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 (LC×LC) 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 LC×LC 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 LC×LC method employed.