Overloading, chromatography

Two case studies will be shown here to demonstrate the development of purification processes in both overload and resolution based separations 51]. The first example summarizes the purification of a synthetic peptide by overload chromatography, or more accurately described as sample self displacement chromatography The techniques applied to this separation are applicable to any molecule and can be applied to all modes of chromatography, with the exception of size exclusion chromatography. 

Lor a particular analytical separation, each biosolute will have an optimal k] value for maximum resolution with a designated column, flow rate, and mobile phase composition. Similar criteria apply in preparative (overload) chromatography with multicomponent mixtures, where resolution is similarly enhanced following optimization of chromatographic selectivity and zone bandwidth. The conventional approach to process purification with low molecular weight solutes has frequently been based on linear scale-up of the performance of an analytical column system. In these cases, high-resolution separations can be achieved often without the burden of conformational or... 

Jandera, P. Komers, D. Guiochon, G. Effects of the composition of the mobile phase on the production rate in reversed-phase overloaded chromatography. J. Chromatogr. A, 1997, 7S7(l-2), 13-25. 

Contemporary development of chromatography theory has tended to concentrate on dispersion in electro-chromatography and the treatment of column overload in preparative columns. Under overload conditions, the adsorption isotherm of the solute with respect to the stationary phase can be grossly nonlinear. One of the prime contributors in this research has been Guiochon and his co-workers, [27-30]. The form of the isotherm must be experimentally determined and, from the equilibrium data, and by the use of appropriate computer programs, it has been shown possible to calculate the theoretical profile of an overloaded peak. 

A chromatographic separation can be developed in three ways, by displacement development, by frontal analysis, and by elution development, the last being almost universally used in all analytical chromatography. Nevertheless, for the sake of completeness, and because in preparative chromatography (under certain conditions of mass overload) displacement effects occur to varying extents, all three development processes will be described. 

Preparative chromatography involves the collection of individual solutes as they are eluted from the column for further use, but does not necessarily entail the separation of large samples. Special columns can be designed and fabricated for preparative use, but for small samples the analytical column can often be overloaded for preparative purposes. Columns can be either volume overloaded or mass overloaded. Volume overload causes the peak to broaden, but the retention time of the front of the peak... 

In all modes of chromatography, high sample loads distort peak shapes and cause an overall decrease in efficiency due to column overload. Sample loads may be increased by using organic solvents to enhance the solubility of the sample or by using higher column temperatures to lower the viscosity of... 

Finally, an officially updated definition of the retardation factor, R, issued by lUPAC is important to the whole field of planar chromatography (the linear and the nonlinear TLC mode included). The importance of such a definition has two reasons. First, it is promoted by the growing access of planar chromatography users for densitometric evaluation of their chromatograms and second, by the vagueness of the present definition in the case of skewed concentration profiles with the samples developed under mass overload conditions. 

The final purification steps are responsible for the removal of the last traces of impurities. The volume reduction in the earlier stages of the separation train is necessary to ensure that these high-resolution operations are not overloaded. Generally, chromatography is used in these final stages. Electrophoresis can also be used, but since it is rarely found in process-scale operations, it is not addressed here. The fin product preparation may require removal of solvent and drying, or lyophilization of the product. 

So far we have considered preparative-scale chromatography when the column is not overloaded and the desired amount of ,sample... 

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