Online, comprehensive two-dimensional liquid chromatography (2D-LC) has become an attractive option for the analysis of complex samples of relevance in various fields (e.g., environmental, food, biology, and polymer sciences). In general, the second-dimension (2D) separation plays a more crucial role than the first (1D) in the total performance of LC × LC systems. Speed and efficiency of the 2D separation are of primary importance. These challenges are amplified in the case of protein separations where mass transfer limitations effect elution kinetics. The research presented here leverages the developments of capillary-channeled polymer (C-CP) fiber stationary phases directed at high-throughput and rapid protein separations, evaluating them as 2D material in comprehensive reversed-phase × reversed-phase (RP × RP) 2D-LC versus conventional, packed-bed columns as 2D. The ability to operate fiber columns at high linear velocities (> 75 mm s-1 at < 1000 psi) without sacrifice in resolution is the first step towards potential 2D implementation, alleviating the need for ultrahigh-pressure pumping (i.e., UHPLC) in that stage. What must be realized are fast elution gradients and rapid re-equilibration to minimize cycling times (modulation periods). Different flow rates and gradient programs were evaluated. Rapid elution allows the use of shorter shift-gradient windows. At the optimized gradient recycling time on this instrument, 36 s, there is no significant degradation in peak capacity. Finally, a graphic comparison of the separation efficiency between seven commercial reversed-phase columns and the polypropylene C-CP fiber column is presented for a synthetic, eleven-protein mixture using the recommended operating conditions for each of the 2D columns. The kinetic advantages of the fiber columns are clearly demonstrated.
Keywords: Capillary-channeled polymer fiber; Protein; RPLC × RPLC; Two-dimensional liquid chromatography.