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The present investigation is based on the evaluation of the performance of a comprehensive two-dimensional liquid chromatography (LC×LC) system during method optimization. The LC×LC set-up, operated in normal phase (NP) mode (adsorption) in the first dimension (1D) and reversed-phase (RP) mode in the second dimension (2D), is equipped with a 1D microbore silica column and a 2D monolithic C 18  column with a 10-port two position valve as the interface. A photodiode array detector is used after the 2D separation. A possible cause of peak distorsion because of the immiscibility of the mobile phases employed in the two dimensions is resolved. The optimization of the analytical run time and flow rate for both dimensions and the initial gradient in the 2D is carried out with various standard compounds. The potential and versatility of this LC×LC approach is demonstrated through the separation of 11 standard components, most of them allergens. The latter, which are characterized by a scattered distribution on the 2D space plane, underwent separation on both a hydrophobicity and polarity basis.

Optimization of a Comprehensive Two-Dimensional Normal-Phase and Reversed-Phase Liquid Chromatography System