Factors that control retention and selectivity

The factors that control separation and dispersion are quite different. The relative separation of two solutes is solely dependent on the nature and magnitude of the Interactions between each solute and the two phases. Thus, the relative movement of each solute band would appear to be Independent of column dimensions or particle geometry and be determined only by the choice of the stationary phase and the mobile phase. However, there is a caveat to this statement. It assumes that any exclusion properties of the stationary phase are not included in the term particle geometry. The pore size of the packing material can control retention directly and exclusively, as in exclusion chromatography or, indirectly, by controlling the access of the solute to the stationary phase in normal and reverse phase chromatography. As all stationary phases based on silica gel exhibit some exclusion properties, the ideal situation where the selective retention of two solutes is solely controlled by phase interactions is rarely met in practice. If the molecular size of the solutes differ, then the exclusion properties of the silica gel will always play some part in solute retention. 

The LC methods discussed before were based mainly on physico-chemical interactions between the solute on the one hand and the two chromatographic phases on the other. Although we have seen that in RPLC the degree of ionization of weakly acidic or basic solutes may be a major factor in the control of retention and selectivity, the ionic species themselves were not exploited purposefully to realize or enhance the separation. In fact, in a typical RPLC system all fully ionized solutes will show little retention and therefore little resolution can be achieved between different ions. The methods described in this section make positive use of the ionic character of solutes to create a chromatographically selective system. 

The chemical structure and the stereoisomerism of a stationary phase are not the only factors that control chiral selectivity. There are two other major factors that also affect selectivity and they are the stationary phase loading on the column and the operating temperature. The effect of these two variables on the retention ratio of a pair of enantiomers has been reported by Supelco and their data is shown in figure 5.9. 

The variables that control the extent of a chromatographic separation are conveniently divided into kinetic and thermodynamic factors. The thermodynamic variables control relative retention and are embodied in the selectivity factor in the resolution equation. For any optimization strategy the selectivity factor should be maximized (see section 1.6). Since this depends on an understandino of the appropriate retention mechanism further discussion. .Jll be deferred to the appropriate sections of Chapters 2 and 4. 

When the amount of the sample is comparable to the adsorption capacity of the zone of the column the migrating molecules occupy, the analyte molecules compete for adsorption on the surface of the stationary phase. The molecules disturb the adsorption of other molecules, and that phenomenon is normally taken into account by nonlinear adsorption isotherms. The nonlinear adsorption isotherm arises from the fact that the equilibrium concentrations of the solute molecules in the stationary and the mobile phases are not directly proportional. The stationary phase has a finite adsorption capacity lateral interactions may arise between molecules in the adsorbed layer, and those lead to nonlinear isotherms. If we work in the concentration range where the isotherms are nonlinear, we arrive to the field of nonlinear chromatography where thermodynamics controls the peak shapes. The retention time, selectivity, plate number, peak width, and peak shape are no longer constant but depend on the sample size and several other factors. 

The MS I group should be required by an administrative procedure to select records which provide a meaningful history of the plant and to retain them throughout the plant s lifetime. Other records that have only a transitory value (such as records on individual components that have been replaced) should be retained either until they cease to serve the purpose for which they were intended or until they are superseded by subsequent records. An important factor that should be considered in selecting records to be retained is their usefulness in assembling reliability data. More information on the retention of records necessary for quality assurance purposes can be found in Ref. [2], in particular in Safety Guide Q3 on Document Control and Records. 

As indicated by these equations, both retention factor and separation factor are controlled by an enthalpic contribution, which decreases with the elevation of temperature, and an entropic contribution, which is independent of the temperature. The selectivity is a compromise between differences in enantiomeric binding enthalpy and disruptive entropic effects. The enthalpy term is a function of overall interactions between each enantiomer and the chiral selector. By plotting ln(o ) vs. 1 /T, all processes that do not contribute to the enantiomeric discrimination cancel out and the plot is linear, the slope being the difference between the enthalpy of association of the enantiomers with the stationary phase. The linear inverse relationship between In a and temperature demonstrates the enhancement of selectivity with a decrease in temperature. There exists a Tiso where - (AG ) = 0 owing... 

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