Retention silica-based reversed-phase materials

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. 

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. 

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. 

The silica gel-based column packings are the active materials of choice for polymer HPLC employing both exclusion and interaction retention mechanisms. These are either bare or bonded with various groups. C-18 alkyls and -CH2-CH2-CH2-NH2 groups are most popular for reversed-phase and normal-phase procedures of polymer HPLC employing the nonpolar and polar interactions, respectively. 

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. 

Carbon-based material on a silica template has been pioneered by Knox (34). It can be used at any pH. However, the mechanism of retention on this support is quite different from that for the average alkyl-bonded silica (35). Further information on reversed-phase retention can be found in Ref. 36. 

Porous graphitic carbon (PGC) has a rigid structure, is chemically stable and has a very hydrophobic surface. It can therefore be used for reversed-phased HPLC, where it displays a similar or greater degree of retention to ODS. It has excellent pH stability and hence ion suppression of either acidic or basic drugs is easily carried out on this material. It displays different selectivity to silica based packings, allowing isomeric separations more typical of unmodified silica to be carried out (Bassler, 1989). 

Ion suppression. By controlling the ionisation of weakly ionic samples they are retained on reversed phase columns for example, a weak acid is retained using a eluent with a pH below the of the acid. But silica-based materials can only normally be used between pH 1.5-7.5 so increased retention for bases cannot usually be obtained in this way. 

HPLC analysis of taxanes is achieved almost exclusively in reversed phase mode on various stationary phases. The normal-phase HPLC mode has been applied in very limited cases and resulted in broad peaks and long analysis times (retention times of 45 min for paclitaxel and 38 min for cephalomannine). The namre of the sample is the main criterion for the choice of the stationary phase. Analysis of plant material is performed mostly on phenyl, biphenyl, and pentafluorophenyl materials, but silica-based cyano, Cig, and Cg materials have been used as well. C18 phases are the most common material utilized in pharmacokinetic studies. Mobile phases typically consist of mixtures of methanol, acetonitrile, and water or buffer (mostly ammonium acetate). Detection is performed by UV, mostly in the low region of 225 -230 nm. Taxanes give similar UV spectra with a minimum at 210-215 nm and a maximum at 225-232 nm. Therefore, detection is performed, preferably at 227-228 nm. Dual/multiple UV detection is performed in both low and upper regions, e.g. 227 and 273 nm 230 and 280 nm 227, 254, and 270 nm, etc. (Fig. 2). 

Both hydrophobic and anionic character are exhibited on bonded silica-based anion-exchange phases. Hydrophobicity is introduced through the alkyl chain portion of the ligand which is buri under the cationic ammonium site. El Rassi and Horvath (47) have studied several different anion-exchange supports under reversed-phase mobile phase conditions of methanol and water. Compared to simple reverscd-phasc materials, similar retention behavior on a homologous series of n-alkybenzenes (CHjfCHjjaQH, where n 0- 3] was observed once phase ratios were normalized. 

Metal contamination of the matrix of the silica, especially by aluminum and iron, also increases the acidity of surface silanols and the heterogeneity of the surface. This is a problem that largely plagues older stationary phases, which are based on silicas derived from inorganic raw materials. Many modem silicas are manufactured from organic silanes. In such processes, a high purity of the silica can be maintained with the appropriate precautions. In turn, this results in reversed-phase bonded phases with superior behavior toward basic compounds, that is, without excessive retention or taOing. 

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