The separation of high-molecular compounds is very difficult, if possible at all, under isocratic conditions. For this 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.
We used high performance liquid chromatography to investigate the reversed-phase chromatographic behaviour of 14 proteins. The first step was the determination of the experimental data, and then these data were used to predict gradient retention times. A water–organic solvent–trifluoroacetic acid system was used to examine the influence of experimental parameters. The chromatographic results from four C18-chain-length supports were comparable.
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