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J Biomol Tech. 2007 February; 18(1): 65.
PMCID: PMC2292019

P188-M Long- and Mixed-Column Nanobore Chromatography for Complex Proteomic Analysis

Abstract

Complex proteomic digests can require a combination of either multi-dimensional biphasic packed beds or longer reverse-phase beds (greater than 15 cm) to achieve adequate separation. Multi-dimensional and extended-length columns are time-consuming and expensive to produce; column failure of one section of the packed bed requires replacement of the entire column. An attractive alternative to a single biphasic or long-bed column is based on efficient column-to-column coupling, as employed with conventional-bore high-performance liquid chromatography. Such connections in nanobore LC are enabled by coupling shorter-bed columns via optically clear high-pressure zero-dead-volume (ZDV) unions. This approach facilitates extended-column configurations and column swap-out during system maintenance. Using two clear ZDV unions to achieve flush connections to column bed termini, two conventional 10-cm-bed nanobore columns were coupled and connected to the bed terminus of a 10-cm nanobore column with integrally fritted tip. Analytical performance of this extended-bed column was then compared with a single 30-cm-bed nanobore column with integrally fritted tip. Chromatographic data from single- and extended-bed configurations were nearly identical, with negligible peak-tailing and resolution loss. The first section of the column is readily removed, replaced, and effectively integrates guard column performance into the analytical column bed.


Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities