Literature Review – Ultrahigh pressure in the second dimension of a comprehensive two-dimensional liquid chromatographic system for carotenoid separation in red chili peppers

Cacciola, F.; Donato, P.; Giuffrida, D.; Torre, G.; Dugo, P.; Mondello, L. Journal of Chromatography A 2012, 1255, 244–251.

This paper is focused on the separation and identification of intact carotenoids in red chili pepper extracts by NP x RP. A micro-bore cyano1D column, dimensions 25 cm x 1.0 mm i.d., 5 μm dp, was paired with a 3 cm x 4.6 mm i.d., 2.7 μm dp superficially porous C18 2D column. The motivation for this work was the application of ultrahigh pressure conditions to the second dimension separation as a means to increase overall analysis peak capacity.

To demonstrate the advantage of UHPLC in the second dimension, a total of three instrumental conditions were evaluated: 1) conventional NP x RP with a modulation time of 1.0 min, 2) NP x UPHLC- RP, with a modulation time of 1.5 min, and 3) NP x UPHLC- RP, modulation time 1.0 min. In the UHPLC separations column length was doubled to 6 cm by serially coupling two 3 cm columns. Each of these instruments was operated with identical 110 min linear 1D gradients providing a 1nc of 45. Overall peak capacity values were calculated for each system, corrected for undersampling, and determined to be 526 for the NP x RP, tm = 1.0 min, and 373 for the NP x RP (UHPLC), tm = 1.5 and 636 for the NP x RP (UHPLC) system with a 1.0 min tm.

Despite the doubling of the 2D column length, with respect to the ‘conventional’ NP x RP set-up, the peak capacity of the 1.5 min modulation time separations were greatly reduced by undersampling. The authors indicate that a lot of work needs to be done to optimize this separation and reduce the detrimental effect of undersampling.

The authors also report this to be the first work incorporating UHPLC conditions in the second dimension and the use of a C18 column with 2.7 μm SPPs.

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