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Novel general expressions are constructed and presented that describe the behavior of the height equivalent of a theoretical plate (plate height), H, as a function of the linear velocity, Vx, along the axis, x, of the column and the kinetic parameters that characterize the mass transfer and adsorption mechanisms in chromatographic columns. Open tube capillaries as well as columns packed with either non-porous or porous particles are studied. The porous particles could have unimodal or bimodal pore-size distributions and intraparticle convective fluid flow and pore diffusion are considered. The expressions for the plate height, H, presented in this work could be applicable to high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC) systems, and could be used together with experimental plate height, H, versus linear velocity, Vx, data to determine the values of the parameters that characterize intraparticle convective fluid flow and pore diffusion. Furthermore, chromatographic systems under unretained as well as under retained conditions are examined. The experimental values of the plate height, H, versus the linear velocity, Vx, for a CEC system involving charged porous silica C8 particles and an uncharged analyte are compared with the theoretical results for the plate height, H, obtained from the expressions presented in this work. The agreement between theory and experiment is good, and the results indicate that the magnitude of the intraparticle electroosmotic flow (EOF) in the pores of the particles is substantial while the pore diffusion coefficient was of small magnitude. But the overall intraparticle mass transfer resistance in these particles was low because of the significant contribution of the intraparticle EOF. Simulation results are also presented (i) for a hybrid HPLC–CEC system, and (ii) for different CEC systems involving open capillaries as well as packed columns having non-porous or porous particles. The analysis of the results indicates (a) the reasons for the superior performance exhibited by the hybrid HPLC–CEC system over the performance obtained when the system is operated only in the HPLC mode, and (b) the operational configuration and the properties that the structure of the porous particles would have to have in CEC systems involving uncharged or charged analytes under unretained or retained conditions in order to obtain high CEC efficiency (low values of the plate height, H). [Copyright &y& Elsevier]

Abstract: In this work, the pore structural parameters and size exclusion properties of LiChrospher strong cation-exchange and reverse phase restricted access materials (RAM) are analysed. The molecular weight size exclusion limit for polystyrenes was found to be about 17.7kDa, while for standard proteins, the molecular weight size exclusion limit was higher, at approximately 25kDa. The average pore diameter on a volume basis calculated from the pore network model changes from 8.5nm (native LiChrospher) to 8.6nm (diol derivative) to 8.2nm (sulphonic acid derivative) to 6.9nm (n-octadecyl derivative). Additional characterisations were performed on restricted access materials with nitrogen sorption at 77K, water adsorption at 25°C, intrusion–extrusion of water (in order to evaluate the hydrophobic properties of the pores of the hydrophobic RAM), and zeta potential measurements by microelectrophoresis. For peptide analysis out of the biofluids, the strong cation-exchange functionality seems to be particularly suitable mainly because of the high loadability of the strong cation-exchange restricted access material (SCX-RAM) and the fact that one can work under non-denaturing conditions to perform effective chromatographic separations. For bacitracin, the dynamic capacity of the SCX-RAM columns does not reach its maximum value in the analysed range. For lysozyme, the dynamic capacity reaches a value of 0.08mg/ml of column volume before column is overloaded. Additionally, the proper column operating conditions that lead to the total effective working time of the RAM column to be equal to approximately 500 injections (depending on the type of sample), is comprehensively described. The SCX-RAM column was used in the same system analysing urine samples for the period of 1 month (∼150 injections) with run-to-run reproducibility below 5% RSD and below 10% RSD for the relative fractions. [Copyright &y& Elsevier]

Abstract: Complex biological samples require very high resolution separation strategies. The platform introduced here capitalises on the hyphenation of liquid chromatographic (LC) and electric potential gradient electrochromatographic multi-dimensional separation genres. First-dimension selectivity is provided by simultaneous size exclusion (SEC) and strong cation exchange (SCX) chromatography modes, while the second dimension comprises reversed phase (RP) characteristics in a dynamic (time-variant) electric field. The time-variant potential gradient with reversal of polarity is applied across the second dimension monolithic capillary throughout the duration of the solvent strength gradient elution. Hence, the platform offers comprehensive on-line sample clean-up (matrix depletion, analyte enrichment), fractionation (first dimention LC), and separation (second dimension LC) with the prospect of altering selectivity via polarity reversal dynamic electric field tuning. [Copyright &y& Elsevier]