## 8. Drying step  
* Cottony shape (can also be finely grinded): freeze drying
* Powder final shape: drying into cold acetone, Buchner filtration against cold acetone, 50\({}^{\circ}\)C drying (oven), final crushing (drop hammer)
* Main strategies for extracting enriched-polysaccharide fractions. Figure 2. Main strategies for extracting enriched-polysaccharide fractions. Overall, four main levels can be defined regarding the level of accuracy and details needed for characterizing carbohydrates (Figure 3), i.e., (i) global composition, (ii) primary composition, (iii) first structural analysis and (iv) full structural investigation. [PERSON] et al. gave an emphasis about the strategies for determining the structure of (exo)polysaccharides and experimenters should strongly read it in its entirely [43]. (i) The total amounts of carbohydrates, neutral sugars and uronic acids, non-carbohydrate substituents and main \"contaminants\" must be quantified, mainly by colorimetric assays. The phenol and/or or orcinol-sulfuric acid are usually used for measuring total carbohydrates content [44]. Uronic acids content is mainly determined by the \(m\)-HDP (meta-hydroxdiphenyl) assay [45] whereas neutral sugars content is monitored based on the resorcinol-sulfuric acid assay method [46]. A corrective formula should be used for enhancing the results accuracy [47]. Non-carbohydrate substituents such as (a) pyruvate, (b) methyl and acetyl, and (c) sulfate groups can be quantified by performing respectively (a) the method of 2,4-dinitrophenylhydrazine [48], (b) High-Performance Liquid Chromatography (HPLC) methodology prior a sapnification step and/or \({}^{1}\)H Nuclear Magnetic Resonance (NMR) (most reliable method for determining methyl and/or acetyl groups) [49], (c) the turbidimetric gelatin/BaCl\({}_{2}\) method [50] and/or the azure A method [51]. Finally, proteins and polyphenols contents can be determined using respectively the \"Smith\", \"Bradford\" or \"Lowry\" methods [52; 53; 54] and the Folin-Ciocalteu assay [55]. Note that the presence of salts, ashes and moisture should be considered. Fourier Transformed InfraRed (FTIR) spectroscopy could also provide useful information concerning vibrationally functional groups of polysaccharides. (ii) Determination of the primary composition requires the release of monosaccharides by glycolysis, mostly by acidic hydrolysis or methanolysis [56]. This is an important step regarding the literature since the hydrolysis conditions (by mineral acids) greatly change the possibility to quantify different types of monosaccharides (ketoses, aldoses, hexosamines and uronic acids) [43]. The released monosaccharides, but also absolute configuration of glycosides, can then be analyzed by various chromatographic methods such as HPLC, high-performance anion-exchange chromatography or Gas Chromatography (GC). GC is used for analyzing low molecular weight compounds which are not thermolabile and can be vaporized. The method is not suitable for polar and/or high molecular compounds and needs preliminary pretreatments (e.g., solvolysis) and derivatization steps to (a) convert polar or non-volatile compounds to relatively nonpolar or volatile products; (b) improve  
Figure 3: Determining the structural features of polysaccharides. thermal stability of target compounds; (c) increase detector response by incorporating functional groups which lead to higher detector signals or improve GC separation performance [57]. Commonly used derivatization reactions include silylation, esterification, acetylation, and alkylation [58]. Note that derivatized monosaccharides are more easily ionized especially if a mass spectrometer is used as a detector with electronic impact (EI). Regarding their abundance, hydroxyl groups give weak volatile properties to polysaccharides. Each released monosaccharide from solvolysis should thus be derivatized. Silylation is probably the most used technique to analyze monosaccharides with GC. The polarity of residues is reduced by switching hydrogens with alkyl-silyl groups.