Mobile phases optimization

Column chromatographic separations depend on the relative affinity of different proteins for a given stationary phase and for the mobile phase. Association between each protein and the matrix is weak and transient. Proteins that interact more strongly with the stationary phase are retained longer. The length of time that a protein is associated with the stationary phase is a function of the composition of both the stationary and mobile phases. Optimal separation of the protein of interest from other proteins thus can be achieved by careful manipulation of the composition of the two phases. 

Mobile phase optimization by the PRISMA system follows the steps [20,38,66,69,70] ... 

On the basis of Snyder s system for characterization of solvents the PRISMA method for mobile phase optimization has been developed. This system enables the optimization of solvent strength and mobile phase selectivity and also the transfer of the optimized mobile phase to different planar chromatographic techniques, in our case the PLC. 

In summary, the use of RPLC is ideal for pharmaceutical analyses because of the broad range of commercially available stationary phases because the most common RPLC mobile phases (buffers with acetonitrile or methanol) have low UV cut-off wavelengths, which facilitate high sensitivity detection for quantitation of low-level impurities and because selectivity can readily be controlled via mobile phase optimization. Additionally, the samples generated for selectivity screening (as detailed above) are typically aqueous based. In subsequent phases of pharmaceutical development, aqueous-based sample solvents are ideal for sample preparation and are, under limited constraints, compatible with MS detection required to identify impurities and degradation products. 

Sz. Nyiredi, C.A.J. Erdelmeier, B. Meier, O. Sticher, The PRISMA mobile phase optimization in thin-layer chromatography Separation of natural compounds, Planta Medica 3 (1985) 241-246. 

HPLC analysis with three-component mobile phase optimization through PRISMA model. 

Biological Zorbax ODS Mobile phase optimization Ultraviolet 235 57... 

In LC we have a choice between optimizing the stationary phase parameters or the mobile phase parameters. Obviously, the latter can be changed more readily. Advantages of mobile phase optimization are ... 

The second approach is the one followed by Drouen et al. [502]. It is based on the experience that only in very few cases does the optimization of a quaternary mobile phase composition in RPLC yield an optimum that is truly quaternary, i.e. contains all four solvents. Hence, the procedure discussed before for ternary solvents usually leads to the global optimum. This argument, correct though it may be, only applies to the particular problem of mobile phase optimization in RPLC, and prohibits the application of the same method to other two-parameter optimization problems [582]. 

Finally, we will summarize here all the correlational equations and experimental solvent parameters required for predictions of solvent strength and selectivity in LSC, and discuss their significance in terms of mobile-phase optimization strategies. 

Figure 5-3. Selected solvents for mobile-phase optimization in NPC. (Reprinted from reference 11, with permission.)...

The variables that are found to be important or significant in a screening design are retained for further optimization. Screening designs have been used in the mobile phase optimization in Refs. [29,48]. In Ref. [48] six variables were screened in a 2 " fractional factorial design. The factors examined were the oi anic modifier content. 

Mobile phase optimization in normal-phase liquid chromatography... 

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. 

Lesellier, E. Saint Martin, P. Tchapla, A. Separation of six polymer additives using mobile-phase optimization software. LC GC Int. 1992, 5, 38-43. 

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