Flow-Reversal Experiments with Macromolecules to Measure Column End Efficiency and Bed Heterogeneity

A flow-reversal method combined with peak parking has been introduced recently to determine the band broadening occurring at the two respective column ends and in the bed of the packing material. Flow-reversal has a peak compression effect, therefore, the peaks observed are always narrower and more symmetrical than the peaks obtained without reversing the flow. This phenomenon can originate from the compensation of the multipath dispersion effects. In the present study, peak parking and flow-reversal measurements were extended to macromolecules and carried out with human insulin. We observed that the peaks of insulin are always narrower with reversed flow than without reversing the flow, and the compression effect can be significantly larger than it is for small molecules. The contributions of the column inlet and outlet to the total band variance have been characterized.