Mobile phases were developed that cover a wide pH-range to ensure sufficient binding and elu9on of AAV5, 6 and 8, which were inves9gated in this study. Separa9on of full/empty mixtures of the three serotypes aeer gradient op9miza9on are shown in Fig. . Notably, all serotypes were analyzed using the same gradient sugges9ng that the chromatographic condi9ons might also be applicable to other serotypes, albeit these were not inves9gated due to material non-availability. All three AAVs elute late in the gradient, which is primarily caused by the high pH at the gradient start, equivalent to a mobile phase B concentra9on of 20%. Star9ng at lower pH was inves9gated but resulted in insufficient column binding or high run-to-run variability. The developed gradient facilitated the separa9on of full and empty capsids, represented by the two dominant peaks in each case, as will be verified later. While AAV 5 and 6 seem base line separated, AAV8 shows par9al separa9on, a clear dis9nc9on of two discrete peaks is however feasible. The mobile phases used were of low ionic strength and composed of MS-friendly components only, allowing for subsequent mass spectrometric interfacing if desired. Despite the rela9vely low ionic strength and limited buffering capacity, full column equilibra9on was achieved in 12 minutes. A flow rate of 0.2 mL/min was employed, which allowed for both swie column equilibra9on as well as full Aeer ini9al method development, the quality of the separa9on was further inves9gated. Elu9on order was determined first by the injec9on of full and empty material independently, in addi9on to the mixtures shown in Fig. . The resul9ng chromatograms are depicted in Fig. . In all cases, surprisingly, full capsids were elu9ng first, which is counterintui9ve considering the apparently higher isoelectric point of the empty par9cle and contrary to what is reported in literature for salt-gradient mediated elu9on.