## 4 Chemical Aspects of Ulvan from _Ulva_ sp.
### Isolation, Purification, and Characterization  
After the stages of the extraction process are carried out, to obtain crude ulvan and pure ulvan, isolation and purification steps are needed. In the isolation of the ulvan from the _U. pertusa_, the separation was carried out using a column chromatography equipped with a QFF column. The polysaccharides were eluted with a linear gradient of 0-2 M NaCl at 2.0 mL/min [16]. There is also isolation using ion column chromatography followed by a depolymerization process on the _U. claratha_[47]. In the purification step, fractionation process is carried out such as to obtain ulvan from _U. lactuca_ and _U. compressa_[33]. The isolation of ulvan from _U. dutnoi_ using the biorefinery method has been reported in other studies [48]. Ulvan isolation from _U. lactuca_ is carried out to separate the desired active components after extraction using a variety of suitable solvents and considering its physicochemical properties. Ulvan isolation separates the polysaccharide components, including uronic acid, sulfated molecules, and sugar components [49; 50]. The isolation process can be carried out by dissolving the extract in water, centrifuging at 8000 rpm, and then separating on a diethylaminoethyl (DEAE)-Sepharose fast flow column (5 \(\times\) 2 cm). It can then be fractionated using sulfuric acid: The higher the concentration of sulfuric acid used, the higher the sulfate content obtained [27]. Figure 3: Schematic flowchart of the ulvan extraction method from _Ulva lactuca_. One study utilized acetone for sulfate fractionation; the sulfate concentrations were 1.07-11.17% [51]. In another study, the sulfate content was 2.21%, in which there was a high content of free sulfate (54%) and total sugar content (15%) [52]. Isolation of sulfated polysaccharides is carried out with NaOH at 90 \({}^{\circ}\)C; there was 17.6% sulfate isolation [53]. The next process is to purify the active components in the multicomponent ulvan into a single component. This review will focus on the purification method and the number of active components obtained [2]. The isolation process will vary depending on a laboratory/factory capabilities and availability or raw material and resources. Extraction yields a crude extract that is purified using a vacuum to remove residual solvents [31]. Another study isolated the ulvan component using 2 N HCl solvent reflux in methanol for 2 h, followed by separating the solvent using vacuum chromatography, then purified the extract using preparative thin layer chromatography (TLC) [13]. Isolation can also be done with dimethyl sulfoxide (DMSO), while other studies have isolated bacteria from algae with the loop inoculation method [54; 55]. Saccharide components, such as glucuronic acid oligosaccharides, have been isolated using acid precipitation after hot water extraction with sodium oxalate [55]. In practice, an optimal strategy for extraction, isolation, and purification procedures should consider the available resources so that the process can be selective and effective. It can also concentrate on evaporation, purification using dialysis, and isolation with precipitation. In resource-rich situations, selective acid extraction is ideal, although less selective extraction processes can be tolerated, with concentration and diafiltration with ultrafiltration. Purification can then be carried out using anion exchange chromatography, and lyophilization or spray drying to isolate pure ulvan. Characterization determines the polysaccharide structure obtained from the extraction, purification, and isolation processes. As mentioned above, the ulvan polysaccharides have various monosaccharides, namely rhamne, arabinose, xylose, mannose, glucose, and galactose [56]. Before advanced ulvan characterization, thermogravimetric analysis (TGA) is used to determine the percent of product loss due to extraction [57]. TGA can also reveal the effect of the extraction temperature on the quality of ulvan obtained.