Taylor-Aris methodology for the experimental determination of molecular diffusion coefficients: Tutorial with focus on large biomolecules

High-Performance Liquid Chromatography (HPLC) is a key technique in the evaluation of biopharmaceuticals. To improve the separation of biopharmaceuticals, it is crucial to improve the fundamental understanding of the parameters governing their band broadening behavior. This can be obtained by a detailed assessment of the individual contributions to their mass transfer. For this purpose, a precise knowledge of the molecular diffusion coefficient (Dm) of biopharmaceuticals is required. Only little experimental data is available for the Dm-values of biopharmaceuticals under HPLC relevant conditions. Furthermore, none of the available equations that can be used to calculate Dm-values, allows to account for any conformational changes that might occur. The Taylor-Aris method is a very simple and absolute method that is often employed to determine Dm-coefficients. The Taylor-Aris method measures the band broadening of an analyte in an open tube under laminar conditions, wherein (1) longitudinal diffusion can be ignored, (2) the sample is fully radially equilibrated and (3) the contribution of the extra-column variance to the total variance is negligible. Moreover, since the open tubes are typically coiled for practical reasons, (4) the influence of secondary flows on the band broadening should be insignificant. In this tutorial paper, the impact of the four conditions mentioned above on the accuracy of the obtained Dm values is revisited. For this purpose, Dm values are measured for two representative compounds (Bovine Serum Albumin and Thiourea), and the obtained values are compared with literature data and theoretical recommendations. Based on these observations, a set of 'rules' for accurate and fast Dm measurements is put forward. Finally, an Interactive Tool (IT), combining these rules in a comprehensive way, is introduced and can be used to set up TA experiments.