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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


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.

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