Theoretical and practical comparison of RPLC and RPLC × RPLC: how to consider dilution effects and sensitivity in addition to separation power?

The objective of this work was to provide an unbiased comparison of one-dimensional reversed-phase liquid chromatography (1D-RPLC) and comprehensive two-dimensional RPLC (RPLC × RPLC), through calculations and experimental verifications. For this purpose, various quality descriptors were evaluated, including peak capacity, analysis time, dilution factor, number of runs in the second dimension, and injection volume. The same strategy was applied to small pharmaceuticals and peptides. Whatever the analysis time between 30 and 200 min, short columns of only 30 × 2.1 mm packed with sub-2-µm particles should be selected in both dimensions of the 2D-LC setup to obtain the best compromise in terms of peak capacity and sensitivity. The peak capacity in RPLC × RPLC vs. RPLC was significantly improved for analysis times beyond 5 min. However, extra-column volume located after the second-dimension column was found to be particularly critical for peptides, and up to 50% lower peak capacity was observed with MS vs. UV detection. Contrary to common belief, higher dilution is not always observed in RPLC × RPLC. With adequate analytical conditions, better sensitivity (in theory fivefold and in practice three- to fivefold) could be achieved in RPLC × RPLC compared to 1D-RPLC, regardless of the analysis time.