Optimization of Chromatographic Conditions for Combined Micropore HPLC/Continuous-Flow Fast-Atom Bombardment Mass Spectrometry

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Abstract
System parameters for microbore high-performance liquid chromatography (HPLC) of proteolytic digests are optimized to provide chromatographic separations amenable to the flow rates (5 to 10 µL/min) used with the continuous-flow fast-atom bombardment mass spectrometer (MS) interface. Results that show the effects of column dimensions, the rate at which solvent gradients are delivered to the microbore column, column flow rates, and the slopes of eluting solvent gradients on these separations are presented. Optimal conditions are achieved using a 1 × 50-mm column at volumetric flow rates of 20 to 25 µL/min combined with precolumn flow splitting that minimizes the delay time for delivery of solvent gradients to the column. Postcolumn splitting (1:4) is used to give optimal interface flow rates and to provide simultaneous MS and UV detection. The LC/MS analysis of the tryptic digest of human apolipoprotein A-I is shown to illustrate the utility of the methods.