## 3 Results and Discussion  
### HF5 Separation of Serum  
Blood serum is a complex sample, containing small molecules, proteins, lipoproteins and micelles and other particles; a simplification is necessary when monitoring selected interactions such as those with free heme. At the same time, it is crucial to preserve the sample native state, to avoid alterations in behavior. The developed method was optimized with the aim of separating serum in its main size components in the shortest analysis time, which was achieved by a combination of isocratic and gradient cross-flow, using isotonic PBS (pH = 7.4) as mobile phase. Recovery, calculated in triplicate from the UV/vis signal at 280 nm, was evaluated for serum, HSA and IgG; in all cases, we obtained quantitative recovery (>99.9%). On the contrary, recovery after injection of free heme was 0% as expected, confirming that only heme binding to proteins was selectively detected, while free heme was effectively filtrated through the hollow fiber pores during the focusing step (see Supplementary Data, Figures S1 and S2). The limit of quantification (LoQ), which could be calculated for reference proteins HSA and IgG, corresponded to 0.0012 pmol and 0.0003 pmol, respectively. The total analysis time resulted to be below 30 min.  
Serum analysis yielded four main bands, peaking at 8.4, 11.0, 15.3 and 20.3 min, respectively (Figure 1a). Absorption intensity decreases from the first to the last peak, while light scattering (which is also sensitive to size) increased as expected, confirming the size differences between the four populations. Characterization and identification of these bands is easily accessible when using HF5-multidetection, since the combination of a concentration detector (UV) and light scattering (MALS) allows for the direct determination of size and molar mass of the eluting species. In this regard, HF5 (and more broadly FFF) with multidetection can offer a standalone technique.  
For what concerns peak attribution, two classes of components can be distinguished, proteins and lipoproteins. Proteins have the strongest absorption at 280 nm, while lipoproteins assemble into tr  
Figure 1: Separation profile of whole serum. (**a**) UV @280 nm (black trace) and LS@90\({}^{\circ}\) (yellow trace). (**b**) UV profile and Molar Mass overlay (black distributions) of the four bands.  
The average molar mass values calculated for the four bands (Figure 1b), using UV absorption at 280 nm as concentration source, were 7.1 (\(\pm\)12%) kDa, 162 (\(\pm\)16%) kDa, 990 (\(\pm\)25%) kDa, 1.6 (\(\pm\)8.5%) MDa. Therefore, the four bands could be attributed to i. mainly BSA (and Haemopexin, which has a similar molar mass) and coeluted HDL [34], ii. IgG (namely 150 kDa) with IgG and IgE (180 and 200 kDa) tailing into IgA (320 kDa), iii. IgM and possible further aggregates of smaller immunoglobulins, iv. LDL and VLDL particles (measured R\({}_{\text{g}}\): 14-35 nm), with a scaling exponent \(\
u=0.89\), compatible with a non-spherical morphology where mass is distributed on the surface of a core-shell discoidal particle. This is in line with literature concerning serum and plasma characterization in similar setups [33; 55].  
The confirmation of the nature of the first two bands also comes from independent injections of standard proteins, such as HSA and IgG with the same method. These two proteins were chosen as example since they together represent the most part of serum protein content and are the most reported in heme binding studies. In the case of HSA, aggregates are also present in the standard sample (Figure 2, blue trace), and the retention time of the monomer corresponds to band 1 maximum. In the case of IgG (Figure 2, green trace), there are no aggregates detected and retention time is in line with band 2. The injection of different amounts of standard protein and following calibration of their content in serum can also act as rapid quantitation of albumin and immunoglobulin and give immediate feedback on healthy/inflammatory/disease conditions.