Slow analyte diffusion effects on the A-term band broadening in macromolecular liquid chromatography separations

Computational methods have been used to study the effect of the molecular diffusivity of analytes on the band broadening and more particular the A-term band broadening, in liquid chromatography. The study was conducted on a two-dimensional mimic of a packed bed with nonporous particles (k″ = 0) and on a hypothetical perfectly ordered packed bed. The advantage of this computational approach is that the molecular diffusion coefficient of the solute in the mobile phase can be varied freely without affecting other parameters. The considered systems hence also formed a perfect test case for Giddings reduced plate height data representation method. The fact that the plate height values for the different Dmol-cases were found to fall exactly on the same single reduced plate height curve hence simultaneously validates Giddings (undisputed) theory and the presently proposed method of chromatographic data simulation. The obtained results are also ideally suited for plate height model discrimination, because they yield highly accurate band broadening data (devoid of any experimental scatter) over a very broad range of reduced velocities (ν up to 9000). The best agreement between simulation data and theory is found for the Knox model in which the Knox coefficient was lowered from the widely used n = 1/3 to a value of n = 0.23. This value agrees well with recent data obtained by Popovici et al. in experimental plate height studies of size exclusion chromatography (SEC) of polymers in a range of reduced velocities amounting up to nearly ν = 105.