Stationary phase examples

There are many examples of the deliberate use of chemical intuition and understanding of forces between molecules in getting selective interactions that enhance separation. An entire book could be written just cataloging examples. It is more appropriate here to give a few stationary phase examples. 

Twenty-eight chiral compounds were separated from their enantiomers by HPLC on a teicoplanin chiral stationary phase. Figure 8-12 shows some of the structures contained in the data set. This is a very complex stationary phase and modeling of the possible interactions with the analytes is impracticable. In such a situation, learning from known examples seemed more appropriate, and the chirality code looked quite appealing for representing such data. 

Figure 8-12. Examples of las eluted enantiomers In a chromatographic separation on chiral HPLC with teicoplanin stationary-phase.

Environmental Analysis One of the most important environmental applications of gas chromatography is for the analysis of numerous organic pollutants in air, water, and wastewater. The analysis of volatile organics in drinking water, for example, is accomplished by a purge and trap, followed by their separation on a capillary column with a nonpolar stationary phase. A flame ionization, electron capture, or... 

An example of a size-exclusion chromatogram is given in Figure 7 for both a bench-scale (23.5 mL column) separation and a large-scale (86,000 mL column) mn. The stationary phase is Sepharose CL-6B, a cross-linked agarose with a nominal molecular weight range of 5000-2 x 10 (see Fig. 6) (31). 

The total stationary-phase volume required to process a given feed stream is proportional to the inlet concentration and volume of the feed. For example, for a typical inlet concentration of protein of 10 g/L, in a 100 L volume of feed, a column volume of at least 100 L is needed for size-exclusion chromatography. In comparison, an ion-exchange column having an adsorption capacity of 50 g/L would only require 20 L of column volume for the same feed. 

Three general methods exist for the resolution of enantiomers by Hquid chromatography (qv) (47,48). Conversion of the enantiomers to diastereomers and subsequent column chromatography on an achiral stationary phase with an achiral eluant represents a classical method of resolution (49). Diastereomeric derivatization is problematic in that conversion back to the desired enantiomers can result in partial racemization. For example, (lR,23, 5R)-menthol (R)-mandelate (31) is readily separated from its diastereomer but ester hydrolysis under numerous reaction conditions produces (R)-(-)-mandehc acid (32) which is contaminated with (3)-(+)-mandehc acid (33). 

Several hundred types of Hquid phases are commercially available. These have been used individually or in combination with other Hquid phases, inorganic salts, acids, or bases. The selection of stationary phases for a particular appHcation is beyond the scope of this article, however, it is one of the most important chromatographic tasks. Stationary phase selection is discussed at length in books, journal articles, and catalogs from vendors. See General References for examples. 

Synthetic chiral adsorbents are usually prepared by tethering a chiral molecule to a silica surface. The attachment to the silica is through alkylsiloxy bonds. A study which demonstrates the technique reports the resolution of a number of aromatic compoimds on a 1- to 8-g scale. The adsorbent is a silica that has been derivatized with a chiral reagent. Specifically, hydroxyl groups on the silica surface are covalently boimd to a derivative of f -phenylglycine. A medium-pressure chromatography apparatus is used. The racemic mixture is passed through the column, and, when resolution is successful, the separated enantiomers are isolated as completely resolved fiactions. Scheme 2.5 shows some other examples of chiral stationary phases. 

It is clear that the separation ratio is simply the ratio of the distribution coefficients of the two solutes, which only depend on the operating temperature and the nature of the two phases. More importantly, they are independent of the mobile phase flow rate and the phase ratio of the column. This means, for example, that the same separation ratios will be obtained for two solutes chromatographed on either a packed column or a capillary column, providing the temperature is the same and the same phase system is employed. This does, however, assume that there are no exclusion effects from the support or stationary phase. If the support or stationary phase is porous, as, for example, silica gel or silica gel based materials, and a pair of solutes differ in size, then the stationary phase available to one solute may not be available to the other. In which case, unless both stationary phases have exactly the same pore distribution, if separated on another column, the separation ratios may not be the same, even if the same phase system and temperature are employed. This will become more evident when the measurement of dead volume is discussed and the importance of pore distribution is considered. 

The stationary phase can be apportioned in a similar manner. For example, with a bonded phase, due to the porous nature of the support, some of the pores will become blocked with stationary phase and so the total amount of stationary phase can be divided into that which is chromatographically available (Vs(A)) and that which is chromatographically unavailable (Vs(u)). 

The induced counter-dipole can act in a similar manner to a permanent dipole and the electric forces between the two dipoles (permanent and induced) result in strong polar interactions. Typically, polarizable compounds are the aromatic hydrocarbons examples of their separation using induced dipole interactions to affect retention and selectivity will be given later. Dipole-induced dipole interaction is depicted in Figure 12. Just as dipole-dipole interactions occur coincidentally with dispersive interactions, so are dipole-induced dipole interactions accompanied by dispersive interactions. It follows that using an n-alkane stationary phase, aromatic... 

Alternatively, using a polyethylene glycol stationary phase, aromatic hydrocarbons can also be retained and separated primarily by dipole-induced dipole interactions combined with some dispersive interactions. Molecules can exhibit multiple interactive properties. For example, phenyl ethanol possesses both a dipole as a result of the hydroxyl group and is polarizable due to the aromatic ring. Complex molecules such as biopolymers can contain many different interactive groups. 

An interesting and practical example of the use of thermodynamic analysis is to explain and predict certain features that arise in the application of chromatography to chiral separations. The separation of enantiomers is achieved by making one or both phases chirally active so that different enantiomers will interact slightly differently with the one or both phases. In practice, it is usual to make the stationary phase comprise one specific isomer so that it offers specific selectivity to one enantiomer of the chiral solute pair. The basis of the selectivity is thought to be spatial, in that one enantiomer can approach the stationary phase closer than the other. If there is no chiral selectivity in the stationary phase, both enantiomers (being chemically identical) will coelute and will provide identical log(Vr ) against 1/T curve. If, however, one... 

There are two ways a solute can interact with a stationary phase surface. The solute molecule can interact with the adsorbed solvent layer and rest on the top of it. This is called sorption interaction and occurs when the molecular forces between the solute and the stationary phase are relatively weak compared with the forces between the solvent molecules and the stationary phase. The second type is where the solute molecules displace the solvent molecules from the surface and interact directly with the stationary phase itself. This is called displacement interaction and occurs when the interactive forces between the solute molecules and the stationary phase surface are much stronger than those between the solvent molecules and the stationary phase surface. An example of sorption interaction is shown in Figure 9. 

It is seen from equation (22) that there will, indeed, be a temperature at which the separation ratio of the two solutes will be independent of the solvent composition. The temperature is determined by the relative values of the standard free enthalpies of the two solutes between each solvent and the stationary phase, together with their standard free entropies. If the separation ratio is very large, there will be a considerable difference between the respective standard enthalpies and entropies of the two solutes. As a consequence, the temperature at which the separation ratio becomes independent of solvent composition may well be outside the practical chromatography range. However, if the solutes are similar in nature and are eluted with relatively small separation ratios (for example in the separation of enantiomers) then the standard enthalpies and entropies will be comparable, and the temperature/solvent-composition independence is likely be in a range that can be experimentally observed. 

When the relationship between the distribution coefficient of a solute and solvent composition, or the corrected retention volume and solvent composition, was evaluated for aqueous solvent mixtures, it was found that the simple relationship identified by Purnell and Laub and Katz et al. no longer applied. The suspected cause for the failure was the strong association between the solvent and water. As a consequence, the mixture was not binary in nature but, in fact, a ternary system. An aqueous solution of methanol, for example, contained methanol, water and methanol associated with water. It follows that the prediction of the net distribution coefficient or net retention volume for a ternary system would require the use of three distribution coefficients one representing the distribution of the solute between the stationary phase and water, one representing that between the stationary phase and methanol and one between the stationary phase and the methanol/water associate. Unfortunately, as the relative amount of association varies with the initial... 

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. 

Other examples of irreversible derivatization on treatment with iodine have been described for phenohc steroids (estrone derivatives [256]), morphine [257] and 23 other pharmaceuticals [258]. These reactions are probably favored by the presence of silica gel as stationary phase and by the influence of light. 

Silica gel and aluminium oxide layers are highly active stationary phases with large surface areas which can, for example, — on heating — directly dehydrate, degrade and, in the presence of oxygen, oxidize substances in the layer This effect is brought about by acidic silanol groups [93] or is based on the adsorption forces (proton acceptor or donor effects, dipole interactions etc) The traces of iron in the adsorbent can also catalyze some reactions In the case of testosterone and other d -3-ketosteroids stable and quantifiable fluorescent products are formed on layers of basic aluminium oxide [176,195]... 

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