Resolving power of high-porosity nanostructured monolithic columns in liquid chromatography for proteomic applications

Particle-packed columns are the first choice as stationary phases to be used in LC for peptide and protein analysis due to its excellent loadability and separation characteristics. Rigid polymer-monolithic stationary phases have emerged as attractive alternative for packed-bed columns. This is due to ease of preparation but also the freedom in structure engineering defining kinetic performance limits and the wide range of surface chemistries available. By fine-tuning the macropore and microglobule size on multiple length scales, the chromatographic performance of current state-of-the-art packed columns can be surpassed. To achieve high resolving power within a short analysis time, monolithic entities in the submicron range need to be synthesized, with macropores ranging between 100 – 500 nm (for fast analysis) or 500 nm – 1 μm (for high-efficiency separations). The limitations and possibilities that exist regarding the effect of structural inhomogeneity on chromatographic dispersion of high-permeability monolithic materials will be a central point of discussion of this contribution. Aiming at high-resolution separation of biomolecules, high-porosity poly(styrene-co-divinylbenzene) monolithic materials featuring nano-sized macropore and globule sizes were developed. The thermodynamic and kinetic properties of the reaction were systematically tuned by varying the porogen ratio, crosslinker density, initiator content, and temperature. Attempts in decreasing the globule and macropore size below a certain threshold led to a point where structural inhomogeneity (A-term) became significant. Optimized polymer monolithic entities yielding separation impedance values <1000 were achieved. High-resolution separations of intact protein were achieved, considering the impact of the gradient volume on the overall performance of these columns. While minimizing the extra-column contribution to band broadening, application for high-throughput analysis is also demonstrated, showing 5 runs of ballistic separation of 6 proteins in a minute (total cycle time of 12 seconds).