(237f) Diffusion Coefficients of DNAs and Pegylated Proteins for Estimating the Performance of Chromatography

Pore diffusion governs the separation or adsorption performance of chromatography. Especially, for large biomolecules such as DNA and PEGylated proteins low pore diffusion coefficients result in low performance of chromatographic separations. Monolithic chromatography, a type of convection-aided chromatography, is best suited for such large biomolecule separations. In order to understand the advantage of monolithic chromatography against porous-bead chromatography quantitatively the diffusion coefficients of large biomolecules are needed. Unfortunately, diffusion data (pore diffusion coefficient D_s and molecular (free) diffusion coefficient D_m) for large biomolecules are not readily available.

In this study, the molecular diffusion coefficient D_m (free diffusion) values were determined by using the Taylor dispersion method for various DNAs and PEGylated proteins as well as standard globular proteins. Necessary conditions for measuring D_m precisely such as tube length, tube diameter, coil diameter and flow velocity were first checked. Single-strand (ss) DNAs showed much lower molecular diffusion coefficients compared with those for globular proteins.

The pore diffusion coefficient D_s values were measured by using isocratic elution at non-binding conditions. D_s of ss-DNA decreased with increasing DNA molecular weight much more sharply compared with D_m. Similar trends were observed for PEGylated proteins. Based on these data we calculated the separation performance of typical porous bead ion-exchange chromatography (IEC) in comparison with that of monolithic IEC.