Stationary-phase selectivity, change

The popularity of reversed-phase liquid chromatography (RPC) is easily explained by its unmatched simplicity, versatility and scope [15,22,50,52,71,149,288-290]. Neutral and ionic solutes can be separated simultaneously and the rapid equilibration of the stationary phase with changes in mobile phase composition allows gradient elution techniques to be used routinely. Secondary chemical equilibria, such as ion suppression, ion-pair formation, metal complexatlon, and micelle formation are easily exploited in RPC to optimize separation selectivity and to augment changes availaple from varying the mobile phase solvent composition. Retention in RPC, at least in the accepted ideal sense, occurs by non-specific hydrophobic interactions of the solute with the... 

Kirkland, J.J. Boyes, B.E. DeStefano, J.J. Changing band spacing in reversed-phase HPLC. Atechnique for varying column stationary-phase selectivity. Am.Lab., 1994, 26(14) (Sept), 36-41 [simultaneous desipramine, doxepin, trimipramine]... 

The nonresolution of the two xylene isomers shows that brnte-force resolving power is not always the most effective solution to a separation problem. A change of stationary-phase selectivity can be a mnch more effective approach to optimization in this case a more polar stationary phase wonld be an appropriate choice. Not all chromatograms are so simple, however. A change of stationary phase may not be snfflcient when there are many closely eluted peaks. Some pairs may experience improved resolution while others may actually be eluted closer together. This kind of complex separation is an ideal candidate for careful optimization of flow and temperature, as discussed later in this chapter. 

When one develops new reversed-phase (RP)-HPLC methods, one usually uses the selectivity of the mobile phase as the primary method development tool. The chromatographic separation can be influenced by the choice of the organic solvent (mainly methanol and acetonitrile), or by variation of pH or buffer type. Schemes for method development using these parameters have been described in the literature [1,2]. Most important are the selectivity changes caused by pH changes, which are well-understood and easily predictable (3). It is well known that the stationary phase influences the selectivity as well, but this effect is often not very well understood. The primary reason for this is the fact that reliable methods for the description of the stationary phase selectivity have only become available fairly recently. In the last few years, several papers have been published that deal with the subject of selectivity in a fimdamental way [4—9] or represent a data collection based on older methods [10-15]. In this chapter, we describe in detail the method used in our laboratory. We then look at our selectivity charts and discuss our results. It needs to be pointed out in advance that selectivity charts only accurately represent the properties of a stationary phase under the conditions of the measurement. If we depart from the mobile phase composition of the test, the relationships between different columns will change, since selectivity arises from a combined effect of the mobile phase and the stationary phase. 

The stationary phase in LC is a fine granular solid such as silica gel. It can be used as such (mainly for nonpolar compounds), or the granules can be modified by a surface-bonded coating that changes (reverses) the polarity of the gel. A very small selection of stationary phases is listed in Table 35.2. 

In the course of mixture separation, the composition and properties of both mobile phase (MP) and stationary phase (SP) are purposefully altered by means of introduction of some active components into the MP, which are absorbed by it and then sorbed by the SP (e.g. on a silica gel layer). This procedure enables a new principle of control over chromatographic process to be implemented, which enhances the selectivity of separation. As a possible way of controlling the chromatographic system s properties in TLC, the pH of the mobile phase and sorbent surface may be changed by means of partial air replacement by ammonia (a basic gaseous component) or carbon dioxide (an acidic one). 

When multiple development is performed on the same monolayer stationary phase, the development distance and the total solvent strength and selectivity values (16) of the mobile phase (17) can easily be changed at any stage of the development sequence to optimize the separation. These techniques are typically fully off-line modes, because the plates must be dried between consecutive development steps only after this can the next development, with the same or different development distances and/or mobile phases, be started. This method involves the following stages ... 

In general, the majority of separations are achieved by exploiting dispersive interactions in the stationary phase and modifying and controlling the absolute and relative retention of the solutes by adjusting the composition of the mobile phase. It is far easier to adjust the mobile phase by selecting different mixtures of water and the solvents methanol, acetonitrile and/or tetrahydrofuran than change from column to column. 

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