Ultra high pressure in the second dimension of a comprehensive two-dimensional liquid chromatographic system for carotenoid separation in red chili peppers

A comprehensive normal-phase × reversed-phase (NP-LC × RP-LC) liquid chromatography system was developed, and applied for analysis of the intact carotenoid composition of red chili peppers, with photodiode array and mass spectrometry detection. A micro-bore cyano column (250 mm × 1.0 mm, 5 μm d.p.) was chosen for the first dimension (1D) separation, interfaced to a second dimension (2D) C18 column (30 mm × 4.6 mm, 2.7 μm d.p.) packed with fused-core particles. Subsequently, two columns of the same stationary phase were coupled serially for second dimension separation, and operated under ultra high pressure LC conditions (UHPLC), within a cycle time of 1.50 or 1.00 min, and equal modulation times. Performances of the three different set-ups were evaluated, in terms of peak capacity values (nc), and afterwards corrected by taking into account both the under-sampling, and the orthogonality effects. After these adjustments, the peak capacity values were estimated as follows: nc 526, for the NP-LC × RP-LC system, nc 373, for the NP-LC × RP-UHPLC system with a 1.50 min modulation time, nc 639, for the NP-LC × RP-UHPLC system, with a 1.00 min modulation time. A total of 33 compounds were separated into 10 different chemical classes in the two-dimensional space, and identified by accurate IT-TOF (ion trap-time of flight) MS detection.