Separation conditions optimization strategies

As in normal phase (see section 3.5.3), the first step in mobile-phase optimization is the determination of the solvent strength that will elute the analytes with a A value between 2 and 10 from the chosen stationary phase. It is not important which modifier is chosen to determine the initial conditions, and methanol-water (50 50, v/v) is a convenient starting place. Once the initial conditions have been established, a variety of techniques may be employed to obtain the optimum separation. Most optimization strategies involve the establishment of the isoelutropic concentrations of methanol-water, acetonitrile-water and tetrahydrofuran-water. The isoelutropic concentrations can be determined by experiment or from tables of isoelutropic mixtures (e.g. Table 3.5) (Wells, 1988). The binary solvent systems A, B, C (Table 3.5, Figure 3.7) define the isoelutropic plane, which is then explored to obtain the optimum combination of water, methanol, tetrahydrofuran, and acetonitrile required for the separation. 

Optimization of the separation of these samples is much more challenging than samples of homologues or oligomers. Basic chromatographic theory (equations 1-4 and related text) provides little direction for these separations, due to peak reversals that occur almost universally when conditions are changed, particularly temperature or composition. Historically, researchers have generally focused on only one or, at most, two experimental variables at a time, and have chiefly used trial-and-error as their optimization "strategy". 

STRATEGIES FOR SELECTING AND OPTIMIZING ISOCRATIC SEPARATION CONDITIONS... 

The plate count required for a resolution of 1.0 using different separation conditions is summarized in Table 1.10 [146]. Practically all chromatographic separations have to be made in the efficiency range of 10 -10 theoretical plates. The importance of optimizing the separation factor and retention factor to obtain an easy separation is obvious from the data in Table 1.10. Easy separations require chromatographic systems that maximize the separation factor and provide at least a minimum value for the retention factor. A common optimization strategy for difficult separations with a limited number of components is to fix the value of the retention factor between 1 and 3 for the two components most difficult to separate in the mixture. 

With the program and derived mathematics, the scientist can calculate the retention times and peak widths for any set of solutes, run under any conditions ranging from Isocratlc to complex gradients with multiple columns. Given this mathematical tool, the problem of determining which chromatographic conditions will achieve the desired separation, within any constraints, becomes the next problem to be approached. The solution to such an "optimization" problem Is not easy, however, the necessary universal mathematical tools are now available for researchers In this area to develop approaches to optimization strategies. 

Many factors may influence the quality of CE separations and contribute to the resolution of protein modifications. Generally, optimization strategies, precautions, and CE conditions applied to peptides are also applicable to the separation of modified proteins. However, optimum parameters for protein separations must still be determined empirically to some extent. In addition to well-known factors influencing resolution in CE like capillary surface and coating, buffer pH, also effects of different... 

As a result, the complementary LC—MS and NMR data of the 12 constituents in the active crude extract were correlated and recovered successfully for simultaneous structure identification by using the NMR/LC—MS PDS method without the need for completed separation and isolation. Five quercetin-based (constituents 1, 2, 3, 7, and 8) and five kaempferol-based compounds (constituent 4, 5, 6, 9, and 10), besides quercetin (constituent 11) and kaempferol (constituent 12), were identified. The NMR/LC-MS PDS technique with the incomplete separation strategy played a more important role in the structure identification of coeluted isomers, such as the coelution of constituents 1, 2, and 3. In this case, the chromatographic separation conditions should be carefully optimized in the combination NMR techniques. Moreover, besides the recovered XICs and NMR signals, relevant spectral information, such as LC—MS/MS data and one-dimensional total correlation spectroscopy (TOCSY) spectra, were collaborated through index of the recovered spectroscopic signals for the unambiguous structure identification. 

Typically, resolution diagrams in MLC are complex, with several local maxima, frequently denoting interaction between factors. For this reason, reliable optimal conditions require considering all factors simultaneously, by applying an interpretive optimization strategy (i.e., based on the description of the retention behavior and peak shape of solutes). In this task, the product of free peak areas or purities has proved to be the best optimization criterion. An interactive computer program is available to obtain the best separation conditions in... 

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