Consequently, all the experiments conducted up to date aimed at understanding the involvement of serum protein for heme scavenging were carried out in an isolated and compartmentalized way, which on one hand facilitates recognition, but on the other hand represent only a partial result that does not necessary fully translate to the real matrix or the physiological state [29, 30, 31, 32]. To examine heme-protein interactions in a more representative scenario, where proteins are simultaneously present in the matrix (in their biological concentrations) and can both concur and compete for binding, it is necessary to develop a detection system that simplifies the sample while preserving its native state. In previous works the ability of F4 (Flow field-flow fractionation) and its miniaturized version HF5 (hollow-fiber F4) platforms to perform this task was demonstrated [33, 34, 35]. F4 (Flow Field Flow Fractionation) techniques are a class of analytical techniques that separate analytes based on their hydrodynamic radius due to the combined action of a laminar flow of a carrier solution, named elution flow, and a flow applied orthogonally to the elution flow, named cross flow. Cross flow drives the analytes into different regions of the laminar flow, resulting in different retention times. Based on the shape of the channel and how the perpendicular flow is applied, different variants of F4 can be defined. HF5 represents the miniaturized version of the common Asymmetrical F4 (AF4) subclass [36; 37]. As the name suggests, the separation device consists in a hollow fiber in which the main flow entering the fiber is split into two components: a flow coaxial to the fiber (the laminar parabolic flow) and a radial flow (the crossflow), as schematized in [38]. When compared to other size-based separation methods, F4 techniques show broader size separation range (from 1 nm up to >1 \(\upmu\)m) and higher flexibility, in terms of mobile phases used (ability to work in native conditions). Moreover, working with an empty channel prevents destruction of the aggregates due to their interaction with the stationary phase, allowing a better evaluation of conjugates formation [39; 40; 41; 42; 43]. HF5, in addition, provides the highest resolutions of the F4 variants, allows to work with lower volumes (both of sample and mobile phase), and the separation device can work in a disposable mode [44]. Thanks to these outstanding advantages, systems derived from the hyphenation of HF5 to uncorrelated detectors such as multi-angle light scattering (MALS), UV/vis Absorption and fluorescence, and Refractive Index have been exploited for the separation and characterization (mass, size and spectroscopic properties) of numerous samples such as biocompatible nanoparticles [45; 46; 47], biological samples of varying complexity [48; 49; 50], and other products of pharmaceutical interest [51; 37; 52]. Within this framework, the aim of this work was the study of the interactions between heme and serum components (like SA and IgG) in a real-life environment. The first step was to successfully separate serum into subcomponents (mainly SA, IgG, IgM and lipoproteins) within the separation method, to correctly identify heme-protein interactions. Then, increasing heme amounts were added to a native serum sample and an IgG-enriched serum sample also simulating an inflammatory response to monitor simultaneous protein-heme interactions [53]. The unbound heme is filtered away during the focus step [44; 45; 38] of the analysis, which acts as a filtration step, thus only the protein-bound heme fraction is selectively detected. The products of heme-protein interaction, along with the other serum components, were separated and evaluated with an HF5 platform coupled with UV/vis and MALS detectors. These detectors respectively allowed monitoring of protein, and heme-protein complexes through their typical absorption wavelengths (280 nm and 405 nm, respectively) and yielded mass and size of the serum components separated through the fiber.