Detailed analysis of the effective and intra-particle diffusion coefficient of proteins at elevated pressure in...

To determine the efficiency that can be obtained in a packed-bed liquid-chromatography column for a particular analyte, a correct determination of the molecular and effective diffusion coefficients (D_m and D_eff) of the analyte is required. The latter is usually obtained via peak parking experiments wherein the flow is stopped. As a result, the column pressure rapidly dissipates and the measurement is essentially conducted at ambient pressure. This is problematic for analytes whose retention depends on pressure, such as proteins and potentially other large (dipolar) molecules. In that case, a conventional peak parking experiment is expected to lead to large errors in D_eff. To obtain a better estimate ofD_eff, the present study reports on the use of a set-up enabling peak parking measurements under pressurized conditions. This approach allowed us to report, for the first time, D_eff for proteins at elevated pressure under retained conditions. First, D_eff was determined at a (average) pressure of about 105 bar for a set of proteins with varying size, namely: bradykinin, insulin, lysozyme, β-lactoglobulin, and carbonic anhydrase in a column packed with 400 Å core-shell particles. The obtained data were then compared to those of several small analytes: acetophenone, propiophenone, benzophenone, valerophenone, and hexanophenone. A clear trend between D_eff and analyte size was observed. The set-up was then used to determine D_eff of bradykinin and lysozyme at variable (average) pressures ranging from 28 bar to 430 bar. These experiments showed a decrease in intra-particle and surface diffusion with pressure, which was larger for lysozyme than bradykinin. The data show that pressurized peak parking experiments are vital to correctly determine D_eff when the analyte retention varies significantly with pressure.