title: High-speed gradient separations of peptides and proteins using polymer-monolithic poly(styrene-co-divinylbenzene) capillary columns at ultra-high pressure.
abstract: 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.
authors.affiliation: Vrije Universiteit Brussel, Department of Chemical Engineering, Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Axel
authors.initials: A
authors.lastname: Vaast
authors.forename: Lucie
authors.initials: L
authors.lastname: Nováková
authors.forename: Gert
authors.initials: G
authors.lastname: Desmet
authors.forename: Björn
authors.initials: B
authors.lastname: de Haan
authors.forename: Remco
authors.initials: R
authors.lastname: Swart
authors.forename: Sebastiaan
authors.initials: S
authors.lastname: Eeltink
mesh_terms: D000818:Animals; D002417:Cattle; D002645:Chickens; D002851:Chromatography, High Pressure Liquid / Q000191:economics / Q000379:methods*; D045304:Cytochromes c / Q000302:isolation & purification; D006736:Horses; D010455:Peptides / Q000302:isolation & purification*; D011108:Polymers / Q000737:chemistry; D011137:Polystyrenes / Q000737:chemistry*; D011506:Proteins / Q000302:isolation & purification*; D040901:Proteomics / Q000191:economics / Q000379:methods; D013997:Time Factors
chemical_list: D010455:Peptides; D011108:Polymers; D011137:Polystyrenes; D011506:Proteins; C432006:divinylbenzene-polystyrene copolymer; D045304:Cytochromes c
keywords: Capillary columns; Proteomics; Repeatability; Ultra-fast separation; Ultra-high pressure