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Asymmetrical flow field-flow fractionation is a versatile chromatographic fractionation method. In combi- nation with at least one detection technique it is used for size-based separation of colloids, biomolecules and polymers. Although often used as pure separation method, a well-elaborated theory is available that allows precise quantification of the translational diffusion coefficient D . Still, current literature suggests different ways to transform this theory into applicable experimental procedures and no “gold standard”for correct data processing exists. While some sources report a direct way to extract diffusion information from the fractogram, others suggest the necessity of an external calibration measurement to obtain the channel width w . In this work, we compare the different approaches and calibration algorithms based on original and literature data using our own open-source AF4 evaluation software. Based on the results, we conclude that available AF4 setups do not fulfill the requirements for absolute measurements of D . We show that the best way to conduct is to consider the area of the channel and D of the calibrant while neglecting the small peak which occurs in the void peak region.