Types of Stationary Phases

In order to accomplish the desired separation, the selection of appropriate stationary phase and eluent system is imperative. The most commonly used stationary phases in normal-phase chromatography are either (a) inorganic adsorbents such as silica and alumina or (b) moderately polar chemically bonded phases having functional groups such as aminopropyl, cyanopropyl, nitrophenyl, and diol that are chemically bonded on the silica gel support [16]. Other phases that are designed for particular types of analytes have also 

Despite the many desirable properties of silica, its limited pH stability (between 2 and 7.5) is also a major issue in NPC when strong acidic or basic mobile-phase additives are used to minimize interactions. Hence, other inorganic materials such as alumina, titania, and zirconia, which not only have the desired physical properties of silica but also are stable over a wide pH range, have been studied. Recently, Unger and co-workers [22] have chosen a completely new approach where they use mesoporous particles based not only on silica but also on titania, alumina, zirconia, and alumosilicates. These materials have been used by the authors to analyze and separate different classes of aromatic amines, phenols, and PAHs (polyaromatic hydrocarbons). 

Bonded stationary phases for NPC are becoming increasingly popular in recent years owing to their virtues of faster column equilibration and being less prone to contamination by water. The use of iso-hydric (same water concentration) solvents is not needed to obtain reproducible results. However, predicting solute retention on bonded stationary phases is more difficult than when silica is used. This is largely because of the complexity of associations possible between solvent molecules and the chemically and physically heterogeneous bonded phase surface. Several models of retention on bonded phases have been advocated, but their validity, particularly when mixed solvent systems are used as mobile phase, can be questioned. The most commonly accepted retention mechanism is Snyder s model, which assumes the competitive adsorption between solutes and solvent molecules on active sites 

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Analytical separations may be classified in three ways by the physical state of the mobile phase and stationary phase by the method of contact between the mobile phase and stationary phase or by the chemical or physical mechanism responsible for separating the sample s constituents. The mobile phase is usually a liquid or a gas, and the stationary phase, when present, is a solid or a liquid film coated on a solid surface. Chromatographic techniques are often named by listing the type of mobile phase, followed by the type of stationary phase. Thus, in gas-liquid chromatography the mobile phase is a gas and the stationary phase is a liquid. If only one phase is indicated, as in gas chromatography, it is assumed to be the mobile phase. 

Figure 8.17 depicts MD-PC performed on three different types of stationary phase (6). The three grafted chromatographic plates (Figure 8.17(a)) are clamped in lap-joint fashion with the edges of their stationary phases in close contact. The manner in which the three plates are prepared and the separation which can theoretically be achieved are also apparent from the schematic diagrams in Figures 8.17(b-d), in which the most polar stationary phase is phase A and the least polar is stationary phase C . 

Bonded phases are the most useful types of stationary phase in LC and have a very broad range of application. Of the bonded phases, the reverse phase is by far the most widely used and has been applied successfully to an extensive range of solute types. The reverse phases are commonly used with mobile phases consisting of acetonitrile and water, methanol and water, mixtures of both acetonitrile and methanol and water, and finally under very special circumstances tetrahydrofuran may also be added. Nevertheless, the majority of separations can be accomplished using simple binary mixtures. 

The two examples of sample preparation for the analysis of trace material in liquid matrixes are typical of those met in the analytical laboratory. They are dealt with in two quite different ways one uses the now well established cartridge extraction technique which is the most common the other uses a unique type of stationary phase which separates simultaneously on two different principles. Firstly, due to its design it can exclude large molecules from the interacting surface secondly, small molecules that can penetrate to the retentive surface can be separated by dispersive interactions. The two examples given will be the determination of trimethoprim in blood serum and the determination of herbicides in pond water. 

There are two types of stationary phases commonly used in exclusion chromatography silica gel and micro-reticulated cross-linked polystyrene gels. A third type of exclusion media is comprised of the Dextran gels. Dextran gels are produced by the action of certain bacteria on a sucrose substrate. They consist of framework of glucose units that can form a gel in aqueous solvents that have size exclusion properties. Unfortunately the gels are mechanically weak and thus, cannot tolerate the high pressures necessary for HPLC and, as a consequence, are of very limited use to the analyst. 

The type of stationary phase, the composition of the mobile phase, the migration distance, the mode of development, and the working temperature may be identical to those of analytical TLC. These procedures have been presented extensively for analytical TLC [5-7] and summarized for PLC [1,2,4,7,8]. 

In gas chromatography the value of the partition coefficient d ends only on the type of stationary phase and the column temperature. It is independent of column type and instrumental parameters. The proportionality factor in equation (l.ll) is called the phase ratio and is equal to the ratio of the volume of the gas (Vg) and liquid (V ) phases in the column. For gas-solid (adsorption) chromatography the phase ratio is given by the volume of the gas phase divided by the surface area of the stationary phase. 

In cases where a mixture has a large number of components, or pure standards are not available, published retention data must be consulted. The uncorrected retention time, tR (p. 86), is not suitable for this purpose because it cannot be compared with data from different columns and instruments. Valid comparisons can be made using relative retention data which are dependent only on column temperature and type of stationary phase. An adjusted retention time, / R, is first obtained by subtracting from tR the time required to elute a non-retained substance such as air (Figure 4.26)... 

Another approach in GC is that of using more power in the separation by doing GCxGC. In this approach, a second column is used with a different type of stationary phase than the primary stationary phase, and fast chromatography using TOF-MS as the detector is carried out [39]. This technique uses only TOF-MS as the detector since it has the most sensitivity for fast-eluting peaks. The method has been applied to complicated matrix analysis. 

Retention in HPLC depends on the strength of the solute s interaction with both the mobile and stationary phases as opposed to GC, where the mobile phase does not contribute to the selectivity. An intelligent selection of the type of stationary phase for the separation is made and... 

For efficient separations it is essential to have very small and regularshaped support media, a supply of mobile phase pumped at a pressure that is adequate to give a suitable constant flow rate through the column and a convenient and efficient detection system. For all types of stationary phases the apparatus required consists of five basic components (Figure 3.5), each available with varying degrees of sophistication. 

It was established that the best separations can be achieved in ODS columns, however the separation capacity depends considerably on the type of stationary phase and on the dimensions of the column [29],... 

Various liquid chromatographic techniques have been frequently employed for the purification of commercial dyes for theoretical studies or for the exact determination of their toxicity and environmental pollution capacity. Thus, several sulphonated azo dyes were purified by using reversed-phase preparative HPLC. The chemical strctures, colour index names and numbers, and molecular masses of the sulphonated azo dyes included in the experiments are listed in Fig. 3.114. In order to determine the non-sulphonated azo dyes impurities, commercial dye samples were extracted with hexane, chloroform and ethyl acetate. Colourization of the organic phase indicated impurities. TLC carried out on silica and ODS stationary phases was also applied to control impurities. Mobile phases were composed of methanol, chloroform, acetone, ACN, 2-propanol, water and 0.1 M sodium sulphate depending on the type of stationary phase. Two ODS columns were employed for the analytical separation of dyes. The parameters of the columns were 150 X 3.9 mm i.d. particle size 4 /jm and 250 X 4.6 mm i.d. particle size 5 //m. Mobile phases consisted of methanol and 0.05 M aqueous ammonium acetate in various volume ratios. The flow rate was 0.9 ml/min and dyes were detected at 254 nm. Preparative separations were carried out in an ODS column (250 X 21.2 mm i.d.) using a flow rate of 13.5 ml/min. The composition of the mobile phases employed for the analytical and preparative separation of dyes is compiled in Table 3.33. 

The selectivity of stationary phase materials can be understood if the method of their synthesis is understood. Differences in the same type of stationary phase material from different manufacturers or even from the same manufacturer depend on the synthetic methods and the quality control that has been employed. Details of the individual synthetic processes from different manufacturers have not been published, but are basically the same.1,2... 

Many manufacturers sell the same types of stationary phase materials,6 but the most popular stationary phase materials, e.g. octadecyl-bonded silica gels, from different sources, even from the same manufacturer, often demonstrate different retention capacities and selectivities. Such differences are due to the aggressive reactivity of the silylation method and the different bonding reactions that are used, as described earlier. 

Example 2 Chromatography of nitroaniline isomers. The elution order of the nitroaniline isomers was ortho, meta, and para in normal-phase liquid chromatography using H-butanol-w-hexane mixtures as the eluent, when the stationary phase material was either silica gel, alumina, an ion-exchanger, polystyrene gel, or octadecyl-bonded silica gel. The results indicate that the separation of these compounds can be performed on a range of different types of stationary phase materials if the correct eluent is selected. The best separation will be achieved by the right combination of stationary phase material and eluent.68... 

The enthalpy of methylphenols was about 2.0 kcal mol- and that of chlorophenols varied from 2.0 to 2.4 kcal mol -1 in the case of pentachlorophe-nol, indicating that the retention difference depended not upon the size but on the 7r-electron density.39 A similar result was obtained for alkylated and halogenated aromatic acids, whose enthalpies were nearly equal, but whose retention factors were different.40 The AH values may depend on the type of stationary phase used and the water content of the eluent.41... 

The ethyl acetate solution of organic species from the pre-treatment scheme shown in Figure 1 is suitable for analysis by this method. In order to cover the range of common explosives several chromatography columns with different types of stationary phase are required to allow for difierent polarities and volatihties. Dimethylsiloxane, phenyl-modified dimethylsiloxane, cyanopropyl- phenyl- vinyl-modified dimethylsiloxane, and polyethylene glycol have been found to represent a useful set of stationary phases. Carefully optimised temperature programming is also needed to obtain the requisite resolution and avoid interferences [19, 20]. 

As the carbon load of a particular type of stationary phase increases we expect both the phase ratio and the contact surface area for a given eluite probe to reach a plateau so that the dependence of k or log k on the carbon load is likely to be hyperbolic over a sufficiently wide range. A key point in this treatment is the accessible hydrocarbonaceous surface area, which is difficult to quantify and may strongly depend on the molecular architecture of the eluite used as a probe. Furthermore, the involvement of the... 

One disadvantage of all silica-based stationary phases is their instability against hydrolysis. At neutral pH and room temperature the saturation concentration of silicate in water amounts to lOOppm. Solubility increases with surface area, decreasing particle diameter drastically with pH above 7.5. This leads also to a reduction of the carbon content. Hydrolysis can be recognized during the use of columns by a loss in efficiency and/or loss of retention. Bulky silanes [32], polymer coating [33], or polymeric encapsulation [34] have been used in the preparation of bonded phases to reduce hydrolytic instability, but most of the RPs in use are prepared in the classical way, by surface silanization. Figure 2.3 schematically shows these different types of stationary phases. 

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