Flow rate dependence on the biopolymer retention in hydrodynamic chromatography. Comparison between the behaviors of proteins and plasmids

Hydrodynamic chromatography (HDC) in a packed column is a useful chromatogr aphic mode for the rapid separation of polymers. This paper compared the re tention of various circular double-stranded DNA molecules (3, 5 and 10 kbp) and proteins (keyhole lumpet hemocyanin, ferritin, thyroglobulin, and cata lase) in a chromatographic system using a C1 stationary phase and a acetoni trile-phosphate buffer mixture as a mobile phase. For a similar molecular w eight, the protein was retained more than the corresponding plasmid. This w as attributed to the difference in the compactness between the two species. As well, the retention dependence on the flow rate (0.03 to 1.5 mL/min) ex hibited a different behavior in relation to the type of biopolymer. The protein retention curve showed a decrease in the relative retention tim e until 0.2 mL/min, followed by an increase for the high size polymers, onl y, while the plasmid retention increased over the entire flow rate range. T his observation confirmed that the migration in HDC was dependent on two an tagonist phenomena, i.e. stress induced diffusion and polymer deformation. This work demonstrated that the HDC separation of protein was optimal for a flow rate equal to 0.2 mL/min.