Optimization gradient separation

Optimizing gradient separations requires the measurement of corresponding isocratic parameters (e.g., k, and 5) for many samples this is more easily done using gradient elution than isocratic elution. 

Computer simulation was first developed for reversed-phase separations. However, computer simulation can also be used for the reliable prediction of ion-exchange separations as a function of gradient conditions [49,50]. For a farther discussion of computer simulation for the systematic development of optimized gradient separations for peptide arid protein samples, see Ref. 51. 

Once the resolution has been optimized as a function of gradient rate, one can continue to fine-tune the separation, raising flow rate and temperature. In a study of temperature and flowrate variation on the separation of the tryptic peptides from rabbit cytochrome c, column performance doubled while analysis time was reduced by almost half using this strategy.97 Commercially available software has been developed to aid in optimization. As a final note, in an industrial laboratory optimization is not completed until a separation has been shown to be rugged. It is a common experience to optimize a separation on one column, only to find that separation fails on a second column of identical type. Reproducibility and rigorous quality control in column manufacture remains a goal to be attained. 

White recently illustrated the use of fast supercritical fluid and EFLC for drug discovery and purification [46]. The optimized isocratic separations used to scale up to preparative-scale separations were often EFL mixtures. For example, Figure 9.13 shows the optimized conditions for the separation of a drug candidate included 30% methanol (with 0.2% isopropyl amine)/C02 on a Chiralcel OJ-H column at 5 mL/min [46]. His work also illustrates by using gradients that start in supercritical conditions and then move into EFL mixture conditions provides efficient and fast separations. 

Combining the appropriate equations for the retention volumes of the solutes 1 and 2 with adjacent bands and Equation 5.5, we obtain Equation 5.6 for resolution in gradient LC [4,6,28], which can be used in the optimization of gradient separations ... 

MUX inlet as possible. A flow rate of 12 ml/min on eight 2.1 x 50 mm Polaris Ci8 columns was optimal for general purposes in our study. This system could analyze more than 3000 compounds per day for a gradient separation with a cycle time of 3.5 min. 

All density gradient separations are carried out in aqueous cesium chloride solutions to which a nonionic surfactant has been added (polyoxyethylene-23-lauryl ether, Brij-35). The surfactant is necessary to disperse the fine coal particles in the aqueous medium. Without complete particle dispersion the separation will most likely not be optimal. 

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