A theoretical basis for parameter selection and instrument design in comprehensive size-exclusion chromatography × liquid chromatography

A novel approach for the selection of the operational parameters (linear velocity, column length) for a comprehensive 2D-LC system is discussed. Starting point for the calculations is a given second dimension (2D) separation and a desired peak capacity for the 2D system. Using the theory developed here the optimum settings for the first dimension (1D) column can be derived. Theory clearly indicates that the choice of the 1D conditions is basically limited to just one set of column lengths and linear velocities. The new method is tested on a comprehensive two-dimensional liquid chromatography system which uses size-exclusion chromatography (SEC) followed by reversed phase liquid chromatography (RPLC). A novel LC/LC interface, using a six-port valve rather than storage loops, joins the two chromatographic dimensions. From a theoretical comparison of continuous low flow and stop-flow operation the latter method was found to be an attractive mode of interfacing. The common idea that stop-flow operation results in additional band broadening is shown to be incorrect. The new interface design operated in the stop-flow mode permits the use of conventional analytical diameter HPLC columns, 7.8 mm for SEC and 4.6 mm for RPLC. The reversed phase chromatography utilizes a monolithic C-18 modified silica column, which produces fast and efficient analyses. As test samples complex mixtures of peptides were analyzed.