# Mini-Review: Potential of Diatom-Derived Silica for Biomedical Applications
## 4 Frustule Cleaning/Purification: Main Techniques and Technical Issues  
Frustules can be thus purified from both living culture-derived algal biomass and diatomic stocks. The impurities of diatom frustules mainly consist of organic matters adhered to their surface [54]. In the case of diatomic samples, impurities are present in larger amounts, and can vary in relation to the local environment and aging conditions of these natural stocks [18]. Diatomic impurities typically contain also clay and metallic oxides, such as aluminium and ferric oxides [55]. Before cleaning procedures, diatomic particles usually undergo a first step of pulverization, in which micrometric powder is grinded to nanoparticles by mechanical crushing and sonication. However, apart from a few exceptions, most studies report purification protocols based on raw material derived from living cultures rather than diatomic, which is currently the only diatomic silica-based marketable product. Organic impurities can be removed from the silica frustule by either a chemical pre-treatment with acids or other oxidative agents, or by exposing the frustules to high temperatures. Some studies, aimed at assessing the efficacy of preliminary hydrochloric acid treatments for organic mass removal, showed that acid concentration greatly influenced both the removal rate of impurities and the state of preservation of the frustule shape, with strong acidic pre-treatments causing frustule erosion [56]. Potassium permanganate can be also used to pre-treat frustules for organic compound removal [57; 58]. However, this procedure is essentially limited to remove impurities outside the frustule, and pre-treatments with acidic solutions are usually applied (even if they are not mandatory) when purification protocols do not foresee acid-based cleaning procedures, such as baking-based purifications [59]. Some preliminary oxidations with acid solutions do not exclude the employment of both acids and high temperatures. Treatment of diatom frustules with sodium permanganate and oxalic acid, for example, is followed by perchloric acid treatments at 100 \({}^{\circ}\)C [57]. Baking (i.e., strong heating of silica cell walls) of diatom frustules at 400-800 \({}^{\circ}\)C is the simplest and least expensive method to remove organic components. However, high-temperature treatments can alter diatom architecture and pore size [60]. Oxygen plasma etching, a procedure consisting of the removal of impurities using ionised gases, was found to be effective to preserve the frustule structure, with a negligible loss of material and without shape alterations [61; 62]. The most commonly used procedure for the removal of organic matter and the purification of diatom biosilica is, however, an oxidative washing treatment. Some protocols require the use of 30% [63; 64; 65; 66; 67] or 15% [68] hydrogen peroxide solutions. The most common washing solvents used in acid-based treatments of diatom frustules are sulphuric [69; 70] and nitric [68; 71] acids. sulphuric acid treatment is rapid (10-30 min) and revealed successful even on small amounts of biosilica [55]. Despite the rapidity of this strong acid-based method, cleaning procedures are time-consuming, since several washes with distilled/deionised water are required for a complete acid removal. However, the effect of acid strength needs to be evaluated in each case, since silica nanostructures can be damaged by the action of acids. For example, frustules from poorly silicified diatom species can be dissolved in strong acid cleaning solutions [70]. To improve the efficiency of biosilica purification, [PERSON] and co-workers [72] set up a vacuum cleaning method in which all the cleaning steps, which are cell extraction, acid treatment and washing, are carried out on polytetrafluoroethylene (PTFE) filter cloths, thus decreasing the processing time. This allows the recycling of the sulphuric acid used for cleaning, decreasing the amount of both the reagent needed for purification and the liquid wastes.