Chemically bonded stationary phases for

The first chemically bonded stationary phases for HPLC were prepared by reacting the surface silanol groups of silica with an alcohol as shown below ... 

The commonly used chemically bonded stationary phases for HPLC, marketed under their trade names by various suppliers, are listed in Table 10.6 (Commercial products presently available under these trade names may have specifications which are different from those given in the Table). 

Chemically bonded stationary phases for high performance liquid chromatography... 

Kuwata, K., M. Uebori, and Y. Yamazaki, Rapid Method for Packing Microparticulate Columns Packed with a Chemically Bonded Stationary Phase for High-Performance Liquid Chromatography, J. Chromatogr. 211, 378-382 (1981). 

SEM has been a primary tool for characterizing the fundamental physical properties of oxide materials for some time. For example, SEM is particularly useful for determining the particle shape and approximate size distribution of various silica materials used as supports in chemically bonded stationary phases for chromatography [8]. The visual images provide resolution at the micron to in some cases the submicron level so that surface morphology can be determined. This information is especially useful when evaluating a new synthetic approach to the formation of oxide materials. For example, a recently developed method... 

Reverse phase chromatography is finding increasing use in modern LC. For example, steroids (42) and fat soluble vitamins (43) are appropriately separated by this mode. Reverse phase with a chemically bonded stationary phase is popular because mobile phase conditions can be quickly found which produce reasonable retention. (In reverse phase LC the mobile phase is typically a water-organic solvent mixture.) Rapid solvent changeover also allows easy operation in gradient elution. Many examples of reverse phase separations can be found in the literature of the various instrument companies. 

Tswett s initial column liquid chromatography method was developed, tested, and applied in two parallel modes, liquid-solid adsorption and liquid-liquid partition. Adsorption ehromatography, based on a purely physical principle of adsorption, eonsiderably outperformed its partition counterpart with mechanically coated stationary phases to become the most important liquid chromatographic method. This remains true today in thin-layer chromatography (TLC), for which silica gel is by far the major stationary phase. In column chromatography, however, reversed-phase liquid ehromatography using chemically bonded stationary phases is the most popular method. 

The great versatility of HPLC lies in the fact that the stability of the chemically bonded stationary phases used in partition chromatography allows the use of a wide range of liquids as a mobile phase without the stationary phase being lost or destroyed. This means that there is less need for a large number of different stationary phases as is the case in gas chromatography. The mobile phase must be available in a pure form and usually requires degassing before use. The choice of mobile phase (Table 3.6) is influenced by several factors. 

This problem was remedied by the discovery of methods for chemically bonding the active stationary phase to the inert support. Most chemically bonded stationary phases are produced by covalent modification of the surface silica. Three modification processes are shown in Equations 3.6-3.8. 

The use of nonpolar chemically bonded stationary phases with a polar mobile phase is referred to as reverse-phase HPLC. This technique separates sample components according to hydrophobicity. It is widely used for the separation of all types of biomolecules, including peptides, nucleotides, carbohydrates, and derivatives of amino acids. Typical solvent systems are water-methanol, water-acetonitrile, and water-tetrahydrofiiran mixtures. Figure 3.15 shows the results of protein separation on a silica-based reverse-phase column. 

Chemically bonded phases (CBP s) are very commonly used in LC, and occasionally also in GC. Such phases cannot be seen as either a solid or a liquid. The common term [201] used for LC involving such phases is bonded phase chromatography (BPC). To be consistent, the stationary phase identification should follow that of the mobile phase in defining the chromatographic system. Hence, LBPC should be used for liquid chromatography using chemically bonded stationary phases. 

For the samples that will be subjected to other (so-called interactive) LC techniques, the next question involves the nature of the solvent in which the sample has been or can be dissolved. If this is a non-polar solvent, such as n-hexane, then the sample solution is compatible with Normal Phase LC (NPLC), in which mobile phases with a relatively low polarity are used in combination with more polar stationary phases (see section 3.2.3). In this form of chromatography solid adsorbents (such as silica or alumina) may be used as stationary phases (LSC). Alternatively, polar chemically bonded stationary phases may be used (see section 3.2.2). 

Polar chemically bonded stationary phases (section 3.2.2.2) may be used as an alternative stationary phase for both RPLC and LSC, if variations in the mobile phase do not result in an adequate separation. If polar CBPs are used in combination with more polar mobile phases (reversed phase mode), then table 3.10c may be used to find the most appropriate optimization parameters. If operated in the normal phase mode, table 3.1 Od... 

The columns used for the GC separation of phytosterols are currently almost exclusively capillary columns with 0.1-0.3 mm internal diameter, and fused-silica capillary columns with chemically bonded stationary phases are commonly used (Abidi, 2001). The best separation of structurally very similar sterols, such as sitosterol and its saturated counterpart sitostanol, is obtained with slightly polar stationary phases like 5% diphenyl-95% dimethylpolysiloxane, and they are currently the most used columns for the separation of phytosterols (Lagarda et al., 2006). For detailed lists of different columns used in sterol analysis, see the papers by Abidi (2001) and Lagarda (2006). 

We now have a fairly adequate understanding of the different properties, including the particle diameter i/p, the pore size, the degree of permeability, and the chemical composition of the surface of the support matrix, to know which type of stationary phase can be successfully used with a particular class of peptides. Most of the HPLC packing materials now in use for peptide separations are based on the wide pore microparticulate silica gels with polar or nonpolar carbonaceous phases chemically bonded to the surface of the matrix. Methods for the preparation of these chemically bonded stationary phases, their available sources of supply. 

Bonded stationary phases for NPC are becoming increasingly popular in recent years owing to their virtues of faster column equilibration and being less prone to contamination by water. The use of iso-hydric (same water concentration) solvents is not needed to obtain reproducible results. However, predicting solute retention on bonded stationary phases is more difficult than when silica is used. This is largely because of the complexity of associations possible between solvent molecules and the chemically and physically heterogeneous bonded phase surface. Several models of retention on bonded phases have been advocated, but their validity, particularly when mixed solvent systems are used as mobile phase, can be questioned. The most commonly accepted retention mechanism is Snyder s model, which assumes the competitive adsorption between solutes and solvent molecules on active sites... 

Fig. 1.8. Chemical modification of silica gel in the preparation of (A) a monomeric and (B) a polymeric non polar alkyl bonded stationary phase for RPC by reaction with mono-, di- and tri-funclional alkylchlorosilanes.

There are several basic rules for the preparation of chemically bonded stationary phases. 

The separation of enantiomers is especially important in the pharmaceutical field, because drag enantiomers may produce different effects in the body. Enantiomer separations by chromatography require one of the components of the phase system to be chiral. This can be achieved by (a) the addition of a chiral compound to the mobile phase, which is then used in combination with a nonchiral stationary phase, or (b) the use of a chiral stationary phase in combination with a nonchiral mobile phase. The chiral phase can either be a solid support physically coated with a chiral stationary phase liquid or a chemically bonded chiral phase. For mobile-phase compatibility reasons, a chiral stationary phase is preferred in LC-MS. However, most chiral stationary phases have stringent demands with respect to mobile-phase compositiorr, which in turn may lead to compatibility problems. Three types of phase systems are applied in LC-MS ... 

The development of chemically bonded stationary phases is one of the major factors that lead to the growth of high-performance liquid chromatography (HPLC) and is responsible for its importance as a separation technique. 

GC on a fused silica capillary column with an MS detector should be used whenever possible for the analysis of organic compounds at trace level in complex mixtures. In fact, it allows the extremely high resolution of GC to be combined with the very high sensitivity and identification power of MS, which makes it possible to determine an analyte at low pg mC levels in the final organic extract. However, GC-ECD is very common for PCB determination since it is both the most sensitive and the less expensive technique for chlorinated compounds (5). PCBs can be separated on a 30-50 m fused silica capillary column with 5% phenyl -95% methylpolysiloxane chemically bonded stationary phase (1). On-column injection is very often used, while several oven temperature programmes have been applied for PCB determinations. The initial temperature is generally 10-15°C lower than the boiling point of the solvent and the final one does not exceed 290-300°C. 

Because the chemically bonded PEG molecules are thought to be arranged on the support surface like "bristles of a brush", Mori1 concluded that the rate of mass transfer should be increased, making chemically bonded stationary phases very suitable for gas chromatographic separations. 

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