Preliminary model for predicting large-molecule separations by gradient elution, particularly for reversed-phase LC

Reduced plate height (h) vs. reduced velocity (v) plots have been measured over a wide range of v for 36 high-performance liquid chromatographic systems. Column type was varied over wide limits and solute capacity factor (k′) values were changed over the range 0.6–22. Resulting data can be accurately described by the Knox equation h = Av1/3 + B/v + Cv, where A is roughly constant (A = 0.5–0.8) for all columns studied, but values of B and C are strongly dependent on column type and solute k′ values. Reduced plate height (h) vs. reduced velocity (v) plots have been measured over a wide range of v for 36 high-performance liquid chromatographic systems. Resulting data can be accurately described by the Knox equation h = Av1/3 + B/v + Cv, where A is roughly constant (A = 0.5–0.8) for all columns studied, but values of B and C are strongly dependent on column type and solute k′ values. 

Analysis of proteins in solution by high-performance liquid chromatography is presented with respect to structural changes in solution, adsorption processes and differentation concerning specific activities. Trypsin and cellulases were taken as examples.

Development of the basic theory relating gradient and isocratic separations, essential to later work on DryLab I and DryLab G.

In Ion-pair reversed-phase chromatography, the retention of ionized analytes on a nonpolar bonded stationary phase is enhanced by the presence of a "hydrophobic" counterion (hetaeron) in the mobile phase. Either ion-pair formation in the mobile phase with relatively strong retention of the complex or the conversion of the stationary phase into an ion-exchanger may explain the phenomenon. Analysis of the pertinent equilibria shows that the observed hyperbolic or parabolic dependence of the capacity factors on the hetaeron concentration cannot shed light on the mechanism. The experimental data obtained for the retention of catecholamlnes by using C4-C10 alkyl sulfates and other similar hetaerons in a wide concentration range, however, could be mechanistically interpreted from the chain length dependence of the parameters for the relationship between the capacity factors and hetaeron concentration. Although the results clearly demonstrate that in the system investigated, ion-pair formation governs retention, ion-exchange mechanism can be operative under certain conditions. Changes in retention upon addition of salt to the eluent are treated both theoretically and experimentally. The effect of organic solvents on the behavior of the chromatographlc system is discussed in view of the proposed theory.

0ver a hundred acidic urinary constituents were separated within 30 min by using 5-µm octadecylsilica columns and gradient elution with increasing acetonitrile concentration in dilute aqueous phosphoric acid solution at 70°. The column effluent was monitored with a UV detector at 280 nm or with a fluorescence detector at 260 nm excitation and 340 nm emission wavelengths. The high sensitivity and speed of analysis, the excellent reproducibility and adequate resolution obtained suggest that this technique may be useful to obtain metabolic profiles in routine clinical work.