Scanning gel chromatography: determination of transport parameters from dynamic profiles measured at low solute...

Direct optical scanning of solute boundaries in large zone gel chromatography experiments provides an accurate means of determining boundary profile shapes and rates of motion. A method has been developed for correcting such boundaries to a constant time frame, eliminating the distortion which arises from finite column scanning rate. Centroids of the corrected profiles can be used to determine the partition cross section for the solute of interest. The partition cross section and flow rate determine translational motion within the column. The axial dispersion coefficient, L, which characterizes rate of boundary spreading may also be calculated from the profiles. In order to explore these procedures a study of four noninteracting solutes was conducted. Partition cross sections determined from rates of motion of boundary centroids were found to be in good agreement with those determined by the equilibrium saturation method on the same column. In order to explore the lowest concentration limits of the technique and to illustrate the boundary characteristics for a self-associating solute, a study of carboxyhemoglobin was conducted over a wide concentration range. From measurements at 220 nm the lowest concentration where useful data could be obtained was 2 micrograms per ml (0.12muM heme). These results establish validity of the procedures used in analyzing the rates of boundary transport and in studying solute transport over a wide range of conditions.