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in the case of a biogenic material, such as an antimicrobial peptide, the understanding of the process of release is more complex since the main interest is in the mass rather than the number of the released molecules. it is therefore assumed that the release is a first-order kinetics, based on an energy barrier model [ 40 ], a faster process than the loading, but slower than the dissociation of the nanocarrier. the study presented in this work is focused on the optimization of af4 fractionation using a ml algorithm to avoid undesirable interactions between nanocarriers and the eluent. it is based on monitoring the size of a population of co-encapsulated liposomes. the aim was to determine the best configuration of the eluent composition and cross-flow rate to prevent liposomes aggregation and to support the excretion of both liposome and drugs in a single fraction.

one of the most common analytical tools employed in the nanoparticle field is the uv–vis spectrophotometer [ 156, 157, 158 ]. as a means of quantification, this technique has its own set of challenges, such as the presence of the matrix, which absorbs light, masking the absorption of the nanoparticles, and light scattering, which increases with the size of the nanoparticles. therefore, the method is not a good candidate for the quantification of nanoformulations, where the particles may be of submicrometric size. this is normally overcome through the employment of light scattering detectors such as dynamic light scattering, which are more suitable for the analysis of nanoparticles, but not able to distinguish between submicrometric particles. another problem is that the standardization of the technique is still in the early stages, and even the very first online methods were not found to have been standardized yet. indeed, the literature has a strong bias towards the use of the offline method, which is not only costly and cumbersome but also requires the storage of the sample in a refrigerator or freezer in order to preserve the integrity of the sample, adding yet another element of error and complexity to the sample preparation and separation phases.

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