The separation of high-molecular compounds under isocratic conditions is very difficult, if possible at all, and thus gradient elution is needed. The theory of gradient elution for small molecules is well established; however, its applications to reversed-phase gradient separations of biopolymers are not straightforward because of specific problems, such as slow diffusion, limited accessibility of the stationary phase for larger molecules, or possible sample conformation changes during the elution.
The first step of our study was the determination of the experimental data, and then these data were used to predict gradient retention times. High performance liquid chromatography was used to investigate the reversed-phase chromatographic behaviour of four proteins. The influence of experimental parameters was examined using a water/organic solvent/trifluoroacetic acid system. Chromatographic results from four Zorbax stationary phases supports were comparable.
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