Dissecting peak broadening in chromatography columns under non-binding conditions

Peak broadening in small columns is dominated by spreading in the extra column volume and not by hydrodynamic dispersion or mass transfer resistances. Computational fluid dynamics (CFD) permits to study the influence of these effects separately. Here, peak broadening of three single component solutes - silica nanoparticles, acetone, and lysozyme - was experimentally determined for two different columns (100 mm × 8 mm inner diameter and 10 mm × 5 mm inner diameter) under non-binding conditions. A mass transfer model between mobile and stationary phases as well as a hydrodynamic dispersion model were implemented in the CFD environment STAR-CCM+^®. The mass transfer model combines a model of external mass transfer with a model of pore diffusion. The model was validated with experiments performed on the larger column. We find that extra column volume plays an important role in peak broadening of the silica nanoparticles pulse in that column; it is less important for acetone and is weakly pronounced for lysozyme. Hydrodynamic dispersion plays the dominant role at low and medium flow rates for acetone because we are in a regime of 1-10 ReSc. Mass transfer is important for high flow rates of acetone and for all flow rates of lysozyme. Then, peak broadening was predicted in the smaller column with the packed bed parameters taken from larger column. The scalability of the prepacked columns is demonstrated for acetone and silica nanoparticles by excellent agreement with the experimental data. In contrast to the larger column, peak broadening in the smaller column is dominated by extra column volume for all solutes. Peak broadening of lysozyme is controlled only at high flow rates by mass transfer and overrides extra column volume and hydrodynamic dispersion. CFD simulations with implemented mass transfer models successfully model peak broadening in chromatography columns taking all broadening effects into consideration and therefore are a valuable tool for scale up and scale down. Our simulations underscore the importance of extra column volume.