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　　On-column injections using packed-tip columns realize optimal chromatographic separation and sensitivity for nanoLC-MS experiments. The unique packed-tip column format adds no post-column volume to the LC plumbing thus providing the sharpest peak shape achievable given the packed column’s specifications (internal diameter and stationary support). Qualitative and quantitative separations of complex biomolecular samples usually depend on long gradients to maximize the separation capacity of the column and improve the recovery of low concentration analytes. While nanobore columns enable nanoliter flow rates, the ability to sample load onto the column is limited by the backpressure of the column. Trap column injection is one approach to enable the ability to sample load at higher flow rates but the resultant peak broadening decreases sensitivity. Here, we present a hardware solution utilizing an automated XYZ translation stage digitally controlled by a graphical user interface. This system enables automated cycling between multiple columns, as controlled by an external device, most commonly the MS or the LC system. Duty cycle improvement is demonstrated by loading, washing and equilibrating one column off-line while the second column gradient elutes. Challenges to the dual column approach include optimizing the flow path to ensure column-to-column reproducibility and creating an optimized autosampler method to control for analyte carryover. Solutions to these challenges are presented for a Thermo LTQ MS connected to a direct flow nanopump using commercially available peptide digests. With each column at voltage during MS acquisition, the presence of cross talk between the two columns was evaluated using syringe infusion.

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