Separation Selectivity

The rate of change in retention of different solutes varies with charge and hydrophobicity of the solute as well as with the length of alkyl chain, charge and concentration of micelles. This causes inversions of elution order that are the result of two competing equilibria: the solute-micelle association, characterized by K2, and the solute-stationary phase interaction, characterized by PSW. The parameters K2 and PSW have a different effect on retention. When PSW increases, retention also increases, but when K2 increases, retention decreases. When the surfactant concentration in the mobile phase increases, the effect that K2 has on retention also increases and reversals in elution order can be obtained if the difference in K2 values for two solutes is large. Therefore, separation selectivity in MLC can be controlled by modifying surfactant nature and concentration. Furthermore, when organic modifiers are added to the mobile phase, the solvent strength parameter for a group of compounds does not have the same ranking for different alcohols owing to the different interaction of these modifiers with the micelles. For these reasons, MLC techniques are very interesting for chromatographic separation.

Although the conditions that optimize separation selectivity in MLC can vary with the nature of the solutes, several workers have shown an increase in separation selectivity for aromatic compounds in MLC with hybrid eluents when the micelle concentration in the mobile phase decreases. However, for a group of amino acids and peptides, an increase in micelle concentration can cause an increase or decrease in selectivity, or even an inversion of the peaks.

The effect of the organic modifier content in the mobile phase seems to be clearer. Generally, separation selectivity in MLC is improved in the presence of an organic modifier and increases with the volume fraction of the modifier in the mobile phase. This result is opposed to that observed in conventional RPLC with aqueous-organic mobile phases in which an increase of the organic modifier content causes a decrease of solute retention and selectivity. The selectivity enhancement observed in MLC when the solvent strength increases has been attributed to the competing partitioning equilibria in micellar systems and/or to the unique abilities of micelles to compartmentalize solutes and organic solvents. For some compounds, however, selectivity can decrease with the content of the alcohol in a micellar (SDS) mobile phase. In this case, for pairs of peaks where the selectivities are reduced by increasing alcohol concentration, a selectivity enhancement is observed with increasing micelle concentration and vice versa. Micelles and alcohols compete to interact with solutes affecting the role of one another in controlling retention and selectivity. The mutual effects of micelles and organic modifiers on each other also require a simultaneous optimization of these two parameters.

The retention mechanism of a solute in MLC can have implications for selectivity. If the retention of a solute in the chromatographic system takes place through a direct transfer mechanism, then the retention factor can be expressed by eqn [3] (Table 1). In this case, and if the surfactant concentration in the mobile phase is high, the selectivity coefficient (a) for a pair of solutes can be calculated from the ratio of their distribution coefficients between the stationary and micellar phases (PSM):

This equation is useful for two reasons. First, because knowledge about the retention mechanism of compounds in the chromatographic system can be enhanced. In fact, if the experimental selectivity coefficient for a pair of solutes is constant and coincides with the ratio of their respective distribution coeffi cients, PSM, it can then be assumed that retention occurs through a direct transfer from the micellar phase to the stationary phase. Second, calculation of the selectivity coefficient from eqn [9] enables prediction of the separation selectivity of two compounds in the chromatographic system, provided the distribution coefficients (PSM) of the solutes are known.

As an example, Figure 3 shows the variation of theoretical and experimental selectivity coefficients as a function of the micellized surfactant concentration in two mobile phases, SDS/5% w-propanol (Figures 3A, 3B and 3C) and hexadecyltrimethylam-monium bromide (CTAB) modified by 5% w-butanol (Figures 3D, 3E and 3F) for three pairs of aromatic solutes. For pyrene/acenaphthene, both of which are highly hydrophobic, a direct transfer mechanism can be assumed for any surfactant concentration in these mobile phases. For pyrene/toluene a direct transfer mechanism can only be assumed for pyrene in all surfactant concentrations. For pyrene/benzamide, benzamide does not experience a direct transfer mechanism except at very high surfactant concentrations. Figure 3 shows that, when both solutes experience a direct transfer mechanism, the experimental and theoretical selectivity coefficients are very similar for all surfactant concentrations in solution, and it is therefore possible to predict the selectivity coefficient from the partition coefficients PSM for the two solutes. When one of the two solutes does not experience a direct transfer mechanism, the theoretical and experimental selectivities are different. This difference decreases under conditions in which the direct transfer mechanism is favoured, i.e. by increasing the solute hydrophobicity, solute-micelle association constants, surfactant concentration in mobile phase and, for mobile phases modified by alcohols, by increasing the polarity of the alcohol. Consequently, the separation selectivity for a pair of solutes shows a tendency to match a limit value close to the ratio of stationary-micellar partition coefficients of two solutes. In this case, the separation selectivity cannot be experimentally modified through a change in the surfactant concentration in the mobile phase.

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