Design and Implementation of a Proof-of-Concept Robotic–Microfluidic\nInterface to Bridge Spatial Comprehensive Three-Dimensional Liquid\nChromatography with Mass Spectrometry

A proof-of-concept system is presented\nfor the hyphenation of spatial\ncomprehensive three-dimensional liquid chromatography (3D-LC) to mass\nspectrometry (MS) detection via a robotic–microfluidic interface.\nA three-dimensional fractal microflow distributor, incorporating 16\nparallel RP monolithic capillary columns arranged in a 4 × 4\nconfiguration, was connected to an X-Y-Z robotic system. This setup\nfacilitated the deposition of successive arrays of microdroplets onto\nan MS target plate. To minimize carryover during droplet deposition,\na strategy was implemented in which the distance between the target\nplate and the capillary was gradually increased during the deposition\nprocess. System-level variation in travel time and subsequent flow\nrates across parallel columns was assessed and translated in retention\nalignment based on injection of a protein standard. The successful\nseparation of intact proteins was demonstrated through a parallel\n4 × 4 column configuration, applying MALDI-MS detection after\nmicrodroplet spotting on an MS target plate. Furthermore, the discussion\nencompasses high-throughput MS imaging detection within the framework\nof spatial 3D-LC.