Mobile phase overlapping-resolution mapping

The present model predicts how solvent selectivity will vary with mobile-phase composition, and this allows the selection of extreme solvents for maximum differences in selectivity. This information plus the ability to calculate solvent strength versus composition of the mobile phase then allows development of a general strategy for optimizing retention of any sample, so as to maximize resolution. This four-solvent approach can be further refined by use of computer-assisted procedures, such as the overlapping-resolution-mapping technique. 

Fig. 1.24. Overlapping resolution map for the separation of a four-component mixture with optimisation of tw

The constants a, b, and m in eqn [3] depend on the solute and on the chromatographic system. b = (ka) " , where ka is the retention factor in a pure nonpolar solvent. Equation [2] or [3] can be used as the basis of optimization of the composition of two-component (binary) mobile phases in NPLC, using a common window diagram or overlapping resolution mapping approach, as illustrated in an example in Figure 3. 

The composition of mixed mobile phases for ternary or quaternary isoselective gradient elution can be optimized using overlapping resolution mapping strategy to adjust optimum separation selectivity based on seven or more initial experiments with solvent mixtures of approximately equal elution strengths. Based on the retention data from the initial experiments, either 3-D diagrams or contour resolution... 

Mobile phase optimization is based both on the analyst s experience and intuition and on modifications of published data. However, as the sample composition becomes more complex, systematic solvent optimization becomes more important (38). The methods used for optimizing isocratic mobile phases in HPLC are generally also applicable, with some modifications, to TLC. Window diagrams have been successfully applied (45-47) for the optimization of TLC mobile phases. Similarly, overlapping resolution maps were used as criteria by Issaq et al. (48) and by Nurok et al. (49). who... 

Besides the advantages of short elution times, excellent resolution, and high recoveries, a further benefit of these reversed-phase HPLC methods for peptide mapping is the ease of sample preparation. In many cases the crude digest can be loaded directly. The problems associated with overlapping peptides in an otherwise straightforward application can often be remedied by small changes in the pH of the mobile phase or, alternatively, variation in the polarity and concentration of the counterion. 

Different regions of the variable space will often be associated with different critical peak-pairs. The resolution of a multicomponent mixture thus requires an analysis involving all components in the whole variable space. Inspection of the contour maps of global resolution will allow the evaluation of the robustness of the optimum. Figure 8.16 shows the contour maps for the separation of a set of fifteen phenols (the same cited previously in this chapter), with mobile phases of CTAB and 2-propanol, where an efficiency N = 2500 was considered for all solutes. For the positional criterion (separation factor), the optimum was found for a mobile phase of 0.12 M CTAB-10% 2-propanol (Fig. 8.16a, upper comer of the variable space), whereas for the valley-to-peak criterion it was 0.102 M CTAB-10% 2-propanol (Fig. 8.16b), and for the overlapped fi-actions, 0.107 M CTAB-10% 2-propanol (Fig. 8.16c). 

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