Polar chemically bonded stationary

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... 

Liquid-solid chromatography (LSC), sometimes referred to as normal phase or straight phase chromatography, is characterized by the use of an inorganic adsorbent or chemically bonded stationary phase with polar functional groups and a nonaqueous mobile phase... 

The influence of the bonded organic moiety on solute retention has not yet been elucidated and only a very small number of papers discuss the properties and use of such phases so far. The numerous advantages of chemically bonded phases make the application of polar chemically bonded phases with nonpolar eluents quite attractive even if the standardization of these phases may pose problems 106) similar to those encountered in the standardization of aidsorbents as well as of polymeric liquid phases in gas chromatography. A detailed discussion of the properties and chromatographic use of bonded stationary phases is given by Melander and Horvath (this volume). 

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. 

Gas Chromatography. The basic components of a gas chromatograph are a carrier gas system, a column, a column oven, a sample injector, and a detector. Very pure helium is the near-universal carrier gas for environmental and many other analyses. Open tubular GC columns are constructed of fused silica with low-bleed stationary phases of varying polarity chemically bonded to the silica surface. Columns are typically 30-75 m in length and have inside diameters (ID) in the range of about 0.25-0.75 mm. The column oven is capable of precise temperature control and temperature programming at variable rates for variable times. 

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. 

In order to accomplish the desired separation, the selection of appropriate stationary phase and eluent system is imperative. The most commonly used stationary phases in normal-phase chromatography are either (a) inorganic adsorbents such as silica and alumina or (b) moderately polar chemically bonded phases having functional groups such as aminopropyl, cyanopropyl, nitrophenyl, and diol that are chemically bonded on the silica gel support [16]. Other phases that are designed for particular types of analytes have also... 

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.

The difficulties encountered in LLC can be overcome by the use of chemically bonded stationary phases or bonded-phases. Most bonded phases consist of organochlorosilanes or organoalkoxysilanes reacted with micro-particulate silica gel to form a stable siloxane bond. The conditions can be controlled to yield monomeric phases or polymeric phases. The former provides better efficiency because of rapid mass transfer of solute, whereas the polymeric phases provides higher sample capacity. BPC can be used in solvent gradient mode since the stationary phase is bonded and will not strip. Both normal-phase BPC (polar stationary, non-polar mobile) and reversed-phase BPC (non-polar stationary, polar mobile) can be performed. The latter is ideal for substances which are insoluble or sparingly soluble in water, but soluble in alcohols. Since many compounds exhibit this behaviour, reversed phase BPC accounts for about 60% of published applications. The main disadvantage of silica bonded phases is that the pH must be kept between 2 to 7.5. However, bonded phases with polymer bases (polystyrene-divinylbenzene) can be used in the pH range of 0 to 14. 

The stationary phases used in reversed-phase chromatography, when it was first introduced, comprised of a non-polar substance (e.g. squalene) coated on to a silica-based support. These are now seldom used. The stability of such systems is low, because the forces holding, say, squalene to even a silylated silica are so weak that the stationary phase is easily washed from the column. A compromise reversed-phase packing material was developed, which had a polymeric hydrocarbon stationary phase on the support, but although quite successful it has now been superseded by a chemically bonded stationary phase of which some examples are discussed below. 

NPLC stationary phases include metal oxides and moderately or strongly polar chemically bonded phases. Unmodified silica gel and silica-based bonded phases are most frequently used nowadays. Considerable effort in the development of new HPLC column packing materials in the past years has resulted in significant improvement of the column efficiency, reproducibility, and increased stability at elevated temperatures and at higher pH, enabling better compatibility with HPLC/mass spectrometry techniques and rapid analyses. Even though the new column technologies were primarily focused on RPLC separations, normal-phase HPLC also benefits from the improved properties of the support materials with uniform small particles and well-defined pore size. 

Recently TLC methods using chemically bonded stationaiy phases in combination with polar eluents have been published (23-27). Although the first official use of this type of capillary action planar chromatography was for the separation of digoxin and its related glycosides in USP (28), the principle has also been recommended by BP88 for the analysis of testosterone esters (29) it can be stated that straight phase (NP/TLC) on chemically bonded stationary phases, or reversed phase (RP/TLC) on chemically bonded stationary phases has not been widely used in steroid analysis. The potential of these techniques is evident, as demonstrated with some examples below. 

The problems of stationary phase erosion can be largely overcome by solvent-generated LLC, where the stationary liquid phase is generated dynamically by the mobile phase, in this approach, one of the phases of an equilibrated liquid-liquid system is applied as a mobile phase to a solid support which is better wetted by the other phase of the liquid-liquid system. The support is usually silica when the stationary phase is aqueous or a polar solvent and a reversed-phase chemically bonded support when the stationary phase is a nonpolar solvent. Under these Conditions a multimolecular layer is formed on the surface of the solid support which has the properties of the liquid phase in... 

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