Experimental validation of the stochastic theory of size-exclusion chromatography: Retention on single and coupled columns

摘要

The experimental validation of the recently developed stochastic model of size-exclusion chromatography (SEC), interpreted as a random process in which macromolecules undergo a continuous exchange process between the moving zone between the packing particles and the stagnant zone inside the particles, is attempted on the basis of an extended data set of experimental chromatograms of polystyrene standards, with molar masses ranging from 0.16 to 2200 kDa, on five different columns with widely different pore sizes. Several features of experimental chromatograms of standards eluting within or outside the permeation range, such as band broadening, peak tailing and peak splitting are qualitatively interpreted at the light of the stochastic theory of SEC. Furthermore, some aspects such as the influence of sample polydispersity on peak broadening and of the exclusion effect in the moving zone (hydrodynamic chromatographic effect) on retention are discussed. A new chemometric procedure was developed for estimating the K-SEC equilibrium distribution constant without a priori assumptions regarding the extraparticle column volume, V-O, and the total pore volume V-p. This procedure allowed to obtain an unified description of the large retention data set over different columns. All retention data belonging to calibration ranges of the various columns, followed quite similar calibration plots, under the form Of K-SEC = (1 - rho)(m), where rho is the ratio of macromolecule size to pore size, with m values close to 3 for all columns. A similar approach was able to represent the behaviour of two connected column in series, with consistent values of the pertinent parameters, which validates the stochastic theory of SEC with two pore types of different sizes. Finally this two-pore model and a mixed SEC-hydrodynamic chromatography model were compared to interpret retention data on single columns around the exclusion limits. The practical application of these approaches in routine analysis, e.g. for monitoring the column degradation during its use, is mentioned. [References: 37]