Peak broadening in protein chromatography with monoliths at very fast separations

Monoliths are stationary phases cast as a continuous medium which are interlaced by flow channels ramified with micropores. Pulse response experiments with bovine serum albumin as a model protein were applied for testing polymethacrylate-based monoliths, resulting in peak broadening that practically was not influenced by the chromatographic velocity. An empirical model was developed to describe peak broadening, allowing a term to account for the pore convection and a term for the pore diffusion. A diffusional distance lower than 10 nm was estimated. This corresponds to values observed with monodisperse 1-microm particles. Systematic investigations by changing the response time of the detector showed that the full potential of the monoliths could not be exploited, since the currently available chromatography systems are the limiting factor regarding the speed of data acquisition and virtual peak broadening by the infinite length of the detector. Inertia of the liquid and synchronization between liquid handling and electronic control introduced an additional disturbance. At the lowest possible response time, reliable peak data could be obtained up to a velocity of 35 cm/min. The pressure drop along the continuous bed was much smaller compared to a conventionally packed bed. Different flow patterns and significantly reduced eddy vortexes may be responsible for the high specific permeability.