Reversed phase chemically bonded

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... 

C. Reversed-phase chemically bonded plates Whatman KC,g plates consist of a layer of sorbent produced by reacting octadecyl monochlorosilane with the surface silanol groups of a special silica gel [30]. This presents a nonpolar hydrocarbon surface to the solutes for separations by reversed-phase TLC. The advantage of this chemically bonded medium compared to conventional impregnated nonpolar phases... 

In reversed-phase TLC, mobile phases are generally water mixed with an organic modifier. Solvent strength increases with increasing concentration and decreasing polarity of the modifier. For separations on reversed-phase chemically bonded Ci8 layers, the empirical solvent strength parameter S can be used as a measure of solvent strength (Snyder et al., 1979). 

Figure 4.4 Separation of SRM 1647 and SRH 869 polycyclic aromatic hydrocarbon test mixtures on a monomeric and polymeric reversed-phase octadecylsiloxane bonded phases by gradient elution. (Reproduced with permission from ref. 69. Copyright American Chemical Society).

Reversed-phase chromatography is the term used to describe the state in which the stationary phase is less polar than the mobile phase. Chemically bonded octadecylsilane (ODS), an -al kane with 18 carbon atoms, is the most frequently used stationary phase. Cg and shorter alkyl chains and also cyclohexyl and phenyl groups provide other alternatives. Phenyl groups are more polar than alkyl groups. 

The highly specific nature of these interactions is due to the fact that the two participating compounds are ideally suited to each other both spatially and electrostatically. One component (ligand) is bonded to a support (in a similar way to a phase chemically bonded to silica) and the other (sample) is adsorbed from solution, the process being reversible (Figure 16.1). 

Permaphase ODS, 1 m X 2.1 mm I.D., reversed-phase partition column with octadecylsilane as stationary phase, chemically bonded to a controlled porous surface ( porosity beads , 20-37 pm) by means of Si-O-Si bonds and contains about 1% stationary phase. Pre-packed Permaphase ODS column has 600 theoretical plates, determined by isocratic elution of anthracene with methanol-water (6 4)... 

D. E. Martire and R. E. Boehm, A unified theory of retention and selectivity in liquid chromatography. 2. Reversed-phase liquid clrromatography with chemically bonded phases , J. Phys. Chem. 87 1045-1062 (1983). 

Quality of the adsorbent layer. Layers for HPTLC are prepared using specially purified silica gel with average particle diameter of 5-15 /mi and a narrow particle size distribution. The silica gel may be modified if necessary, e.g. chemically bonded layers are available commercially as reverse-phase plates. Layers prepared using these improved adsorbents give up to about 5000 theoretical plates and so provide a much improved performance over conventional TLC this enables more difficult separations to be effected using HPTLC, and also enables separations to be achieved in much shorter times. 

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. 

Reversed-phase PLC precoated plates are based on silica gel matrices with chemical modifications in such a manner that the accessible polar, hydrophilic silanol groups at the silica gel surface are replaced by nonpolar, hydrophobic alkyl chains via silicon-carbon bonds. For preparative purposes, up to now only PLC precoated RP plates with C-18 modification are available. This abbreviation is often also designated as RP-18, meaning that an octadecyl alkyl chain is chemically bonded to the silica gel surface. 

Solvent strength determines the value, but not the selectivity. The mobile phase can be established by using the polarity index P proposed by Snyder. The highest values of P represent the strongest solute adsorbed in conventional TLC but represent the weakest for the separation in reversed phases. Sometimes aqueous polar mixtures cannot totally wet the chemically bonded layer. For this reason, checking... 

Chemically bonded stahonary phases, e.g. alkylamide silica reversed phases, were also developed. Despite a generally good stability and good quality of resolution and less interachons with free silanol groups, correlahons between log Pod and log kw are relahvely poor compared to a number of other stahonary phases [26]. Finally, monolithic silica stahonary phases have also been applied for Upo-philicity determinahon of a series of P-blockers [27]. 

Buszewski, B., Gadza-la-Kopciuch, R. M., Markuszewski, M. L, Kaliszan, R. Chemically bonded silica stationary phases synthesis, physicochemical characterization, and molecular mechanism of reversed-phase HPLC retention. Anal. Chem. 1997, 69, 3277-3284. 

The PRISMA model was developed by Nyiredy for solvent optimization in TLC and HPLC [142,168-171]. The PRISMA model consists of three parts the selection of the chromatographic system, optimization of the selected mobile phases, and the selection of the development method. Since silica is the most widely used stationary phase in TLC, the optimization procedure always starts with this phase, although the method is equally applicable to all chemically bonded phases in the normal or reversed-phase mode. For the selection of suitable solvents the first experiments are carried out on TLC plates in unsaturated... 

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