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Overview: High duty cycle nanoLC-MS analyses can be achieved using two columns that alternate between on-line MS analysis and off-line washing and re-equilibration. Using columns with closely matching performance, peptide separations with near identical retention times can be achieved with up to 2X improvement in throughput. Introduction: Inter-column reproducibility is difficult to achieve for capillary columns that operate at nanoliter flow rates. Variations in column ID, inlet- and outlet- frits, column lengths and packing methods, contribute to variations in performance between columns. Chip columns are fabricated to very tight tolerances and as a result achieve closely matched performance. In addition using a recently developed connection system to these chips, variations due to poor connections are eliminated. Near identical performance between columns creates the possibility for increasing throughput in proteomic experiments by alternating between multiple columns during the analysis. In this poster we will be reporting on a microfluidic based method to maximize throughput in LC-MS peptide analyses by alternating between two chip columns. Materials/Equipment: NanoLC-Ultra nanoLC system with cHiPLC nanoflex (Eksigent Technologies, Dublin, CA) in direct injection mode. 15 cm × 75 μm ID ChromXP C18 3μm cHiPLC column (Eksigent Technologies) LTQ iontrap MS (Thermo Scientific, San José, CA) with New Objective (Woburn, MA) nanospray source. Bovine serum albumin reduced/alkylated and digested with trypsin. Summary of Results: We will summarize the inter-run reproducibility of peptide retention times and peak shapes using a dual chip based column set-up over long periods of time and multiple columns. Conclusions: A chip LC platform enables the acquisition of high quality, highly reproducible data. The increased sample throughput obtained using alternating chip based columns and a dual gradient nanoLC pump makes the described system very attractive for studies with larges amounts of samples while at the same time conserving expensive MS analysis time.