Stationary phase, description

USP Nomenclature Code USP Stationary-Phase Description Similar Stationary Phases... 

Hydrodynamic volume refers to the combined physical properties of size and shape. Molecules of larger volume have a limited ability to enter the pores and elute the fastest. A molecule larger than the stationary phase pore volume elutes first and defines the column s void volume (Vo). In contrast, intermediate and smaller volume molecules may enter the pores and therefore elute later. As a measure of hydrodynamic volume (size and shape), SE-HPLC provides an approximation of a molecule s apparent molecular weight. For further descriptions of theoretical models and mathematical equations relating to SE-HPLC, the reader is referred to Refs. 2-5. 

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). 

The protein recovery was found to be 95% of the amount injected, whereas, on the untreated carrier they were almost totally irreversibly adsorbed. Meanwhile, some reduction in the pore volume of the carrier could be deduced from the results of the chromatographic test. The calculated pore volume available for phtalic acid was 0.67 cm2/g (V) whereas for cytochrome C — 0.5 cm2/g. A detailed description of the experiment allows the evaluation of the effective thickness (teff) of the polymeric stationary phase. The tcff calculated as V/Ssp is 2.3 nm. The value... 

General Description. Liquid chromatography encompasses any chromatographic method in which the mobile phase is a liquid (c.f. gas chromatography). A variety of stationary phases and retention mechanisms are available such that a broad range of modes of separation are possible. It is worthwhile to briefly describe the important modes that find use in clinical chemistry. 

The fourth type of mechanism is exclusion although perhaps inclusion would be a better description. Strictly, it is not a true sorption process as the separating solutes remain in the mobile phase throughout. Separations occur because of variations in the extent to which the solute molecules can diffuse through an inert but porous stationary phase. This is normally a gel structure which has a small pore size and into which small molecules up to a certain critical size can diffuse. Molecules larger than the critical size are excluded from the gel and move unhindered through the column or layer whilst smaller ones are retarded to an extent dependent on molecular size. 

This derivation shows that retention time is dependant on three factors temperature, energies of intermolecular interactions and flow rate. Temperature and flow rate are controlled by the user. Energies of intermolecular interactions are controlled by stationary phase choice. This theory is also the basis for the popular software programs that are available for computer-assisted method development and optimization [4,5,6,7]. More detailed descriptions of the theory behind retention times can be found in the appropriate chapters in the texts listed in the bibliography. 

Since the interaction of the mixture components with the liquid stationary phase plays the key role in the separation process, the nature of the stationary phase is obviously important. Several hundred different liquids useful as stationary phases are known. This means that the analyst has an awesome choice when it comes to selecting a stationary phase for a given separation. It is true, however, that relatively few such liquids are in actual common use. Their composition is frequently not obvious to the analyst because a variety of common abbreviations have come to be popular for the names of some of them. Table 12.3 lists a number of common stationary phases, their abbreviated names, a description of their structures, and the classes of compounds (in terms of polarity) for which each is most useful. 

Normal phase HPLC consists of methods that utilize a nonpolar mobile phase in combination with a polar stationary phase. Adsorption HPLC actually fits this description, too, since the adsorbing solid stationary phase particles are very polar. (See discussion of adsorption columns in Section 13.5.3.) Normal... 

Adsorption HPLC is the classification in which the highly polar silica particles are exposed (no adsorbed or bonded liquid phase). Aluminum oxide particles fit this description too and are also readily available as the stationary phase. As mentioned earlier, this classification can also be thought of as normal phase... 

As can be concluded from this short description of the factors influencing the overall reaction rate in liquid-solid or gas-solid reactions, the structure of the stationary phase is of significant importance. In order to minimize the transport limitations, different types of supports were developed, which will be discussed in the next section. In addition, the amount of enzyme (operative ligand on the surface of solid phase) as well as its activity determine the reaction rate of an enzyme-catalyzed process. Thus, in the following sections we shall briefly describe different types of chromatographic supports, suited to provide both the high surface area required for high enzyme capacity and the lowest possible internal and external mass transfer resistances. 

The multicomponent Langmuir adsorption isotherm given in Eq. (7) is the simplest model for the description of non-linear, multicomponent, adsorption equilibrium. At high concentration, the model predicts saturation of the stationary phase and overload of the chromatographic column. At low concentration (high dilution) the behavior can be correctly described by the non-competitive linear adsorption isotherm ... 

The retention of analyses in RP-HPLC markedly depends on the adsorption of the organic constituent of the mobile phase on the surface of the stationary phase. The excess adsorption isotherms of ACN, THF and methanol were measured on silica support modified with C, C6, C8, C10, C12 and C18 monomeric phase and a model was developed for the description of the retention of solutes from the binary mobile phase. The dependence of the retention factor on the partition coefficient can be described by... 

A discussion on the theories of retention mechanisms and the type of forces generating retention and selectivity is not intended in this chapter. The interactions between solute, eluent components, and stationary phases with inhomogeneous surfaces are based on molecular recognition, where our understanding is still very limited. Therefore, an empirical description of retention and selectivity is preferred. 

A complete description of the protein-stationary phase interaction involves many complications and a general model is extremely difficult to formulate. The slab model described above is very simple, yet it gives interesting physical insights and may be a useful starting point for more elaborate theories. Here, we shall only briefly discuss some of the challenges a more complete model meets. For more complete discussions see Refs. [1,24]. 

However, because of the complicated geometry of the protein and the stationary phase interaction, it is impossible to find a general geometrical description. 

Despite the low reproducibility and short life time of the NP columns, they are widely used owing to their better selectivity and resolution power, which enable the separation of (3- and y-isomers easily. The most used stationary phases are silica, aminopropyl- or diol-bonded [476], A more accurate description of the column used can be found in a review about tocol-derivatives analysis by Abidi [477], Kamal-Eldin et al. [478] compare the performance of new silica-type columns, six different silica columns, three amino columns, and one diol column. The new generation column results are much more repeatable and therefore suitable for vitamers analysis. 

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