High-speed gradient separations of peptides and proteins using polymer-monolithic poly(styrene-co-divinylbenzene) capillary columns at ultra-high pressure

• Polymer monolithic capillary columns were operated at ultra-high pressures (80 MPa).
• Monoliths display adequate robustness and stability at such pressures (> 800 runs).
• Ultra-fast (≪1 min) gradient separations of bio-molecules could be realized.
• Potential for both implementation as 2D column in 2D-LC and hyphenation with MS.

For the first time, polymer monolithic capillary columns have been employed at ultra-high-pressure liquid chromatographic conditions (UHPLC) to investigate their potential for high-speed separations of peptides and intact proteins. In comparison to conventional flow rates and gradient conditions, a substantial decrease in analysis time (>factor 4) can be achieved when operating monolithic columns such as ultra-high-pressure conditions while scaling the gradient volume. The effects of flow rate and column length on the peak capacity and the gradient performance limits were assessed for the separation of peptide and protein mixtures applying the maximum system pressure (80 MPa) and a fixed gradient steepness. The potential for ultra-fast gradient separations of large biomolecules was further demonstrated for very steep gradients (gradient times ≪ 1 min). A tryptic digest of cytochrome c was separated using a gradient time of only 1 min. Finally, the run-to-run repeatability and column robustness were assessed at ultra-high pressure conditions (after > 800 runs) with consecutive steep 1 min separations of peptides, yielding RSD values below 0.12% in retention time.