Abstract
OBJECTIVE: Short-chain triacylglycerols (TAGs) in lipid extracts of biological samples are not sufficiently resolved using conventional reversed-phase separation on two C18 columns in series, or using a two-dimensional chromatographic separation with a silver ion column as the second dimension ((2)D). An additional dimension of separation was required. RESULTS: The hardware and software components to allow a second second-dimension ((2)D) separation and three total separation dimensions were developed. Two contact closure (CC) activated 4-port, 2-position valves (4P2PVs) for ultra-high performance liquid chromatography (UHPLC) were joined together and used for one of two second dimensions in comprehensive two-dimensional liquid chromatography (2D-LC) coupled to four mass spectrometers simultaneously in parallel in an LC1MS2 × (LC1MS1 + LC1MS1) = LC3MS4 configuration. A timed contact closure circuit (TCCC) controlled the two UHPLC valves, operated by repetitive CCs for the 4P2PVs. The TCCC-controlled 4P2PVs were used to direct a portion of the (1)D eluent to one of the two (2)D's for separation by a quaternary UHPLC system that was not allowed by the commercial 2D-LC system. The (1)D separation was a non-aqueous reversed-phase HPLC instrument used for separation of TAGs; the commercial 2D-LC (2)D binary UHPLC was used for silver-ion chromatography of unsaturated TAGs; and the CC-controlled second (2)D was used for separation of short-chain (SC) saturated TAGs.