The online preconcentration of analytes onto low volume trapping columns is a commonly used injection strategy in nano- and microbore LC-MS/MS analysis of complex peptide mixtures. Compared to direct injection onto the analytical column, a sample trapping approach provides several advantages. By effectively desalting and preconcentrating the analytes of interest onto the trap column, analytical column lifetime and workflow throughput can be improved. The trapping configuration allows effective removal of sample matrix components such as salts and contaminants which can interfere with downstream MS analysis, thus increasing analytical column lifetime and at the same time improving detection sensitivity and the spectral quality that is generated for a sample. It also allows loading higher volume and dilutes samples at a much higher flow rate than what is feasible when working with a direct injection approach, having a positive effect on the LC-MS/MS duty cycle.
However, apart from providing sample clean-up and preconcentration, it is also of paramount importance to maintain chromatographic performance when combining an analytical column with a trapping column.
In this application note, PharmaFluidics demonstrates how µPAC™ trapping columns can be effectively used to perform large volume (≥5 µl) peptide sample injections on µPAC™ analytical columns with minimal impact on the total analysis time. The unique design of these trapping columns allows loading samples at high flow rates (10-20 µl/min) and this at very low operating pressures (20-40 bar). The surface morphology of these columns is designed to match µPAC™ analytical nano-LC columns (50 and 200 cm long nano-LC versions available). Minimal loss of chromatographic performance was observed when comparing the “in-valve” trapping setup to a direct injection strategy. For 15 PRTC peptides, on average 5% increase in peak width (FWHM) was observed.
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