Hydrodynamic chromatography of macromolecules using polymer monolithic columns.

ABSTRACT

The selectivity window of size-based separations of macromolecules was tailored by tuning the macropore size of polymer monolithic columns. Monolithic materials with pore sizes ranging between 75 nm and 1.2 µm were prepared in situ in large I.D. columns. The dominant separation mechanism was hydrodynamic chromatography in the flow-through pores. The calibration curves for synthetic polymers matched with the elution behavior by HDC separations in packed columns with 'analyte-to-pore' aspect ratios (λ) up to 0.2. For large-macropore monoliths, a deviation in retention behavior was observed for small polystyrene polymers (M(r)<20 kDa), which may be explained by a combined HDC-SEC mechanism for λ<0.02. The availability of monoliths with very narrow pore sizes allowed investigation of separations at high λ values. For high-molecular weight polymers (M(r)>300,000 Da) confined in narrow channels, the separation strongly depended on flow rate. Flow-rate dependent elution behavior was evaluated by calculation of Deborah numbers and confirmed to be outside the scope of classic shear deformation or slalom chromatography. Shear-induced forces acting on the periphery of coiled polymers in solution may be responsible for flow-rate dependent elution.