Sample trapped column injection is an injection strategy commonly employed in nanobore LC/MS based analysis of complex peptide mixtures. The approach of sample trapped column injection provides several inherent advantages. By effectively desalting and concentrating samples on-line, sample traps improve analytical column longevity and throughput. Additionally, the ability to load samples onto a trap column at a much higher flow rate than is feasible for a typical 75μm ID nanobore analytical column is a particularly attractive feature of this approach. Duty cycle can be significantly improved when the sample loading flow rate is decoupled from the gradient flow rate. Here we investigate the role sample trap column injection has on analyte retention and overall chromatographic performance. Using a direct flow nano-LC pump with the ability to deliver flow rates ranging from 50 nl/min to 20 μ/min coupled to an autosampler and commercially available peptide standards and protein digests, we evaluated the relationship between flow rate, analyte concentration and analyte composition to determine the effect on chromatographic performance.