The main component of algal cell wall and extracellular polysaccharide is mucopolysaccharide, the monosaccharide composition of mucopolysaccharide of a part of cyanobacteria has been investigated clearly, and what kind of relationship between the biological activity of polysaccharides and their structure has attracted much attention. (Source: Biochemistry and Molecular Biology Laboratory Guide, Shao Xueling et al, Wuhan University Press, 2003)
Operation method
High performance liquid chromatography (HPLC) method
Materials and Instruments
Macroalgae Move (1) The polysaccharide was purified by centrifugation of 1000 ml of culture solution, concentrated to 50 ml by vacuum at 40 ℃, and then transferred to a dialysis bag and dialyzed in distilled water at 4 ℃ for three days. The distilled water was replaced once in the morning and evening, and the liquid in the dialysis bag was poured into a beaker, and 4 times the volume of 99% ethanol was added while stirring, and the solution was centrifuged at 11000 r/min to obtain the precipitate, and then freeze-dried to obtain the crude polysaccharide. The crude polysaccharide was further purified by DEAE-sephacel column chromatography and gradient elution with 0~2 mol/L NaCl, and the purity was identified by acetate film electrophoresis [1]. Common Problems [1] Nihonkagakukai , Saccharo-chemstry (lower) , Tokyoukagakudojin, 148-149 , 1976 For more product details, please visit Aladdin Scientific website.
NaNO; Na2HPO4-12H2O; EDTA
Polarization photometer; High performance liquid chromatograph
(2) Molecular weight was determined by high performance liquid chromatography (HPLC) using TSK-GEL.G300SWXL (7.8 mm × 300 mm column) GPC. The mobile phase was 1% trihexylamine and the detection was performed using an oscillometric refractometer (YRU-880RI-UI detector) with Pulluna-ShodexSTANDARDP-82 (molecular weights of 5.9, 11.8, 22.8, 47.8, 112, 212, 404, and 788 kU, respectively) under the same conditions.
(3) Viscosity was determined using a TokimecEMD rotational viscometer under the conditions of 25 °C, a rotary cone variety of 1°34′, and a sugar concentration of 0.5%.
(4) The rotational light was measured using a JASCO.DIP-370 polarimeter under the conditions of 16.9 ℃, and the sugar concentration was 0.24%.
(5) Elemental analysis was performed by atomic spectral absorption.
(6) Sulfate-based quantification was performed using the Rhodizonicacid method [2].
(7) Thin-layer chromatography was performed by completely hydrolyzing the polysaccharide, taking 0.5 ml of Dowex1 (acetic acid type) column with trace resin, first eluting with pure water to collect the neutral part, then eluting with 0.2 mol/L acetic acid to collect the acidic part, and then spotting the sample on a silica gel plate (Kieselgel60, 10×10 cm) and unfolding it using an unfolding agent (ethyl acetate: acetic acid:water=2∶1:1), 50% sulfuric acid spraying, 100 ℃, and then unfolding it using an unfolding agent (ethyl acetate: acetic acid:water=2:1:1). After the sample was sprayed with 50% sulfuric acid, it was charred at 100 ℃, and the sugars were identified according to the Rf value of the charred spots.
(8) Gas chromatography was carried out after 1 mg of polysaccharide was completely hydrolyzed with trifluoroacetic acid to make a sugar alcohol-acetylation inducer, and the experimental conditions were TC-I column (0.25 mm×20 mm), gasification chamber temperature: 230 ℃, column temperature: 140~210 ℃, heating at 1.3 ℃/min, carrier gas was nitrogen, and hydrogen ionitis detection.
(9) Quantification of glucuronic acid was determined by Sulfatedacid-carbazole reaction method [3].
(10) H1-NMR spectroscopy was performed by dissolving pure polysaccharides in heavy water, freeze-drying, and repeating twice, using acetone δ2.23 as an internal standard. Spectra were determined on an INOVA-600 spectrometer fully equipped with proton irradiation and Ferrin transformation, 600 MHz, 20 °C.
The different fractions of polysaccharides or oligosaccharides were analyzed for biological activity, and those that were active were analyzed for structure.
[2 ] Louis J . Silverstri , Analysis of sulfate in complex carbohydrates, Ana . Biochen . , 123 , 303～309 , 1982
[3 ] Midori K . Physics and Chemistry Dictionary , IwanamiBookshop , 47 , 1981