An integrated multipath liquid chromatography–mass spectrometry system for the simultaneous preparation,separation, and detection of proteins and small molecules

A multipath liquid chromatography with mass spectrometry instrument was constructed with the help of restricted access media to online segregate small and large molecules. This liquid chromatography system was custom built with five pumps and three two‐position six‐port valves to control the flow in a multipath system for the simultaneous analysis of small molecules and proteins. On separate chromatographic channels, small molecules trapped and proteins excluded from the online restricted access media were analyzed downstream using high‐efficiency columns and a triple quadrupole mass spectrometer. A model sample, which included five proteins and 22 small molecules with different physicochemical properties, was used to evaluate the system. Following injection, the complete multipath separation and detection was performed in 22 min. Protein exclusion by the restricted access media was not quantitative. Four commercial trap columns were evaluated for their exclusion efficiency toward the proteins. Exclusion efficiency varied from <50% to only a maximum of 75% exclusion across the trap columns tested. An attempt was made to optimize the exclusion efficiency using different flow rates, flow rate gradients, and different additives both in the sample and the mobile phases. Protein exclusion was still erratic and generally nonquantitative.