Peak capacity optimization of peptide separations in reversed-phase gradient elution chromatography: fixed column format

The optimization of peak capacity in gradient elution RPLC is essential for the separation of multicomponent samples such as those encountered in proteomic research. In this work, we study the effect of gradient time (tG), flow rate (F), temperature (T), and final eluent strength (phi(final)) on the peak capacity of separations of peptides that are representative of the range in peptides found in a tryptic digest. We find that there are very strong interactions between the individual variables (e.g., flow rate and gradient time) which make the optimization quite complicated. On a given column, one should first set the gradient time to the longest tolerable and then set the temperature to the highest achievable with the instrument. Next, the flow rate should be optimized using a reasonable but arbitrary value of phi(final). Last, the final eluent strength should be adjusted so that the last solute elutes as close as possible to the gradient time. We also develop an easily implemented, highly efficient, and effective Monte Carlo search strategy to simultaneously optimize all the variables. We find that gradient steepness is an important parameter that influences peak capacity and an optimum range of gradient steepness exists in which the peak capacity is maximized.