1 mm ID poly(styrene-co-divinylbenzene) monolithic columns for high-peak capacity one- and two-dimensional liquid chromatographic separations of intact proteins
✍ Scribed by Sebastiaan Eeltink; Sebastiaan Dolman; Frederik Detobel; Gert Desmet; Remco Swart; Mario Ursem
- Publisher
- John Wiley and Sons
- Year
- 2009
- Tongue
- English
- Weight
- 834 KB
- Volume
- 32
- Category
- Article
- ISSN
- 1615-9306
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✦ Synopsis
Abstract
The LC performance of a 1×50 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/×/RPLC separation of intact Escherichia coli proteins was performed within 6 h yielding a maximum theoretical peak capacity of 4880.