## 4 Chemical Aspects of Ulvan from _Ulva_ sp.
### Isolation, Purification, and Characterization
For the first stage of ulvan extraction, temperatures below 160 \({}^{\circ}\)C are associated with initial mass loss due to moisture volatilization [22]. Analysis of the structural content in ulvan is mostly done by a turbidimetric assay. Specific structural analyses employ chromatographic methods, such as high-performance liquid chromatography, coupled with conductivity or Fourier transform infrared (FTIR) spectroscopy [58; 59]. FTIR spectra of dry cellulose fractions show peaks in the range of 400-600 cm\({}^{-1}\) at room temperature [53; 57]. There are absorption bands at 3412, 1645, 1251, 1080, and 852 cm\({}^{-1}\) caused by hydroxyl groups, uronic acid, SO stretching (sulfate) vibrations, CO hand COC glycosidic linkages, and COS sulfate flexural vibrations in the axial and/or equatorial position (in C-2 or C-3 rhamne), respectively [58; 60]. Some monosaccharide contents have been analyzed using chromatographic techniques, namely gas chromatography (GC), high performance liquid chromatography (HPLC), and liquid chromatography (LC) [21; 61]. Other studies have employed high performance anion exchange chromatography (HPAEC) and HPLC to determine the number of monosaccharides contained in ulvan [41; 62]. HPAEC determines the carbohydrate composition and purity of glucose. Most of the carbohydrate content of ulvan is dominated by glucose and xylose [57]. HPAEC can selectively measure those monosaccharides and determine the quantity of acid and sugars present in ulvan without further derivatization [63]. GC has been used to measure volatile compounds contained in ulvan, such as alditol [41]. Bond structures, link positions, and substitution patterns may be analyzed with a two-dimensional nuclear magnetic resonance (2D NMR) instrument [41; 57; 64]. Signals at 65 and 63 ppm correspond to the carbon C-6 in crystals and amorphous regions in cellulose. Strong signals at 76 and 73 ppm correspond to the carbons C-2, C-3, and C-5 [57]. In addition to the uronic acid component, 2D NMR can analyze hydrolyzed saccharide fragments. Analysis of sulfate groups can be seen from the resonance of H and C atoms [22; 55]. Molecular weight were analyzed by using size exclusion chromatography (SEC), which can measure the molecular weight of ulvan. SEC is also used to measure particle size distribution; it is performed during the final stages of endoglucanase purification. An SEC is equipped with a refractive index and ultraviolet (UV) detector [21; 65]. X-ray diffraction can be used to determine the crystalline structure of the obtained ulvan. It typically displays four characteristic peaks of cellulose at 14.6\({}^{\circ}\), 16.5\({}^{\circ}\), 22.9\({}^{\circ}\) and 34.7\({}^{\circ}\)[66; 67]. The important point of characterizing extracted ulvan is to pay close attention to the equipment components and the time required for analysis. For fast ulvan content analysis, a colorimetric assay approach can be performed to determine the sugar content, such as uronic acid, rhamnose, and xylose, as well as the protein content [41]. This colorimetric approach can also be used to quantitatively determine the sugar content [68]. To analyze the sulfate content, a turbidimetric assay method can be performed. The combination of two methods, namely chemometric assay and spectral analysis such as IR, NIR, and NMR, is very effective to quickly and accurately measure single-component polysaccharides [69]. The biological activity of ulvan can be identified from characterization of spectral analysis such as IR and NMR, which describe the structural and physicochemical properties of ulvan. Based on the published literature, sugar content analysis using colorimetric analysis, which provides fast and accurate results, can be combined with spectral analysis.