Molecular diffusion of biomolecules: a database for representative model biomolecules in reversed-phase liquid...

The Taylor-Aris (TA) method is used to establish a database of experimental molecular diffusion (D_m) coefficients for a representative set of biomolecules with a wide variety in molecular weights, to aid the fundamental investigation of band broadening phenomena of biomolecules in LC. To speed up the D_m-measurements, the validity of the TA method is first studied in capillaries with reduced lengths and inner diameters, at the highest possible flow rate. It is demonstrated that results with an accuracy within 10 % of reported literature values can be obtained by increasing the flow rate up to the transitional flow rate (F_trans) at which centrifugal forces in the coiled tube come into play. However, since the calculation of F_trans requires a first estimate of the molecular diffusion coefficient and these estimates can be up to 50 % off, a more prudent maximum flow rate of 0,7 × F_trans is suggested instead. An optimized and validated set-up consisting of a PEEK capillary with a length of 460 cm and an I.D. of 260 µm is subsequently used to construct a database of D_m-values under typical conditions encountered in reversed-phase liquid chromatography conditions. The measured D_m-values are in close agreement with the theoretically expected D_m·ηT = constant relation. On the other hand, and especially for the larger biomolecules, the measured D_m-values do not correlate very strongly with their molecular weights. It is hypothesized that this is due to conformational changes that occur under specific experimental conditions of mobile phase composition (pH, % ACN in the mobile phase). As such, the TA could be used to inspect the conformational structure of biomolecules under relevant chromatographic conditions.