Optimum conditions for high-resolution gradient analysis

The effect of experimental variables in the photometric scanning of centrifuged sucrose gradients on the apparent resolution was studied. Better resolution was obtained with a flow cell with a comparatively large diameter flow path that was designed for bulk flow than with a flow cell with a comparatively small diameter flow path that was designed for laminar flow. Degradation of resolution caused by an increase in the flow rate was more apparent with the laminar flow cell than with the bulk flow cell. The resolution increased as the illuminated volume of the flow cell decreased but was relatively insensitive to illuminated volume at the lower values tested. Resolution was determined by the number of pea plyribosomes resolved and the shape of their peaks and by the deterioration in shape of peaks given by zones of southern bean mosaic virus during repetitive scanning. It was found that resolution of ribosomal peaks is not a good quantitative method for characterizing instrumental performance.