1 mm ID poly(styrene‐co‐divinylbenzene) monolithic columns for high‐peak capacity one‐ and two‐dimensional liquid chromatographic separations of intact proteins

Abstract

The LC performance of a 1x50 mm polymer monolithic column format was demonstrated with high-peak capacity one- (1D) and offline two dimensional (2D) LC separations of intact proteins. After optimizing the RP 1D-LC conditions, including column temperature, flow rate and gradient time, a peak capacity of 475 was achieved within a 2-h analysis. The suitability of the monolithic column was also demonstrated for fast 1 min protein separations yielding 1 s peak widths determined at half peak height. In addition, an offline 2D-LC method was developed using the micro-fraction collection capabilities of the autosampler allowing automatic fractionation of intact proteins after the weak-ion-exchange (WAX) separation, and re-injection of the fractions onto the second-dimension RP monolithic column. The best peak capacity-to-analysis time ratio was obtained when applying 10 min second-dimension RP gradients. At optimized conditions, the WAX/x/RPLC separation of intact Escherichia coli proteins was performed within 6 h yielding a maximum theoretical peak capacity of 4880.

New trends in reversed-phase liquid chromatographic separations of therapeutic peptides and proteins: Theory and applications

“…The use of a 5 cm-long poly(styrene-co-divinylbenzene) monolithic 1 mm I.D. column for the separation of intact proteins was previously reported [121]. When a 1 min-long gradient span was applied, peak widths at half height of only 1 s were achieved.…”

Evolution and Current Trends in Liquid and Supercritical Fluid Chromatography

“…Finally, 5 cm-long poly(styrene-co-divinylbenzene) monolithic column of 1 mm I.D. were employed for the separation of intact proteins [51]. Using short capillary poly(styrene-codivinylbenzene) monolith coupled to an LTQ Orbitrap XL mass spectrometer, a limit of detection in the low femtomol range was achieved after injecting a mixture of nine proteins with molecular weights ranging from 5.7 to 150 kDa [52].…”

Recent advances on multidimensional liquid chromatography–mass spectrometry for proteomics: From qualitative to quantitative analysis—A review

“…The first online 2D-LC system coupled SCX with RPLC for protein separation was proposed by Jorgenson and co-workers [50], and then various online multidimensional HPLC separation methods have been reported for intact protein profiling [51][52][53][54]. Recently, Zhou et al [55] proposed an online 2D-capillary isoelectric focusing (CIEF)-RPLC-ESI-MS system for intact protein profiling.…”