Overload gradient elution

Analytical Solution for Volume-overloaded Gradient Elution Chromatography. 703... 

The Pr xY objective fxmction can successfully be applied to the optimization of overloaded gradient elution chromatography [40,43]. Figure 18.22 compares the chromatograms obtained rmder the optimum conditions given by the Pr and... 

Felinger, A. Guiochon, G. Multicomponent interferences in overloaded gradient elution chromatography. J. Chromatogr. 1996, 724, 27-37. 

While analyses made in gradient elution often involve the use of small and dilute samples, the column is often overloaded in preparative gradient elution chromatography. This causes the adsorption isotherms to be nonlinear and competitive. Therefore, interference effects become important. Furthermore, the mass transfer resistances can be very significant, especially for macromolecules. Various dispersive effects, such as axial dispersion and the mass transfer resistances often coimter-balance the thermodynamic effects of adsorption and desorption... 

Later Antia and Horvath [39] discussed the numerical solution of overloaded linear gradient elution. They calculated numerical solutions of the mass balance equations for single components and binary mixtures. These authors assumed ... 

Vacuum chromatography is simple, rapid, and convenient. Optimum sample loads are similar to flash chromatography. However, it is not unusual to use sample overload conditions to separate simple mixtures by stepwise gradient elution, or to simplify mixtures for further separation. Under these conditions the sample loads may reach 10 % (w/w), or even higher, of the bed mass. 

The effect on chromatography is to complicate the separation greatly. If we consider a reverse-phase separation, the first thing we notice is an almost irreversible binding of protein to the column. Even after protein removal, we find polar peaks, which overload the early part of the chromatogram and tail into the compounds of interest. The components that are more nonpolar than our compounds of interest adhere to the column and must be washed off before the next injection. To ensure polar elution before our target compounds and nonpolar removal afterwards, we are almost forced to run solvent gradients. 

In cases where mass transfer is rapid, as is the case with most small molecule separations, then isocratic elution can offer advantages such as automatic fraction reprocessing and solvent recycle. However, with larger synthetic objectives the rate of mass transfer is comparatively low so isocratic elution leads to band broadening and subsequently to recovery of the peptide at high dilution. Most preparative HPLC based peptide separations are carried out under gradient and overload conditions that allow for maximum throughput in terms of time and quantity. 

A generic gradient recommended for preparative separations is to start the elution at a concentration of B that corresponds to a value 10% earlier that the anticipated desorption then apply a change in concentration of B corresponding to 5% over 15 minutes. To exemplify this approach Table 5.2 shows a gradient, applied to an analytical column, used to purify a peptide that is anticipated to desorb at 20% B under overload conditions. 

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