Solvent precipitation method

Separation is achieved by taking advantage of the differences in the solubility of polysaccharides in different solvents. For instance, when ethanol is added to the polysaccharide extract, most of the polysaccharides will precipitate in the ethanol solution of a certain concentration, while impurities such as proteins remain in the solution. Usually, after concentrating the extract, slowly add 4 to 5 times the volume of anhydrous ethanol, stir evenly, let it stand overnight at 4℃, centrifuge to collect the precipitate, and the polysaccharide can be initially purified.

Gel column chromatography

Separation is based on the different sizes of polysaccharide molecules. Commonly used gels include Sephadex gel and Sepharose gel, etc. When the polysaccharide mixture passes through the gel column, the macromolecular polysaccharides cannot enter the interior of the gel particles and flow out rapidly with the eluent. Small molecule polysaccharides enter the pores of gel particles, with a slow elution rate, thus achieving separation. For instance, polysaccharides extracted from shiitake mushrooms can be processed through gel column chromatography to obtain different components based on molecular size.

Ion exchange column chromatography

Separation is carried out by taking advantage of the charge differences carried by polysaccharide molecules. If polysaccharides contain acidic or basic groups, corresponding ion exchange resins can be selected, such as strongly acidic cation exchange resins (such as Dowex 50), strongly basic anion exchange resins (such as Dowex 1), etc. Negatively charged polysaccharides will combine with anion exchange resins. By changing the ionic strength or pH value of the eluent, different polysaccharides can be eluted in sequence, achieving the purpose of purification.

Enzymatic hydrolysis method

Protease, nuclease and other enzymes are used to remove impurities such as proteins and nucleic acids from the polysaccharide extract. For instance, when an appropriate amount of trypsin is added to the polysaccharide extract and the reaction is carried out under suitable temperature (such as 37℃) and pH conditions for a certain period of time, the protein is enzymatically hydrolyzed into small peptide segments or amino acids. Then, the enzymatic hydrolyzed products are removed through subsequent separation methods (such as centrifugation or dialysis), thereby enhancing the purity of the polysaccharide.

Membrane separation method

The polysaccharide solution was separated by using membranes with different pore sizes. Ultrafiltration membranes can retain large molecule polysaccharides and allow small molecule impurities (such as salts, monosaccharides, etc.) to pass through, achieving initial purification and concentration. Reverse osmosis membranes can further remove even smaller impurity molecules and improve the purity of polysaccharide solutions. For instance, from the extract of Ganoderma lucidum polysaccharides, first use an ultrafiltration membrane to remove large molecular impurities such as proteins, and then use a reverse osmosis membrane to desalize, to obtain a high-purity Ganoderma lucidum polysaccharide solution.

Quaternary ammonium salt precipitation method

Quaternary ammonium salt compounds (such as cetyltrimethylammonium bromide, CTAB) can form complex precipitates with acidic polysaccharides. When an appropriate amount of CTAB solution is added to the polysaccharide extract, acidic polysaccharides will combine with it to form precipitates, while impurities such as neutral polysaccharides remain in the solution. After the precipitate is collected by centrifugation, it is washed with an organic solvent (such as acetone), and finally dissociated with a dilute acid solution to release polysaccharides, achieving the purpose of purifying acidic polysaccharides.