Development of an online capillary comprehensive 2D-LC system for the analysis of proteome samples

In the present contribution, a fully automated capillary comprehensive two-dimensional LC (LCxLC) method, for proteomic analysis, was developed for the first time. The investigated platform was characterized by the coupling of high-pH RP with low-pH RP separations thus ensuring the generation of high peak capacity despite the employment of identical stationary phases. The use of capillary columns in both dimensions allowed to reduce mobile-phase consumption and enhance sensitivity. Fraction transfer from the first to the second dimension was performed by means of two 2-position 6-port nano-switching valves, under stop-flow conditions. Values as high as 1208 and 955 were obtained for the theoretical and practical peak capacity, respectively. The investigated LCxLC system showed good retention time repeatibility with RSD values ranging from 0.8 to 6.0% for the first dimension and from 1.0 to 3.0% for the second dimension, respectively. RSD peak area values below 9.5% were also attained, thus demonstrating the precision of the LCxLC method employed.