Preparative gradient elution chromatography of chemotactic peptides.

Experimental and computational studies are carried out on the separation of a mixture of chemotactic peptides by reversed-phase gradient elution on commercial octadecyl silica supports with acetonitrile as the modulator. The solubility of this mixture is found to be a complex function not only of mobile phase composition, but also of the order in which the various constituents of the mobile phase are mixed together. In certain cases, the feeds seem to reach a metastable state in which they are fully soluble for several hours: this is exploited here for preparative separations. Separations are also carried out by stepwise and nonlinear isocratic elution, and the yields and productivities compared to those from gradient elution. Predictive simulations of all these separations are run using independently measured single component feed isotherms. Good agreement with experiment is found when multicomponent (nonlinear) feed interactions are accounted for, but not when the usual assumption of linear feed isotherms is made. Simultaneous concentration and separation of these feeds is easily achieved by gradient elution. Simulations indicate that the combination of the focusing power of the gradient with the multicomponent feed interactions is likely to give good separations even at high feed loadings.