*3.4. Purification*

Despite the previously mentioned purification steps, residuals of co-extracted contaminants are still present, and resulting fucoidans are still crude-type. [27]. Therefore, further selective purification steps are needed to obtain a high-quality product for reproducible and accurate biological investigations. Some researches adopted simple, non-chromatographic steps, such as bleaching of the crude fucoidans (NaClO2 in dilute HCl) followed by precipitation with cetyltrimethylammonium bromide [135] or by cold overnight incubation in aqueous buffered solution of calcium acetate (20 mM, pH = 6.5 -7.5) followed by dialysis [136]. In addition, membrane filtration was reported to produce fucoidans fractions of different molecular weight [137].

However, other chromatographic purification techniques were discussed in our previous publications [41,53,98,102]. Almost all the chromatographic techniques are based on the permanent negatively charged sulfate ester groups distributed on the polymer backbone which allow selective

fucoidans capture. However, carboxylated (e.g., alginate) and phosphorylated (e.g., nucleic acids) compounds might interfere [138,139]. Therefore, the pH value of the applied solvents is critical during chromatographic purification. One option for this uses anionic exchange resins (e.g., diethylaminoethyl cellulose or DEAE-cellulose), which was performed at pH 7.2 using 0.1 M sodium phosphate buffer [140]. An alternative is cationic dyes (e.g., toluidine blue- or perylene diimide derivative), modified resins or chitosan functioning in buffered solutions [27,102]. Both anion exchange and dye affinity chromatography involve the use of highly concentrated NaCl elution solvents. As a result, a subsequent purification step using chromatographic gel permeation [141] or dialysis [140] is required to remove salts, increasing the production costs. Other methods based on the use of biological macromolecules, such as lectins and anti-thrombin III, were also reported [53].

Novel innovative purification techniques were recently developed, such as selective solid phase extraction for purifying fucoidans and other complex seaweeds polymers by molecularly imprinted polymers (MIP) [142,143] or MIP modified by deep eutectic solvents [142,143]. Abdella et al., developed a green and time-saving purification protocol using genipin cross-linked toluidine blue immobilized-chitosan beads employing fucoidans affinity to cationic thiazine dyes [102].
