In this case, check that the mass of G-25 Sephadex® is enough to run this separation and that there are no air bubbles throughout the column. If this happens, you should clean the Sephadex® completely with the external solution and then reseparate the collected vesicles, contaminated with non-encapsulated HPTS, through the size separation column. If the column is clean, in good condition, and the sample is added carefully (i.e., without disturbing the stationary phase), separation should be successful (as seen in Figure ). This is only if the reading must be taken manually. If this happens, you are not recording the spectrofluorometer reading along with the experiment. In addition, it is not possible to know when the assay started and, consequently, when to add detergent to lyse the vesicles. Therefore, the results from that run are not valid.