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Application of size exclusion chromatography in polysaccharides analysis of adriatic sea macroalgae Acetabularia acetabulum and Padina pavonica

Bioactive polysaccharides are valuable molecules that have been highly used in biomedical and pharmaceutical applications. Extensive research has been done to ensure their efficient extraction from marine algae using specific techniques to isolate and purify them. Marine macroalgae contain various polysaccharides such as frame polysaccharides, mucopolysaccharides, and storage polysaccharides. In our study we used common extraction methods, which include various steps of pretreatment, using solvents for extraction, precipitation, and size exclusion chromatography (SEC). Pretreatments are necessary to remove chlorophylls, mannitol, salts, and other small compounds. Dry algal powder was suspended in ethanol, and the mixture was continuously stirred with a mechanical stirrer at room temperature. After filtration the same procedures were applied using acetone and dichloromethane. The residual material was then extracted with 0.1 M HCl (60 °C, 2 hours), centrifuged at 4500 rpm for 40 minutes, and the supernatant precipitated with ethanol (overnight, 2-8 °C). The precipitated polysaccharides were filteredoff, washed with ethanol and acetone, air-dried and analysed. The molecular weights were determined by HPLC on Agilent 1260 Infinity II instrument, equipped with a refractive indeks detector G7162A. Analysis was performed using a PL aquagel-OH size-exclusion column (8 µm, 300 x 7, 5 mm) and a PL Aquagel-OH Guard (8 µm, 50 x 7, 5 mm) from Agilent Technologies. The column was calibrated with pullulan polysaccharide calibration kit (MW 180- 700 kDa, Agilent) and a standard curve was then established.

size exclusion chromatography ; polysaccharides ; macroalgae ; Acetabularia acetabulum ; Padina pavonica

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