Requirements of a Stationary Phase

Separations in GLC are the result of selective solute-stationary phase interactions and differences in the vapor pressure of solutes. The main forces that are responsible for solute interaction with a stationary phase are dispersion, induction, orientation, and donor-acceptor interactions (52-54), the sum of which serves as a measure of the polarity of the stationary phase toward the solute. Selectivity, on the other hand, may be viewed in terms of the magnitude of the individual energies of interaction. In GLC, the selectivity of a column governs band spacing, the degree to which peak maxima are separated. The following parameters influence selectivity  

Differences in selectivity are significant because they permit the separation of solutes of similar or even the same polarity by a selective stationary phase. 

The stationary-phase requirements of selectivity and higher thermal stability then became more clearly defined the process of stationary-phase selection and classification became logical after the studies of McReynolds (55) and Rohrschneider (56, 57) were published, both of which were based on the retention index (58). The 

Kovats retention index procedure and the McReynolds and Rohrschneider constants are discussed in detail in the following sections. The Kovats index remains a widely used technique for reporting retention data, and every stationary phase developed for packed and capillary gas chromatography has been characterized by its McReynolds constants. 

The stationary phases designated for the USP methods are listed in Table 2.8. Also listed are equivalent stationary phases recommended by other chromatographic suppliers. 

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The primary requirements of a stationary phase are to provide separation of the sample with reasonable column life. Therefore, in addition to having suitable selectivity, the phase should have reasonable chemical and thermal stability. Many catalogs list upper temperature limits for stationary phases, but these should be used only as approximations because the true limit depends upon the type of detector used and the amount of column bleed one can tolerate to get the job done. Even if a phase is stable to 250°C, the column will last much longer if the temperature is limited to 200°C. Excessive temperatures result not only in shorter column life, but also in more rapid fouling of the detector. 

The HcReynolds abroach, which was based on earlier theoretical considerations proposed by Rohrschneider, is formulated on the assumption that intermolecular forces are additive and their Individual contributions to retention can be evaluated from differences between the retention index values for a series of test solutes measured on the liquid phase to be characterized and squalane at a fixed temperature of 120 C. The test solutes. Table 2.12, were selected to express dominant Intermolecular interactions. HcReynolds suggested that ten solutes were needed for this purpose. This included the original five test solutes proposed by Rohrschneider or higher molecular weight homologs of those test solutes to improve the accuracy of the retention index measurements. The number of test solutes required to adequately characterize the solvent properties of a stationary phase has remained controversial but in conventional practice the first five solutes in Table 2.12, identified by symbols x through s have been the most widely used [6). It was further assumed that for each type of intermolecular interaction, the interaction energy is proportional to a value a, b, c, d, or e, etc., characteristic of each test solute and proportional to its susceptibility for a particular interaction, and to a value x, X, Z, U, s, etc., characteristic of the capacity of the liquid phase... 

The selection of a stationary phase depends largely on trial and error or experience, with consideration given to the polar nature of the mixture, as noted in Table 12.3 or a similar table. The usual procedure is to select a stationary phase, based on such literature information, and attempt the separation under the various conditions of column temperature, length, carrier gas flow rate, etc., to determine the optimum capability for separating the mixture in question. If this optimum resolution is not satisfactory (see Section 12.6), then an alternate selection is apparently required. 

From this, the basic equation of the stationary-phase retention process can be derived, a number of assumptions and complex theoretical treatments being required. Taking as example the planetary centrifuge of Type J, the average cross-sectional area of a stationary-phase layer has been estimated for hydrophobic liquid systems, which are characterized by high values of interfacial tension y, low values of viscosity r], and low hydrodynamic equilibrium settling times ... 

Retention differences are controlled by the extent to which sample components can diffuse through the pore structure of the stationary phase, which depends on the ratio of molecular dimensions to the distribution of pore-size diameters. Since no separation will result under conditions where the sample is completely excluded from the pore volume, or can completely permeate the pore volume the essential requirement for a stationary phase is that the fraction of the pore volume accessible to sample components must be different in a size-dependent manner. Compared with the other liquid chromatographic retention mechanisms discussed so far, a unique feature of SEC is that there are no enthalpic (attractive) stationary phase interactions involved. The driving force for retention is the difference in entropy of the solute within the pore volume compared with that in the moving mobile phase. For SEC the separation is complete when a volume of mobile phase equivalent to the conventional hold-up volume has passed through the column. In other liquid chromatographic techniques, retention is measured as the difference between the elution volume and the hold-up volume, and is always... 

Two main pattern types are produced in GCxGC plots. Globular clusters correspond to groups of isomers with very similar properties, and then with similar retention times in both D and D columns. In these cases, separation will require the use in ID or 2D of a stationary phase that is more selective toward the small structural differences among isomers, or use of a separation technique of higher dimensionality (see Chapter 6). 

CC is used mainly in the separation of a mixtore of carotenoids at semipreparative or preparative scale, although subsequently TLC is required. The choice of a stationary phase in which the carotenoid mixture is adsorbed and separated depends on its selectivity and nonreactivity with the pigments or mobile phase. Table 6.6 shows some of the stationary phases most commonly used in CC of carotenoids. Pigment... 

In common with all multidimensional separations, two-dimensional GC has a requirement that target analytes are subjected to two or more mutually independent separation steps and that the components remain separated until completion of the overall procedure. Essentially, the effluent from a primary column is reanalysed by a second column of differing stationary phase selectivity. Since often enhancing the peak capacity of the analytical system is the main goal of the coupling, it is the relationship between the peak capacities of the individual dimensions that is crucial. Giddings (2) outlined the concepts of peak capacity product and it is this function that results in such powerful two-dimensional GC separations. 

However, in LC solutes are partitioned according to a more complicated balance among various attractive forces solutes interact with both mobile-phase molecules and stationary-phase molecules (or stationary-phase pendant groups), the stationary-phase interacts with mobile-phase molecules, parts of the stationary phase may interact with each other, and mobile-phase molecules interact with each other. Cavity formation in the mobile phase, overcoming the attractive forces of the mobile-phase molecules for each other, is an important consideration in LC but not in GC. Therefore, even though LC and GC share a considerable amount of basic theory, the mechanisms are very different on a molecular level. This translates into conditions that are very different on a practical level so different, in fact, that separate instruments are required in modern practice. 

In addition to the development of the powerful chiral additive, this study also demonstrated that the often tedious deconvolution process can be accelerated using HPLC separation. As a result, only 15 libraries had to be synthesized instead of 64 libraries that would be required for the full-scale deconvolution. A somewhat similar approach also involving HPLC fractionations has recently been demonstrated by Griffey for the deconvolution of libraries screened for biological activity [76]. Although demonstrated only for CE, the cyclic hexapeptides might also be useful selectors for the preparation of chiral stationary phases for HPLC. However, this would require the development of non-trivial additional chemistry to appropriately link the peptide to a porous solid support. 

In Chapter 14, one of the least-used applications of TLC and PLC is described, namely inorganics and organometallics. These separations in the analytical mode often require quite unusual stationary phases (e.g., inorganic ion exchangers and impregnated and mixed layers) combined with a variety of diverse mobile phases. This means that the use of the analogous systems in the preparative mode represents an unusually difficult challenge. 

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