Bonded stationary phases principle

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 most popular and versatile bonded phase is octadecylsilane (ODS), n-C18H37, a grouping that is non-polar and used for reverse phase separations. Octylsilane, with its shorter chain length, permits faster diffusion of solutes and this results in improved peak symmetry. Other groups are attached to provide polar phases and hence perform normal phase separations. These include cyano, ether, amine and diol groups, which offer a wide range of polarities. When bonded stationary phases are used, the clear distinction between adsorption and partition chromatography is lost and the principles of separation are far more complex. 

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 two examples that have been given are simple and basic, and illustrate the principles of a TLC separation. Ion exchange material can also be bonded to the silica, allowing ionic interactions to be dominant in the stationary phase and, thus. 

Over 100 stationary phases of various types have been described in the literature for packed columns, which are slowly being abandoned. However, for bonded phase capillary columns the choice of stationary phase is limited because the generation of the film at the surface of the column requires a different principle than impregnation. Generally, two families of compounds are used to modify the polarity polysiloxanes and polyethylene (silicones) glycols. Very special phases such as cyclodextrins can be used for enantiomeric separations. Stationary phases can be used between a minimum temperature under which equilibrium is too slow to occur and a maximum temperature above which degradation of the polymer occurs. The maximum temperature depends on the film thickness and the nature of the polymer. 

In this section an overview of the numerous methods and principles for the discrimination of enantiomers is given. First, the interaction principles of the polymer-based methods adapted from chromatographic procedures are illustrated. The discrimination of enantiomers was achieved some decades ago by using different types of stationary materials, like cyclodextrins or polymer-bonded amide selectors. These stationary-phase materials have successfully been appointed for label-free optical sensing methods like surface plasmon resonance (SPR) or reflectometric interference spectroscopy (RIfS). Furthermore, various successful applications to optical spectroscopy of the well-established method of fluorescence measurements for the discrimination of enantiomers are described. 

One of the main principles for chiral separation used in modern capillary GC is the bonding of the optically active compounds via hydrogen bridges to a stationary-phase material. Feibush and Gil-Av [8] suggested a rapid and reversible formation of association complexes between carbonyl and amide functions of selector and selectand. The formation of diastereomeric associates yields complexes of different stability, depending on the relative configuration. The introduction of dipeptide and diamide phases leads... 

In principle, the composition of the stationary phase may be varied by using mixed phases . Phases which incorporate different functional groups in a given ratio have been synthesized (see for example ref. [348]). However, retention may not be expected to vary linearly with the composition of such mixed phases in a manner similar to what is observed with mixed liquid phases in GLC (section 3.1.1). This, combined with the complications involved in preparing mixed phases and the irreversibility of bonding reactions, excludes the composition of mixed CBPs as a practical parameter for optimization purposes. 

The interaction by ji coordination of Ag(I) between silver ions and C=C double bonds or aromatic groups is the basic principle for many effective chromatographic separations. Silver trifluoroacetate and silver trifluoromethanesulfonate are very soluble in poly(methylphenylsiloxane), due to the presence of phenyl groups. Such stationary phases were studied for the GC separation of benzene-cyclohexene-cyclohexane mixtures. The salting-out effects and formation constants of the complex of Ag(I) with various olefinic and aromatic compounds were estimated based on retention time measurements271. 

In this chapter stationary phases whose interaction with compounds to be separated is significant are examined interactions based on the formation of hydrogen bonds are not examined here, although it is in principle a particular case of chemical interactions. Separations on stationary phases with the formation of hydrogen bonds were examined by logansen and Kurkchi [5]. The separation of diastereoisomers of a number of organic substances is based on the selective formation of intermolecular hydrogen bonds [6]. 

In reversed-phase chromatography, a nonpolar stationary phase is used in conjunction with polar, largely aqueous mobile phases. Between 70 and 80% of all HPLC applications utilize this technique. Its popularity is based largely on its ease of use equilibration is fast, retention times are reproducible, and the basic principles of the retention mechanism can be understood easily. Most stationary phases are silica-based bonded phases, but polymeric phases, phases based on inorganic substrates other than silica, and graphitized carbon have found their place as well. 

In liquid chromatography, separations will occur based on one of two principles partition and adsorption. With partition (liquid-liquid chromatography), a liquid stationary phase is used. This phase will be coated onto or chemically bonded onto a finely divided inert support (the latter is more common). With all forms of liquid chromatography, as the name suggests, we have a liquid mobile phase. 

Wahlund applied ion-pair principles using alkyl-bonded silica as the support for pentanol as liquid stationary phase [4]. Tetrabutylammonium was used as counter-ion in the mobile aqueous phase for the separation of anionic compounds such as barbiturates, carboxylates, sulfonamides and sulfonates [4, 50). Hydrophobic amines were separated as ion-pairs with inorganic anions, with long-chain ammonium ions added to the mobile phase to improve peak symmetry [23]. The content of penlanol in the mobile phase had a decisive influence on the retention. The value found for approached that calculated (eqn. 8) when the mobile phase was saturated with pentanol. At lower concentrations of pentanol, adsorption onto the hydrophobic support had a strong influence on kj.. [51]. 

The basic principle of SFC is partition between the supercritical mobile phase and the stationary phase, which is either a packing of silica particles on the surface of which are bonded a variety of functional groups (cf. high-... 

In ion pair separations with a hydrophobic solid stationary phase, most often alkyl-bonded silica, the binding capacity is limited. The principles of retention in such a system can be expressed as follows (1) the retention of an ionic solute, HA, is accompanied by the displacement of a system ion of the same charge or by the binding of a counterion, X and (2) the solute and the components in the mobile phase may compete for the binding capacity of the adsorbing solid phase. 

The basic principle of SPE technology is a variation of chromatographic techniques based around small disposable cartridges packed with silica gel, or bonded phases that are in the stationary phase. The sample is loaded in one solvent, generally under reduced pressure rinsed, where most of the contaminants are removed and eluted in another solvent [37],... 

Figure 4.14 Schematic illustration of the principles underlying design of Pirkle-type chiral stationary phases (CSPs). (a) Illustration of the concept of reciprocity a single enantiomer of a racemate which separates well on the CSP shown on the left, when used to produce a second CSP shown at the right, will usually afford separation of the enantiomers of analytes that are structurally similar to the chiral selector of the first CSP. Reproduced from Pirkle et al, J. Org. Chem. 57 (1992), 3854, Copyright (1992), with permission of the American Chemical Society, (b) Two CSPs that exhibit reciprocal behavior, and (c) enantiomeric recognition model for the more stable diastereomeric complex between (S)-naproxen dimethylamide and the Whelk-0-1 (3R,4R) analog. Note that hydrogen atoms bonded to carbons are omitted for clarity. Reproduced from Wolf and Pirkle (2002), Tetrahedron 58, 3597, copyright (2002), with permission from Elsevier.

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