Stationary phase affinity chromatography

Stationary phase in chromatography, the porous solid or hquid phase through which an introduced sample passes. The different affinities the stationary phase has for a sample allow the components in the sample to be separated, or resolved. 

Two models have been proposed to describe the process of retention in liquid chromatography (Figure 3.3), the solvent-interaction model (Scott and Kucera, 1979) and the solvent-competition model (Snyder, 1968 and 1983). Both these models assume the existence of a monolayer or multiple layers of strong mobile-phase molecules adsorbed onto the surface of the stationary phase. In the solvent-partition model the analyte is partitioned between the mobile phase and the layer of solvent adsorbed onto the stationary-phase surface. In the solvent-competition model, the analyte competes with the strong mobile-phase molecules for active sites on the stationary phase. The two models are essentially equivalent because both assume that interactions between the analyte and the stationary phase remain constant and that retention is determined by the composition of the mobile phase. Furthermore, elutropic series, which rank solvents and mobile-phase modifiers according to their affinities for stationary phases (e.g. Table 3.1), have been developed on the basis of experimental observations, which cannot distinguish the two models of retention. 

In laboratory practice, an affinity matrix (stationary phase) is tailor-made for the protein to be purified and filled into a chromatography column. For process applications many different affinity gels are commercially available. The raw extract is then passed through the column by using a physiological buffer as the mobile phase. In this process, the desired product is adsorbed selectively by the ligand and all unwanted components are washed away. 

Schematic illustration of elution chromatography. Three solutes are separating depending on the affinity to stationary phase at different times.

Affinity Chromatography. This technique involves the use of a bioselective stationary phase placed in contact with the material to be purified, the ligate. Because of its rather selective interaction, sometimes called a lock-and-key mechanism, this method is more selective than other lc systems based on differential solubiHty. Affinity chromatography is sometimes called bioselective adsorption. 

Elution Chromatography The components of the mobile phase supphed to the cohimn ter feed introduction have less affinity for the stationary phase than any of the feed solutes. Under trace conditions, the feed solutes travel through the cohimn as bands or zones at different velocities that depend only on the composition of the mobile phase and the operating temperature and that exit from the cohimn at different times. 

FIG. 16-30 Operational steps in displacement chromatography, The column, initially equilibrated with a carrier solvent at time 0, is loaded with feed until time tp and supplied with displacer for a time to + tp. Development of the displacement train occurs during the time to and elution of the separated products ends at time tp. tp is the time required to remove the displacer from the column and restore the initial conditions, Components are niimhered in order of decreasing affinity for the stationary phase, [Reference Horoath et at, J, Ghromatogr, 2i8, 365 (1981). Reprinted with peimission of], Ghromatogr,]... 

TABLE 16-15 Concentrations and h-Function Roots for Displacement Chromatography of a Mixture of M-1 Components Numbered in Order of Decreasing Affinity for the Stationary Phase (Adapted from Frenz and Horvath/ 1985). 

Ion-exchange chromatography involves an electrostatic process which depends on the relative affinities of various types of ions for an immobilised assembly of ions of opposite charge. The stationary phase is an aqueous buffer with a fixed pH or an aqueous mixture of buffers in which the pH is continuously increased or decreased as the separation may require. This form of liquid chromatography can also be performed at high inlet pressures of liquid with increased column performances. 

In a chromatographic separation, the individual components of a mixture are moved apart in the column due to their different affinities for the stationary phase and, as their dispersion is contained by appropriate system design, the individual solutes can be eluted discretely and resolution is achieved. Chromatography theory has been developed over the last half century, but the two critical theories, the Plate Theory and the Rate Theory, were both well established by 1960. There have been many contributors to chromatography theory over the intervening years but, with the... 

The coupling of supercritical fluid extraction (SEE) with gas chromatography (SEE-GC) provides an excellent example of the application of multidimensional chromatography principles to a sample preparation method. In SEE, the analytical matrix is packed into an extraction vessel and a supercritical fluid, usually carbon dioxide, is passed through it. The analyte matrix may be viewed as the stationary phase, while the supercritical fluid can be viewed as the mobile phase. In order to obtain an effective extraction, the solubility of the analyte in the supercritical fluid mobile phase must be considered, along with its affinity to the matrix stationary phase. The effluent from the extraction is then collected and transferred to a gas chromatograph. In his comprehensive text, Taylor provides an excellent description of the principles and applications of SEE (44), while Pawliszyn presents a description of the supercritical fluid as the mobile phase in his development of a kinetic model for the extraction process (45). 

Chemical forces are normally irreversible in nature (at least in chromatography) and thus, the distribution coefficient of the solute with respect to the stationary phase is infinite or close to infinite. Affinity chromatography is an example of the use of chemical forces in a separation process. The stationary phase is formed in such a manner that it will chemically interact with one unique solute present in the sample and thus, exclusively extract it from the other materials... 

Affinity chromatography A form of chromatography in which separation is achieved by utilizing highly specific biochemical interactions, such as steric-or charge-related conditions, between me analyte and a molecule immobilized on a column. It is different from most forms of chromatography in mat analytes do not continuously elute from me column - only mose mat interact wim me stationary phase are retained and mus separated from omer components of me mixture under investigation. These immobilized materials are eluted from me column after all omer materials have been removed. 

Crude chloroform-methanol-water (30 60 8, v/v) extracts of immunostainedTLC bands were analyzed without further purification by nanoelectrospray low-energy mass spectrometry. The authors showed that this effective PLC/MS-joined procedure offers a wide range of applications for any carbohydrate-binding agents such as bacterial toxins, plant lectins, and others. Phenyl-boronic acid (PBA) immobilized on stationary support phases can be put to similar applications. This technology, named boronate affinity chromatography (BAC), consists of a chemical reaction of 1,2- and 1,3-diols with the bonded-phase PBA to form a stable... 

Ion-exchange chromatography (lEC) is used mainly for the separation of ions and easily ionized substances (e.g., substances that form ions by pH manipulation or complexation) in which one of the principal contributions to retention is the electrostatic attraction between mobile phase ions, both sa le and eluent, for immobilized ion centers of opposite charge in the stationary phase. The sample ions are separated based on differences in their relative affinity for the stationary phase ion centers compared to that of the mobile phase counterions in a dynamic exchange system, in which sample ions and eluent ions interact with multiple stationary phase ion centers as they pass through the column. Ion-... 

Affinity chromatography involves precisely the same kind of electrostatic, hydrophobic, dipolar, and hydrogen-bonding interactions described above, but the specificity of binding is extraordinarily high. Demands on the homogeneity of the stationary phase and on the rigidity of the support are often... 

The elution of [60]- and [70]fullerenes was measured in water-methanol as a function of temperature on a poly(octadecylsiloxane) phase.67 The retention was shown to be dependent on the surface tension of the stationary phase through a simple geometrical model in which the solute formed a cavity in the stationary phase. In affinity chromatography, it was demonstrated that low ligand density may be a requirement for specificity of binding.68... 

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