RP-LC x RP-LC analysis of a tryptic digest using a combination of totally porous and partially porous stationary phases

Different chromatographic separation modes have been combined, in off-line and on-line approaches, attempting to achieve enhanced separation power for the analysis of complex proteome samples. Among these, the use of RP-LC x RP-LC brings in the added advantages of high peak capacity in both dimensions, capability of full automation, and the likelihood of direct hyphenation to MS. In this contribution, we achieved enhanced resolution in the first dimension, by serially coupling Bio wide pore C18 columns at basic pH, at moderate pressure. Selectivity was tuned using one short (3 cm) C18 column packed with partially porous particles for fast second-dimension analysis, operated at acidic pH. The comprehensive system was configured around electronically activated two-position, ten-port valve for within-loop automated fraction collection/re-injection. A theoretical peak capacity of 3796 was calculated for the set-up employing one basic column in the first dimension, while a peak capacity of 10 686 was attained with four times the length of stationary phase, even at the expense of longer analysis time. The system showed very good reproducibility of retention times and chromatographic peak areas (RSD less than 1.2 and 4%, respectively).