 to 5 jxm packing materials have been developed for biological sample separations... 

The CEC phases must be capable of carrying a charge to generate an EOE and appropriate moieties to facilitate the chromatographic processes. Silica-based reversed-phase packing materials have been most widely used in CEC. The use of polymeric and mixed-mode bonded particles has also been reported. Eor the silica-based phases, the carbon chains bonded on the silica surface provide the retention and selectivity for analytes, and the residual silanol groups on the surface of the silica are ionizable and generate the EOF. 

In addition to the silica-based, reversed-phase particles, polymeric particles are available that utilize a hydrophobic stationary phase, e.g., cross-linked polystyrene divinylbenzene. In these polymeric stationary phases, the backbone of the particle provides the opportunity for hydrophobic interactions. Although these packing materials are available in high-performance particles, the particles cannot withstand the high pressure that silica particles can and thus are typically used in medium- to low-pressure operations. The polymeric stationary phases usually have a series of aromatic... 

Polymeric resins have several advantages over the commonly used silica-gel based reverse phase materials. The cost of polystyrene-based resins is considerably less than bonded phase silica gel adsorbents. Also, the potential... 

The choice of reverse phase packing material will depend on the amount of information available on the component of interest and on other sample components. Initial tests such as solvent partitioning behavior, solubility m various solvents, and others see Chapter 1) can be used to estimate polarity and hence be of use in initial column/mobile phase selection. The most retentive of the silica-based reverse phase supports, Cl8 and C8, are a sensible first choice, as the retention of polar compounds is maximized, while the retention of nonpolar materials can be easily modulated by choice of eluent. If the compound of interest is very nonpolar (or the sample contains components that bind very strongly to retentive phases such as C8/C18), a shorter chain alkyl-bonded phase such as C6 or C4 may be more suitable. 

Knowledge of the materials surface chemistry is crucial. The required adsorbents are silicas with a chemically modified surface carrying bonded n-octadecyl or n-octyl groups. The long n-alkyl ligands invert the surface polarity from hydrophilic (native adsorbent) to hydrophobic. Silica-based reversed-phase columns are operated with aqueous/organic eluents and inversion of the elution order takes place (hydrophilic components elute first). More details are given in Table 3.15. 

A unified theory recently proposed to explain the manner of sorption and the form of sorption isotherm in gas, liquid, and ion-exchange chromatography is presented in some detail. Selectivity in reversed-phase high-pressure liquid chromatography is explored at length. Several chapters deal with characterization of bonded phases, relationship of column-packing structure and performance, variability of reversed-phase packing materials, and the differences between silica-based reversed-phase and poly(styrene-divinylbenzene) columns. A short review is included to cover various approaches used in HPLC to achieve the desired selectivity for resolution of enantiomeric compounds. 

In fact, every silica-based reversed phase represents a mixed-mode column, because besides the alkyl chains it has silanol groups, which are groups capable of weak cation exchange. Some C18 materials of the first generation, such as Partisil ODS from Whatman or Spherisorb ODSl from Waters, have only a very low carbon content (<5%) and a high number of free silanol groups. With these... 

A wide variety of SPE materials and cartridges are commercially available for example, alkyl-diol silica-based restrictive access materials (RAMs) and a variety of silica- and polymer-based SPE materials of different binding abilities and capacities. Reversed-phase, size-exclusion, ion-exchange SPE, and turbulence flow methods will be discussed in this chapter related to real-world applications. 

Ion pairing interactions need charged analytes to operate. Let us focus on the dependence of basic analyte retention as a function of pH. The hydrophobic retention of ionogenic bases at pH values two units above the basic analyte pK is the highest on reversed phase materials because the analyte is predominantly neutral. Separations at these pH levels may not be feasible because the pH stability thresholds of common silica-based stationary phases are exceeded, as discussed in Chapter 5. 

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