Supercritical fluid chromatography x ultra high pressure liquid chromatography for red chilli pepper fingerprinting by photodiode array, quadrupole-time-of-flight and ion mobility mass spectrometry (SFC x RP-UHPLC-PDA-QToF MS-IMS).

The coupling of normal phase and reversed phase liquid chromatography (NP-LC x RP-LC) is one of the most effective ways to increase orthogonality in two-dimensional comprehensive separations, being the two retention mechanisms truly independent. However, such coupling is not straightforward to implement, due to immiscibility of the mobile phases, poor peak focusing at the head of the secondary column (D-2), signal interferences and peak deterioration. In this research, supercritical fluid chromatography (SFC) was used in the first dimension (D-1), coupled to ultra high pressure LC (UHPLC), to alleviate incompatibility issues and provide a number of advantages related to the use of supercritical CO2. An on-line SFC x RP-UHPLC system was implemented in an automated fashion, around two 2-position, six-port switching valves equipped with octadecyl silica cartridges for effective trapping and focusing of the analytes eluted from D-1. A water make-up flow added to the SFC effluent permitted to efficiently focus the solutes on the sorbent material and to reduce interferences of the expanded CO2 on the D-2 separation. In addition to photodiode array and quadrupole-time-of-flight mass spectrometry detection, ion mobility separation based on analyte mass, shape and size added a fourth separation dimension for carotenoid fingerprinting in a red chilli pepper sample.