RP‐LC×RP‐LC analysis of a tryptic digest using a combination of totally porous and partially porous stationary phases

Abstract

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).

Stop-flow comprehensive two-dimensional liquid chromatography combined with mass spectrometric detection for phospholipid analysis

“…Therefore, in this work, for the 1 D separations, a 15 cm-long, silica HILIC column, built on the Fused core technology, was used. Such a stationary phase was successfully employed in previous researches, either in one-dimensional or multidimensional LC × LC set-ups [8,[25][26][27][28][29][30][31]. Fig.…”

Effects of first dimension eluent composition in two-dimensional liquid chromatography

“…In LCxLC there is an absolute need for maximally different selectivities in the two dimensions to produce the highest effective peak capacity. The most direct approach to achieving this is to use radically different modes of separation as discussed above; nonetheless, we and others have used RPLC in both dimensions [26, 43–45] but do so using pairs of reversed phases with radically different selectivity [46,47] or different mobile phase [48,49]. Here, we explore the possibility of changing selectivity by applying different pHs in the mobile phase of the first dimension.…”

Comprehensive two‐dimensional liquid chromatography with evaporative light‐scattering detection for the analysis of triacylglycerols in Borago officinalis

“…In particular, the monolithic column employed in the D2 was replaced by a partially porous column (fused core technology) [23][24][25]. In this regard, recent studies have demonstrated the possibility to use these columns in the D2 of comprehensive LC systems in order to obtain very fast D2 gradient elution separations [19,20,26].…”