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J Chromatogr A. 2013 Aug 23;1304:177-82. doi: 10.1016/j.chroma.2013.07.040. Epub 2013 Jul 13.

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

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Vrije Universiteit Brussel, Department of Chemical Engineering, Pleinlaan 2, B-1050 Brussels, Belgium.


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 (80MPa) and a fixed gradient steepness. The potential for ultra-fast gradient separations of large biomolecules was further demonstrated for very steep gradients (gradient times≪1min). A tryptic digest of cytochrome c was separated using a gradient time of only 1min. Finally, the run-to-run repeatability and column robustness were assessed at ultra-high pressure conditions (after>800 runs) with consecutive steep 1min separations of peptides, yielding RSD values below 0.12% in retention time.


Capillary columns; Proteomics; Repeatability; Ultra-fast separation; Ultra-high pressure

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