Impact of biomolecule solute size on the transport and performance characteristics of analytical porous polymer monoliths

• Elution performance of porous polymer monoliths for small and large biomolecules.
• Modulated gel porosity by elution experiments at varying mobile phase compositions.
• Performance at nonretained conditions and biomolecule molecular weight is addressed.
• Plate height curves taper off at high flow speed and pre-asymptotic dispersion.
• Retention and efficiency depend strongly on mobile phase composition and flow rate.

Porous monolithic poly(styrene-co-divinylbenzene) stationary phases in 4.6 mm ID analytical format have been investigated with respect to their transport properties probed by solutes of biological origin varying vastly in size. Elucidation of several properties of these benchmark and robust materials gave complementary insight. These are: (i) the porous polymers’ apparent dry-state microscopic appearance, (ii) the columns porosity probed by the biomolecules and modulated by mobile phase solvent composition, (iii) the impact of probe solute size on apparent retention at varying mobile phase solvent compositions, and (iv) the elution performance under both nonretained and retained elution conditions. By varying the volume percentage of acetonitrile in the mobile phase, it is demonstrated that the monolithic scaffold shows a variable porosity experienced in particular by the larger sized solutes, while the smaller solutes are gradually less affected. The nanoscale swelling and solvation of porous monolithic adsorbents resulting in gel porosity varied with mobile phase solvent composition was, therefore, indicated. The plate height curves for the solutes under nonretained conditions show a moderate increase at increased flow velocity while approaching plateau values. These plateau values were in conjunction with a trend of a decreased performance at an increased molecular weight of the solute. The systematic shape of the plate height curves at increased flow velocity indicates pre-asymptotic dispersion. This is because the column bed aspect ratio of length-to-diameter is equal or smaller than 10. Imposing retention on the solutes at a constant flow velocity deteriorates isocratic elution performance, more pronouncedly for the larger sized solutes at even weak retention. This is explained with slow pore fluid–gel interface diffusion. Additionally, the apparent retention factor for elution of the probe solutes becomes a function of flow rate, consequently a function of imposed pressure experienced by the scaffold.