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2021 - Gaseous heptanethiol removal by a fe3+-phenanthroline-kaolinite hybrid material [Articolo su rivista]
Bernini, F.; Castellini, E.; Brigatti, M. F.; Bighi, B.; Borsari, M.; Malferrari, D.

Kaolinite functionalized by the μ-oxo Fe3+-phenanthroline complex (Fe+3Phen) was selected to test its ability to efficiently remove and store gaseous heptanethiol (HPT). Spectroscopic techniques, elemental analysis, and thermal analysis coupled with evolved gas mass spectrometry were employed to characterize the material before and after the exposure to the gas and to define the adsorption process. The amount of HPT trapped by the functionalized kaolinite after 60 days is 0.10940 moles per 100 g of kaolinite which, considering the amount of adsorbed Fe+3Phen (0.00114 moles per 100 g of kaolinite), means a thiol/Fe3+Phen molar ratio of about 100:1, a value much higher than those found in the past for Fe+3Phen functionalized montmorillonite and sepiolite. In addition, the process was found to be efficient also beyond 60 days. This significant removal of the smelly gas was explained by considering a continuous catalytic activity of Fe3+ toward the oxidation of thiol to disulfide.

2021 - Molecular structure and ammonia gas adsorption capacity of a Cu(II)-1,10-phenanthroline complex intercalated in montmorillonite by DFT simulations [Articolo su rivista]
Sainz-Diaz, C. I.; Castellini, E.; Escamilla-Roa, E.; Bernini, F.; Malferrari, D.; Brigatti, M. F.; Borsari, M.

A hydrated complex of 1,10-phenanthroline with Cu2+ cation was intercalated in the interlayer space of montmorillonite. This intercalation occurs initially by through a cation exchange mechanism in which the charge of the complex cation compensates the excess of the negative charge of the interlayer, then, once the cation exchange capacity (CEC) value has been reached, by direct adsorption of the sulfate salt of this complex (i.e. the cation together with its sulfate counterion). This material has showed interesting entrapping properties of gaseous phases and a peculiar chemical reactivity. However, the complete characterization and explanation of the formation of these materials is difficult with only experimental techniques. Hence, we used computational methods at atomic level to know how are the molecular structure of these complexes and their adsorption capacity of ammonia inside the interlayer confined space of montmorillonite for a better understanding of the experimental behaviour. First Principles calculations were performed based on Density Functional Theory (DFT). The intercalation of the phenanthroline-Cu(II) complex inside the nanoconfined interlayer of montmorillonite is energetically favourable in the relative proportion observed experimentally, being a cation exchange process. The further adsorption of the sulfate salt of the phenanthroline-Cu complex is also energetically possible. The adsorption of ammonia molecules in these montmorillonite-phenanthroline-Cu complexes was also favourable according with experimental behaviour.

2021 - Self-Assembled Structures from Solid Cd(II) Acetate in Thiol/Ethanol Solutions: a Novel Type of Organic Chemical Garden [Articolo su rivista]
Bernini, Fabrizio; Castellini, Elena; Sebastianelli, Lorenzo; Bighi, Beatrice; Ignacio Sainz-Díaz, Claro; Mucci, Adele; Malferrari, Daniele; Ranieri, Antonio; Franca Brigatti, Maria; Borsari, Marco

Cd(II) acetate in tablet form, immersed in ethanol solutions with a high concentration of heptanethiol (30-80 mM) was able to develop self-assembled sail-shaped structures. The solution in which the self-assembled structure is formed is totally organic (both solute and solvent), thus representing a unique case among organic Chemical Gardens. The constituting material for this new Chemical Garden (CG) is made of a single phase, namely a cadmium heptanethiolate with a microcrystalline structure consisting of a central cluster [CdnSn]. The morphological and structural features were studied using different techniques (SEM-EDS, elemental analysis, FT-IR, 113Cd MAS NMR, XRPD). A crystalline cell for the Cd(II) heptanethiolate cluster was obtained that completely differs from that of the solid phase precipitated by mixing an ethanol solution of Cd(II) acetate with heptanethiol. It follows that the conditions under which the formation of Cd(II) thiolate occurs (slowly from solid, quickly from solution) play a critical role in determining the nature and structure of the precipitating phase.

2021 - The Copper Chemical Garden as a Low Cost and Efficient Material for Breaking Down Air Pollution by Gaseous Ammonia [Articolo su rivista]
Castellini, Elena; Bernini, Fabrizio; Sebastianelli, Lorenzo; Bighi, Beatrice; Ignacio Sainz-Diaz, Claro; Mucci, Adele; Malferrari, Daniele; Ranieri, Antonio; Gorni, Giulio; Marini, Carlo; Brigatti, Maria Franca; Borsari, Marco

Chemical garden (CG) from copper(II) sulfate, nitrate and chloride (CG CuSO4, CG Cu(NO3)2, CG CuCl2) were grown and characterized from the structural and compositional point of view using scanning electron microscopy, X-ray powder diffraction, elemental analysis, thermogravimetric analyses coupled with mass spectrometry, and DR (Diffuse Reflectance) UV-Vis-Nir spectroscopy. The main crystalline phases, controlled by the anion of the starting salt, were brochantite and kobyashevite for CG CuSO4, gerhardtite, rouaite and anthonyite for CG Cu(NO3)2, and atacamite for CG CuCl2. The materials were then exposed to ammonia vapors to test the effectiveness of their entrapping property. All materials proved to be very efficient and rapid in the uptake of ammonia which invariably results in the formation of a Cu(II)/NH3 complex. However, after a few tens of minutes, CG Cu(NO3)2 and CG CuCl2 release water and get wet, thereby resulting unsuitable for applications. Only CG CuSO4 keeps dry for at least 25 hours. This makes it a valid candidate for building devices for trapping ammonia and possibly other gases capable of interacting with Cu (II). The entrapment of ammonia by this material was also characterized by 1H and 29Si MAS-NMR XAS spectroscopies.

2020 - A new material based on montmorillonite and Cu(II)-phenanthroline complex for effective capture of ammonia from gas phase [Articolo su rivista]
Castellini, E.; Malferrari, D.; Bernini, F.; Bighi, B.; Mucci, A.; Sainz-Diaz, I. C.; Serrano, A.; Castro, G. R.; Brigatti, M. F.; Borsari, M.

The intercalation of [Cu(Phen)(H2O)2]2+ (CuPhen) in montmorillonite (Mt) produces a stable hybrid material that is very efficient in removing NH3 from gas phase even at extremely low pressures. The process was studied by elemental analysis, X-ray powder diffraction, thermal analysis coupled with evolved gas mass spectrometry and DR UV–Vis, NMR and X-ray absorption spectroscopy. The adsorption of CuPhen on Mt consists of two consecutive steps. During the first one, CuPhen intercalates alone into Mt through a cation exchange process, afterwards CuPhen and SO42− ions entry jointly into the mineral interlayer. The two-steps adsorption process is described by a VI-type isotherm, successfully fitted by two independent Frumkin isotherms. NH3 trapping is long-lasting, easy, fast even at extremely low gas pressure and reversible under mild conditions. Mt containing CuPhen always results well performant in removing ammonia from gas phase, but an appreciably higher adsorption capacity of NH3 is obtained when SO42− ion is absent from the interlayer. This hybrid montmorillonite is thus a promising material to be used in industrial or environmental contexts, as an efficient air-cleaner.

2020 - Electrochemical data on redox properties of human Cofilin-2 and its Mutant S3D [Articolo su rivista]
Pignataro, M.; Di Rocco, G.; Lancellotti, L.; Bernini, F.; Subramanian, K.; Castellini, E.; Bortolotti, C. A.; Malferrari, D.; Moro, D.; Valdrè, G.; Borsari, M.; del Monte, F.

The reported data are related to a research paper entitled "Phosphorylated cofilin-2 is more prone to oxidative modifications on Cys39 and favors amyloid fibril formation" [1]. Info about the formation and redox properties of the disulfide bridge of a protein is quite difficult to obtain and only in a few cases was it possible to observe a cyclic voltammetry (CV) signal [2,3]. Human cofilin-2 contains two cysteines (Cys39 and Cys80) which can be oxidized in suitable conditions and form a disulfide bridge [1]. For this purpose, CV measurements were carried out on human cofilin-2 WT and its mutant S3D immobilized on a gold electrode coated by an anionic self-assembled monolayer (SAM), after a pre-oxidation time which was fundamental for observing a CV signal relating to the oxidation/reduction process of the disulfide bridge of the proteins. The data include CV curves obtained with and without electrochemical pre-oxidation and after oxidation with H2O2. In addition, the plot of the cathodic peak current vs. electrochemical pre-oxidation time and the pH dependence of the formal potential (E°’) are reported. The data obtained by CV measurements were used to determine the time required to form the disulfide bridge for the immobilized proteins and, consequently, to observe the CV signal, to calculate the E°’ values and analyse the pH dependence of E°’. The electrochemical data were provided which will be useful for further electrochemical investigations regarding proteins bearing disulfide bridge(s) or cysteines prone to oxidation.

2020 - Interlayer-confined Cu(II) complex as an efficient and long-lasting catalyst for oxidation of H2s on montmorillonite [Articolo su rivista]
Castellini, E.; Bernini, F.; Sebastianelli, L.; Sainz-Diaz, C. I.; Serrano, A.; Castro, G. R.; Malferrari, D.; Brigatti, M. F.; Borsari, M.

Removal of highly toxic H2S for pollution control and operational safety is a pressing need. For this purpose, a montmorillonite intercalated with Cu(II)-phenanthroline complex [Cu[(Phen)(H2O)2]2+ (Mt-CuPhen) was prepared to capture gaseous H2S under mild conditions. This hybrid material was simple to obtain and demonstrated an outstanding ability to entrap H2S at room temperature, retaining high efficiency for a very long time (up to 36.8 g of S/100 g Mt-CuPhen after 3 months of exposure). Sorbent and H2S uptake were investigated by elemental analysis, X-ray powder diffraction measurements, diffuse reflectance (DR) UV–Vis and infrared spectroscopy, thermal analysis and evolved gas mass spectrometry, scanning electron microscopy equipped with energy-dispersive X-ray spectrometer, and X-ray absorption spectroscopy. The H2S capture was studied over time and a mechanism of action was proposed. The entrapping involves a catalytic mechanism in which [Cu[(Phen)(H2O)2]2+ acts as catalyst for H2S oxidation to S0 by atmospheric oxygen. The low cost and the long-lasting performance for H2S removal render Mt-CuPhen an extremely appealing trap for H2S removal and a promising material for many technological applications.

2020 - Phosphorylated cofilin-2 is more prone to oxidative modifications on Cys39 and favors amyloid fibril formation [Articolo su rivista]
Pignataro, M.; Di Rocco, G.; Lancellotti, L.; Bernini, F.; Subramanian, K.; Castellini, E.; Bortolotti, C. A.; Malferrari, D.; Moro, D.; Valdre, G.; Borsari, M.; Monte, F. D.

Cofilins are small protein of the actin depolymerizing family. Actin polymerization/depolymerization is central to a number of critical cellular physiological tasks making cofilin a key protein for several physiological functions of the cell. Cofilin activity is mainly regulated by phosphorylation on serine residue 3 making this post-translational modification key to the regulation of myofilament integrity. In fact, in this form, the protein segregates in myocardial aggregates in human idiopathic dilated cardiomyopathy. Since myofilament network is an early target of oxidative stress we investigated the molecular changes induced by oxidation on cofilin isoforms and their interplay with the protein phosphorylation state to get insight on whether/how those changes may predispose to early protein aggregation. Using different and complementary approaches we characterized the aggregation properties of cofilin-2 and its phosphomimetic variant (S3D) in response to oxidative stress in silico, in vitro and on isolated cardiomyocytes. We found that the phosphorylated (inactive) form of cofilin-2 is mechanistically linked to the formation of an extended network of fibrillar structures induced by oxidative stress via the formation of a disulfide bond between Cys39 and Cys80. Such phosphorylation-dependent effect is likely controlled by changes in the hydrogen bonding network involving Cys39. We found that the sulfide ion inhibits the formation of such structures. This might represent the mechanism for the protective effect of the therapeutic agent Na2S on ischemic injury.

2020 - Tuning of halobenzenes uptake in montmorillonite from gas phase through a functionalization process involving Cu(II)-phenanthroline and heptanethiol [Articolo su rivista]
Castellini, Elena; Malferrari, Daniele; Bernini, Fabrizio; Bighi, Beatrice; Mucci, Adele; Sainz Diaz, Ignacio Claro; Serrano, Aida; Castro, German R.; Brigatti, Maria Franca; Borsari, Marco

In this paper, montmorillonite functionalized with a Cu(II)-phenanthroline complex (Mt-CuPhen) and then exposed to heptanethiol vapors (Mt-CuPhen-HPT) was tested for the capture of halobenzenes in gas phase. The treatment of Mt-CuPhen with heptanethiol vapors produced important compositional changes involving Cu(II) reduction, oxidation of thiol to disulfide and dehydration of the mineral interlayer space. Entrapping experiments were carried out on fluorobenzene, chlorobenzene and bromobenzene at 25 and 45°C. The hybrid material proved to be very efficient for all halobenzenes and especially for fluorobenzene. Observed performances are slightly affected by temperature. Halobenzene desorption occurred even at 100°C, a temperature quite lower than that required for decomposition of Mt-CuPhen-HPT. The Mt-CuPhen-HPT hybrid material, therefore, can be reused several times and requires low energy costs for regeneration. An alternative regeneration procedure based on disulfide removal has been proposed as well. Mt-CuPhen-HPT was extensively characterized before and after halobenzenes immobilization through elemental analysis, UV-Vis measurements, thermogravimetric analyses, FTIR, NMR and X-ray absorption spectroscopies.

2019 - Structural properties of adsorbent phyllosilicates rule the entrapping ability of intercalated iron-phenanthroline complex towards thiols [Articolo su rivista]
Castellini, E.; Malferrari, D.; Bernini, F.; Mucci, A.; Borsari, M.; Brigatti, M. F.

The interaction of volatile organic sulfur derivatives, such as 1-heptanethiol (C7H16S), with clay minerals treated with a μ-oxo Fe3+-phenanthroline 1:1 complex results strongly affected by crystal chemical properties of pristine mineral phases. In particular, two sepiolite clays with different structural features demonstrated significantly different ability to immobilize the Fe3+-phenanthroline complex at two pH values (pH = 5.4 and pH = 2.3). The most effective binding was obtained with sepiolite with higher structural disorder at pH 5.4. Accordingly, the resulting hybrid material showed also the greatest efficiency in removal of thiol in gas phase. A direct correlation can be established between the adsorption of the Fe3+-phenanthroline complex and the gas binding process at room temperature. In fact, 1-heptanethiol entrapping occurs via redox reactions between Fe3+ and a first thiol molecule to give the reduced Fe2+-phenanthroline complex and disulfide, followed by the binding of further thiols to the reduced metal centre. The extremely high amount of thiol immobilized by the hybrid material also suggests the co-presence of a catalytic mechanism that guarantees the reoxidation of Fe+2 to Fe+3 and the restoration of redox reactions with thiol. Investigation and conclusions were supported by the several experimental techniques: elemental analysis, X-ray powder diffraction analyses, UV–Vis measurements, FT-IR and NMR spectroscopies, thermogravimetric analyses.

2019 - Sulfur bearing and aromatic compound trapping by layered silicates: a great start for innovative technological applications [Abstract in Atti di Convegno]
Malferrari, D.; Bernini, F.; Bighi, B.; Borsari, M.; Brigatti, M. F.; Castellini, E.; Mucci, A.; Sebastianelli, L.

2019 - Trapping at the Solid-Gas Interface: Selective Adsorption of Naphthalene by Montmorillonite Intercalated with a Fe(III)-Phenanthroline Complex [Articolo su rivista]
Castellini, E.; Malferrari, D.; Bernini, F.; Sainz Diaz, C. I.; Mucci, A.; Sola, M.; Brigatti, M. F.; Borsari, M.

In this study, stable hybrid materials (Mt-Fe(III)Phen), made by the μ-oxo Fe(III)-phenanthroline complex [(OH 2 ) 3 (Phen)FeOFe(Phen)(OH 2 ) 3 ] 4+ (Fe(III)Phen) intercalated in different amounts into montmorillonite (Mt), were used as a trap for immobilizing gaseous benzene and naphthalene and their mono chloro-derivatives at 25 and 50 °C. The entrapping process was studied through elemental analysis, magic angle spinning NMR spectroscopy, thermal analysis, and evolved gas mass spectrometry. Naphthalene and 1-chloronaphthalene were found to be immobilized in large amount at both temperatures. Molecular modeling allowed designing of the structure of the interlayer in the presence of the immobilized aromatic molecules. Adsorption is affected by the amount of the Fe complex hosted in the interlayer of the entrapping hybrid materials. On the contrary, under the same conditions, benzene and chlorobenzene were not adsorbed. Thermal desorption of naphthalenes was obtained under mild conditions, and immobilization was found to be reversible at least for 20 adsorption/desorption cycles.

2018 - Chemical trapping of gaseous H⁠2S at high and low partial pressures by an iron complex immobilized inside the montmorillonite interlayer [Articolo su rivista]
Malferrari, Daniele; Castellini, Elena; Bernini, Fabrizio; Serrano Rubio⁠, Aida; Rafael Castro, German; Ignacio Sainz-Díaz⁠, Claro; Caleffi, Matteo; Brigatti, Maria Franca; Borsari, Marco

A stable hybrid material (Mt-Fe(III)Phen) formed by intercalation of the μ-oxo Fe(III)-phenanthroline complex [(OH⁠2)⁠3(Phen)FeOFe(Phen)(OH⁠2)⁠3]⁠4+ (Fe(III)Phen) in montmorillonite (Mt) is able to immobilize H⁠2S in gaseous phase with high efficiency even at extremely low pressures. DR UV–vis and I.R. spectroscopies, elemental analysis, X-ray powder diffraction, thermal analysis coupled with evolved gas mass spectrometry, and X-ray absorption spectroscopy show that the material has high adsorption capacity, performs fast H⁠2S trapping and is long-lasting. Moreover, even extremely low levels of H⁠2S can be removed easily and quickly from gaseous phase using a suitable amount of the trapping material. The immobilization mechanism likely involves a redox reaction between iron (III) and one S⁠2− ion, followed by the binding of a second S⁠2− ion to the metal centre. The process takes place at room temperature, is reversible for several cycles, and does not require pre-treatment of neither gaseous H⁠2S nor the adsorbent material. Therefore, this modified montmorillonite is a promising material to get rid of H⁠2S in processes of environmental interest and to obtain gaseous (and gasifiable) high quality hydrocarbons in fuels refineries.

2018 - Experimental and Theoretical Investigation of Intercalation and Molecular Structure of Organo-Iron Complexes in Montmorillonite [Articolo su rivista]
Sainz-Díaz, C. I.; Bernini, F.; Castellini, E.; Malferrari, D.; Borsari, M.; Mucci, A.; Brigatti, M. F.

The intercalation of the μ-oxo Fe(III)-phenanthroline 1:1 complex [(OH2)3(Phen)FeOFe(Phen)(OH2)3]+4 inside montmorillonite yielded a nanostructured material with strong and selective entrapping ability toward thiol molecules and hydrogen sulfide. In this work, experiments and computational molecular modeling by means of quantum mechanical calculations has been applied to study the molecular structure and interactions between this complex and the interlayer of montmorillonite. This approach allowed the identification of the geometrical disposition of the complexes inside the interlayer, the characterization of the hydration and coordination water molecules, and the explanation of the physico-chemical properties of these functionalized materials. The antiferromagnetic spin configuration of the Fe(III) ions results in the most stable state. Two conformers of the complex have been considered, having the phenanthroline rings in twisted or in parallel planes, respectively, and the transition of one conformer into the other has been explored by molecular dynamics simulations. The conformer with phenanthroline rings in parallel planes is found to be the favored species for intercalation in montmorillonite. Both experimental nuclear magnetic resonance analysis and adsorption isotherms are consistent with the modeling results. Different complex amount, equal and double of the cation exchange capacity (CEC) of montmorillonite, and hydration states inside the interlayer have been investigated reproducing faithfully the experimental d(001) spacing of the montmorillonite in the different conditions. The complex molecules intercalated over the CEC of montmorillonite adopt a disposition of the phenanthroline rings perpendicular to that of the complex already introduced by cation exchange.

2017 - Baseline studies of The Clay Minerals Society Source Clay montmorillonite STx-1b [Articolo su rivista]
Castellini, Elena; Malferrari, Daniele; Bernini, Fabrizio; Brigatti, Maria Franca; Castro, German Rafael; Medici, Luca; Mucci, Adele; Borsari, Marco

For more than forty years, The Clay Minerals Society has dispensed a set of source clays which have enabled a large number of researchers to work on identical materials. Many of these source clays remained unchanged over the years but, conversely, other clays have gone out of stock and thus were replaced. This was the fate of montmorillonite STx-1a, which was replaced by STx-1b. Although STx-1a and STx-1b share many basic chemical and mineralogical features, some minor differences exist that can affect behavior. A baseline characterization of the source clay STx-1b, which was the objective of this study, was, therefore, necessary to provide researchers a tool useful not only for new investigation but also to compare new results obtained on STx-1b with literature data on STx-1a. This characterization was gained using traditional and advanced methods that include: 1) chemical composition (major and trace elements); 2) cation exchange capacity determination; 3) thermal analyses coupled with evolved gas mass spectrometry; 4) quantitative mineralogical characterization using powder X-ray diffraction and Rietveld-RIR (Reference Intensity Ratio) refinement; 5) X-ray absorption spectroscopy at the Fe K-edge; 6) diffuse reflectance ultraviolet-visible and infrared spectroscopies; 7) 29Si, 27Al, and 1H magic-angle spinning nuclear magnetic resonance measurements. According to this multi-analytical approach, the montmorillonite chemical formula is [4](Si7.753 Al0.247) [6](Al3.281 Mg0.558 Fe0.136 Ti0.024 Mn0.002) [12](Ca0.341 Na0.039 K0.061) O20 (OH)4.

2017 - Corrigendum: Pre-amyloid oligomers budding:a metastatic mechanism of proteotoxicity [Articolo su rivista]
Bernini, F.; Malferrari, D.; Pignataro, M.; Bortolotti, C. A.; Di Rocco, G.; Lancellotti, L.; Brigatti, M. F.; Kayed, R.; Borsari, M.; Del Monte, F.; Castellini, E.

2017 - Crystal chemical characterization and computational modeling of a μ-oxo Fe(III) complex with 1,10-phenanthroline clarify its interaction and reactivity with montmorillonite [Articolo su rivista]
Brigatti, Maria Franca; Díaz, Claro Ignacio Sainz; Borsari, Marco; Bernini, Fabrizio; Castellini, Elena; Malferrari, Daniele

This work provides a systematic study of the μ-oxo-di-fac-[triaqua-(1,10-phenanthroline-κ2N,N′)-iron(III)]bis(sulfate), [(OH2)3(phen)FeOFe(phen)(OH2)3] (SO4)2 (phen = phenanthroline). Crystal structure is determined by single-crystal X-ray diffraction data and refined to R = 0.039. The crystal structure is monoclinic (Z = 2), space group P21 with unit cell dimensions a = 8.5157(3), b = 17.6434(5), c = 9.9678(3) Å, β = 90.133(2)°, V = 1497.62(8) Å3. The triaqua(1,10-phenanthroline)iron(III) parts are linked through one oxo-bridge. Both Fe(III) cations show a distorted octahedral coordination. The single-crystal data are complemented by computational chemistry modeling at quantum mechanical level, X-ray powder diffraction at room and high temperature conditions and by thermal analysis. Molecular modeling suggests that the role of the crystallization water molecules is critical to establish the intermolecular interactions for the stability of the crystal structure.

2017 - Effective and Selective Trapping of Volatile Organic Sulfur Derivatives by Montmorillonite Intercalated with a μ-oxo Fe(III)-Phenanthroline Complex [Articolo su rivista]
Bernini, Fabrizio; Castellini, Elena; Malferrari, Daniele; Castro, German Rafael; Sainz Díaz, Claro Ignacio; Brigatti, Maria Franca; Borsari, Marco

The mu-oxo Fe(III) phenatithroline Complex [(OH2)(3)(Phen)FeOFe(Phen) (OH2)(3)](+4) intercalated in montmorillonite provides a stable hybrid material. In this study, the ability and efficiency of this material to immobilize thiols in gas phase, acting as a trap at the solid-gas interface, were investigated. Aliphatic thiols containing both hydrophilic and hydrophobic end groups were chosen to test the selectivity of this gas trap. DR-UV vis, IR, elemental analysis, thermal analysis and evolved gas mass spectrometry, X-ray powder diffraction, and X-ray absorption spectroscopy techniques were employed to characterize the hybrid material before and after thiol exposure and to provide information on the entrapping process. Thiol immobilization is very large, up to 21% w/w for heptanethiol. In addition, evidence was obtained that immobilization occurs through the formation of a covalent bond between the iron of the complex and the sulfur of the thiol. This provides an immobilization process characterized by a higher stability with respect to the method's based on physi-adsorption. Thiol immobilization resulted thermally reversible at least for 20 adsorption/desorption cycles. Unlike standard desulfurization processes like hydrotreating and catalytic oxidation which work at high temperatures and pressures, the present system is able to efficiently trap thiols at room temperature and pressure, thus saving energy. Furthermore,we found that the selectivity of thiol immobilization can be tuned acting on the amount of complex intercalated in montmorillonite. In particular, montmorillonite semisaturated with the complex captures both hydrophobic and hydrophilic thiols, while the saturated montmorillonite shows a strong selectivity toward the hydrophobic molecules.

2016 - Pre-amyloid oligomers budding:a metastatic mechanism of proteotoxicity [Articolo su rivista]
Bernini, Fabrizio; Malferrari, Daniele; Pignataro, Marcello; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Lancellotti, Lidia; Brigatti, Maria Franca; Kayed, Rakez; Borsari, Marco; Del Monte, Federica; Castellini, Elena

The pathological hallmark of misfolded protein diseases and aging is the accumulation of proteotoxic aggregates. However, the mechanisms of proteotoxicity and the dynamic changes in fiber formation and dissemination remain unclear, preventing a cure. Here we adopted a reductionist approach and used atomic force microscopy to define the temporal and spatial changes of amyloid aggregates, their modes of dissemination and the biochemical changes that may influence their growth. We show that pre-amyloid oligomers (PAO) mature to form linear and circular protofibrils, and amyloid fibers, and those can break reforming PAO that can migrate invading neighbor structures. Simulating the effect of immunotherapy modifies the dynamics of PAO formation. Anti-fibers as well as anti-PAO antibodies fragment the amyloid fibers, however the fragmentation using anti-fibers antibodies favored the migration of PAO. In conclusion, we provide evidence for the mechanisms of misfolded protein maturation and propagation and the effects of interventions on the resolution and dissemination of amyloid pathology.

2015 - Human Cofilin2: Towards the Comprehension of the Molecular Mechanism [Abstract in Atti di Convegno]
DI ROCCO, Giulia; Pignataro, Marcello; Bortolotti, Carlo Augusto; Castellini, Elena; Lancellotti, Lidia; Borsari, Marco; Sola, Marco; Del Monte, F.

Cofilin is an evolutionarily highly conserved protein which belongs to the ADF/cofilin family involved in the regulation of actin-filament dynamics depolymerizing and/or severing actin filaments. Phosphorylation on serine 3 inactivates cofilin [1,2] by generation of a charge repulsion between cofilin and actin, which is thought to occur without altering the protein structure [3]. In terms of physiological functions, cofilin- 2 is the least understood member of this protein family, which is present predominantly in skeletal and cardiac muscle [4-6]. In reducing media, even phosphatidylinositol 4,5-bisphosphate-bound cofilin is active, leading to actin dynamics in the vicinity of the plasma membrane. This mechanism has been proposed to explain why dendritic cells that are able to increase the thiol pool in antigen-specific T cells enable T cell activation even under oxidative stress conditions. On the contrary, cofilin is inactivated by oxidation, provoking T-cell hyporesponsiveness or necrotic-like programmed cell death [7]. In this study we present the production, the physico-chemical characterization and the modelled structure of the wt and the phosphorylated-mimicking S3D variant of the human cofilin2. The study allowed the evaluation of the structural differences between the active and the inactive protein while an electrochemical and fluorometric approach provided new data to increase in the understanding of the cofilin-action mechanism. 1. Agnew BJ, Minamide LS, Bamburg JR. J Biol Chem 1995; 270:17582–17587. 2. Moriyama K, Iida K, Yahara I. Genes Cells 1996; 1:73–86. 3. Blanchoin L, Robinson RC, Choe S, Pollard TD. J Mol Biol 2000;295:203–211. 4. Bernstein BW, Bamburg JR. Trends Cell Biol 2010;20(4):187–95. 5. Agrawal PB, Joshi M, Savic Tetal.. Hum Mol Genet. 2012 May 15; 21(10): 2341–2356 6. C. Thirion et al. (Eur. J. Biochem. 268)-2001 7. Y. Samstag, I. John, G. H. Wabnitz Immunological Reviews 256 (2013) 30-47.

2015 - Solvent tunes the peroxidase activity of cytochrome c immobilized on kaolinite [Articolo su rivista]
Castellini, Elena; Bernini, Fabrizio; Berto, Marcello; Borsari, Marco; Sola, Marco; Ranieri, Antonio

The adsorption process and the peroxidase activity of yeast cytochrome c (ycc) immobilized on kaolinite (Kaol) were investigated in mixed ethanol/water solutions. The protein strongly adsorbs on the surface of the clay mineral and the thermodynamic adsorption constant increases with increasing ethanol concentration. The adsorption parameters suggest that in ycc a conformational transition from molten globule to helical state occurs in solution for ethanol concentration above 20%. The peroxidase activity of ycc immobilized on Kaol increases from 0% to 20% ethanol (v/v), then it progressively decreases and almost vanishes in pure ethanol. The catalytic properties of adsorbed yccwere studied in 20 and 40% ethanol solutionswhich correspond to the molten globule and to the helical state, respectively. In both cases, catalysis adheres to theMichaelis–Menten model. Themolten globule state, which binds more weakly to kaolinite than the helical state, was found to be more catalytically active. This study is meant to identify the physicochemical factors that modulate the catalytic activity of this kaolinite-based interface of broad applicability.

2015 - Stepwise structuring of the adsorbed layer modulates the physico-chemical properties of hybrid materials from phyllosilicates interacting with the μ-oxo Fe+3-phenanthroline complex [Articolo su rivista]
Bernini, Fabrizio; Castellini, Elena; Malferrari, Daniele; Borsari, Marco; Brigatti, Maria Franca

The -oxo Fe+3-phenanthroline 1:1 complex [(OH2)3(Phen)FeOFe(Phen)(OH2)3]+4 (hereafter Fe+3Phen) was successfully immobilized on montmorillonite (Mt) and kaolinite (Kt) minerals. Adsorption data on both minerals described an adsorption isotherm of VI type and were successfully fitted using two independent Frumkin isotherms. The interaction between the complex and the minerals is strong and yields two stable hybrid materials: Kt-Fe+3Phen and Mt-Fe+3Phen. DR UV-Vis, elemental analysis, TGA-MSEGA, temperature-controlled XRPD techniques were used to characterize the structural properties of the hybrid materials. These investigations showed that the Fe+3Phen adsorption occurs stepwise via the formation of a bilayer structure. The first layer is the result of a cation exchange process involving the negative charges of the mineral while the second one probably forms through stacking interaction and/or sulphate ions bridging. XRD diffraction measurements show that in Mt the second layer formation is accompanied by a super- structuring of the interlayer that changes the thermal and chemical properties of the composite material. In particular, interesting catalytic properties are observed in Mt-Fe+3Phen samples, but they are completely suppressed with the formation of the Fe+3Phen bilayer structure.

2013 - Enhancing Biocatalysis: The Case of Cytochrome c Unfolded Immobilized on Kaolinite [Articolo su rivista]
Castellini, Elena; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Bernini, Fabrizio; Ranieri, Antonio

Urea-unfolded wild-type cytochrome c and its variants immobilized on kaolinite show peroxidase activity that is significantly higher than that of the folded wild-type protein. The accessibility of the substrate to the metal center and the influence of strategic amino acidic residues on the surface of the protein are discussed. This approach sheds light on the factors affecting the catalytic activity of a new versatile biocatalytic interface.

2013 - Sodium hexametaphosphate interaction with 2:1 clay minerals illite and montmorillonite [Articolo su rivista]
Castellini, Elena; Berthold, C.; Malferrari, Daniele; Bernini, Fabrizio

The interaction of an efficient deflocculant, sodium hexametaphosphate (NaHMP), with illite and montmorillonite samples of different origins was investigated analyzing the HMP uptake (adsorption on mineral surface and/or intercalation inside the mineral interlayer) and the ion release mechanisms. HMP adsorption isotherms on a standard Na-saturated illite were determined at different temperatures providing the thermodynamic parameters of the process and the maximum adsorbed amount of the ion on this mineral. The data indicate that the adsorption process is hindered compared to that on kaolinite and are consistent with the hypothesis that HMP is chemiadsorbed on the aluminol edge sites, forming Al-O-P linkages. The effect of HMP on the natural illite and montmorillonite samples was analyzed using different techniques. Inductively coupled plasma optical emission spectroscopy (ICP-OES) measurements were used to detect the concentration of P, Si, Al, Ca, Mg and K in deflocculant solutions wetting the clay minerals in order to detail the immobilization, dissolution and exchange reactions associated to the deflocculant activity. Thermal analysis and XRD measurements were used as well to gain information on the properties of the HMP-treated clay mineral samples. Montmorillonite samples show a peculiar ability to immobilize HMP. Intercalation processes involving the formation of Ca2+/HMP complexes inside the mineral interlayer are proposed to explain this result and could be the reason of the well known decrease in the deflocculant efficiency of HMP in ceramic slurries rich in montmorillonite.

2012 - The Met80Ala point mutation enhances the peroxidase activity of immobilized cytochrome c [Articolo su rivista]
Ranieri, Antonio; Bernini, Fabrizio; Bortolotti, Carlo Augusto; Castellini, Elena

The effects of replacement of the axial methionine 80 hemeligand with a non-coordinating alanine on the peroxidaseactivity of kaolinite-immobilized cytochrome c wereinvestigated at different pH values. The catalytic activity10 of the adsorbed mutant was found remarkably higher thanthat of wild-type cytochrome c. The pH dependence ofVmax and KM values is discussed in term of accessibility ofthe substrates to the metal center and surface charge ofkaolinite. Our approach, based on the combined use of15 adsorption on kaolinite and protein engineering, endowsthis bioinorganic interface with remarkable catalyticproperties.

2011 - pH-dependent peroxidase activity of yeast cytochrome c and its triple mutant adsorbed on kaolinite [Articolo su rivista]
Ranieri, Antonio; Bernini, F.; Bortolotti, Carlo Augusto; Bonifacio, A.; Sergo, V.; Castellini, Elena

The peroxidase activity of wild type yeast cytochrome c and its triple mutant K72AK73AK79A adsorbed onto kaolinite was investigated as a function of pH and temperature. Both adsorbedproteins displayed an appreciable catalytic activity, which remained constant from pH 7 to 10, decreased below pH 7 and showed a remarkable increase at pH values lower than 4. In the whole pH range investigated the catalytic activity of the adsorbed wild type cytochrome c was higher than that of the mutant. Both diffuse-reflectance UV-Vis and Resonance Raman spectroscopies applied on solid samples were used to probe the structural features responsible for the catalytic activity of the immobilized proteins. At neutral and alkaline pH values a six-coordinate low-spin form of cytochrome c was observed, while at pH < 7 the formation of a high-spin species occurred whose population increased at decreasing pH. The orientation and exposure of the haem to the substrate - strictly dependent on adsorption - was found to affect the peroxidase activity.

2011 - Sodium hexametaphosfate interactions with illite and montmorillonite: preliminary observation on thermal behavior [Abstract in Rivista]
Castellini, Elena; M., Bert; C., Berthold; Brigatti, Maria Franca; Malferrari, Daniele

The interaction of an efficient deflocculant, sodium hexametaphosphate (NaHMP), with two different illites (IMt-1 from Silver Hill, Montana USA and from Schwäbisch Hall, Würtenberg Germany) and two different Ca-montmorillonites (STx-1 from Gonzales County, Texas, USA and from Santa Rita, New Mexico, USA) was investigated.Adsorption measurements of the NaHMP anion were performed onNa-exchanged illite IMt-1 as a function of temperature by means of inductively coupled plasma technique (I.C.P.). The thermodynamic parameters of the adsorption process, together with an estimation of the maximum adsorbed amount on the Na-illite were obtained and compared to the corresponding data previously obtained for Na-kaolinite.The effect of NaHMP on the natural illites and montmorillonites was analyzed using different techniques. I.C.P. measurements of the concentration of P, Na, Si, Al, Ca, Mg, and K of the deflocculant solutions in contact with the clays were performed in order to detail the adsorption, dissolution and exchange phenomena which accompany the deflocculant activity.Thermal analysis (thermo-gravimetric, thermal-differential) and XRD measurements at non ambient temperature conditions were used to characterize the clay solid samples after the interaction with NaHMP.Thermo-gravimetric analyses indicate that NaHMP-treated illites and montmorillonites are less hydrated than natural samples, whereas the dehydroxilation reaction seems to be not influenced by the NaHMP treatment. Basal periodicity was measured via XRPD diffraction on (00l) oriented mounts of the air-dried samples in the temperature range 25 - 400°C (heating rate 2 °C/min) using a powder diffractometer equipped with an area detector. No significant variation of layer periodicity was observed, thus indicating that NaHMP molecules are adsorbed on the edge and/or in the interlayer without modifying natural layer periodicity.

2011 - The Reorganization Energy in Cytochrome c is Controlled by the Accessibility of the Heme to the Solvent [Articolo su rivista]
Bortolotti, Carlo Augusto; Siwko, Magdalena Elzbieta; Castellini, Elena; Ranieri, Antonio; Sola, Marco; S., Corni

Elucidation of the molecular determinants of the reorganization energy λ is central to the understanding of fundamental biological processes based on energy transduction pathways. Here, we use a combined experimental/theoretical approach to electrochemically determine the reorganization energy for a number of cytochrome c variants and compute structure-related properties relevant to the kinetics of the electron transfer process through molecular dynamics simulations. We find that the exposure of the heme group to solvent controls the reorganization energy of the investigated proteins. Therefore, fine-tuning of the kinetics of the electron transfer process can be achieved through modulation of the accessibility of the iron to the surrounding water. Our findings lead the way for a new strategy for the design of protein-based bioelectronic materials, requiring fast and efficient electron transfer.

2009 - Thermodynamic Aspects of the Adsorption of Cytochrome c and Its Mutants on Kaolinite [Articolo su rivista]
Castellini, Elena; Ranieri, Antonio; Domenico A., Simari; Di Rocco, Giulia

The adsorption of native, wild-type and engineered cytochrome c on sodium-exchanged kaolinite was investigated by spectroscopic means. The variants of yeast cytochrome c were obtained replacing surface lysines in position 72, 73 and 79 with alanine residues. All proteins are strongly adsorbed onto kaolinite. In particular, the presence of the lysine residue in position 73 remarkably favors adsorption. A detailed characterization of the thermodynamic aspects of the adsorption process has been performed. Most notably, adsorbed cytochrome c maintains its moderate peroxidase activity against guaiacol. This investigation is prodromal to the exploitation of the catalytic activity of engineered cytochrome c immobilized on a polydisperse system.

2008 - Deflocculant effect on the surface properties of kaolinite investigated through malachite green adsorption [Articolo su rivista]
Castellini, Elena; Andreoli, Roberto; Malavasi, Gianluca; Pedone, Alfonso

The adsorption of malachite green (MG) on sodium-exchanged kaolinite and on the same clay treated with two widely used deflocculants, sodium hexametaphosphate (NaHMP) and sodium disilicate (NaDIS), was investigated. The results indicate that the deflocculants affect the adsorption process. In particular, DIS treatment increases physisorption, whereas HMP treatment almost completely prevents it. The Frumkin isotherm accurately describes MG adsorption for all of the adsorbents investigated and the Frumkin inter- action parameter is found to be repulsive, in keeping with the cationic nature of the adsorbed species. TheKeywords: temperature dependences of the adsorption constant and the related thermodynamic parameters H◦ adsAdsorption and S◦ ads were also determined and discussed.

Andreola, Nora Maria; Castellini, Elena; Lusvardi, Gigliola; Menabue, Ledi; Romagnoli, Marcello

Chem. investigations have been performed on deflocculated suspensions of kaolinite previously treated with two widely employed dispersants: sodium hexametaphosphate (NaHMP) and sodium disilicate (NaDIS). F. Andreola et al. (2006) showed remarkable differences in the rheol. behavior between untreated and treated samples. The release of ions by kaolinite dispersed in deflocculant solns. results strongly affected by the nature of the treatment. The chem. anal. of the soln. phase sepd. from the suspensions of treated and untreated kaolinite provides relevant information about the deflocculation mechanism of HMP and DIS and gives reason of the obsd. differences in the rheol. responses.

2006 - Effect of sodium hexametaphosphate and ageing on the rheological behaviour of kaolin dispersions [Articolo su rivista]
Andreola, Nora Maria; J. M. F., Ferreira; S., Olhero; Romagnoli, Marcello; Castellini, Elena

Concentrated dispersions (35% v, v) of ceramic grade kaolin dispersed with sodium hexametaphosphate (NaHMP) were prepared at three different experimental conditions: in distilled water at 30 and 60 degrees C, and in a 5 * 10(-3) M Ca(NO3)(2)(.)4H(2)O solution, at 30 degrees C. The dispersions were kept at constant temperatures for different ageing times (2, 4, 6, 18, 24, 48 h). After each ageing period they were submitted to rheological measurements and centrifuged to measure the concentration of HMP- ion in the supernatant solution by ICP. Measurements of the electrophoretic mobility in dispersions containing Ca(NO3)(2)(.)4H(2)O and KCl as background electrolyte were also performed both in the presence and in absence of NaHMP. The results allow a better understanding of the roles played by calcium ions and ageing of kaolin dispersions in the presence of NaHMP. They also shed light on the deflocculating mechanism of NaHMP. Correlations between theological parameters and chemical properties of the dispersions could be established.

2006 - Role of the surface treatment in the deflocculation of kaolinite [Articolo su rivista]
Andreola, Nora Maria; Romagnoli, Marcello; Castellini, Elena; Lusvardi, Gigliola; Menabue, Ledi

Suspensions of untreated and properly treated Na-exchanged kaolinite have been subjected to rheological investigations using sodium hexametaphosphate (NaHMP) or sodium disilicate (DIS) as deflocculants. The treatment of the clay consisted of a 24 h contact with solutions of NaHMP or DIS and separation of the resulting solid phase. This was then used to form the suspensions subjected to the rheological measurements. The results indicate that the dispersion ability of each deflocculants is remarkably influenced by the type of treatment. In particular, with respect to the untreated kaolinite, the efficiency of DIS is dramatically increased when the clay has been previously treated with a NaHMP solution. On the other hand, a decrease in the dispersant ability of NaHMP is observed from the untreated kaolinite to the one previously treated with DIS.

2005 - Thermodynamic aspects of the adsorption of hexametaphosphate on kaolinite [Articolo su rivista]
Castellini, Elena; Lusvardi, Gigliola; Malavasi, Gianluca; Menabue, Ledi

The adsorption of hexametaphosphate ion, an important deflocculant used in the ceramic industry, from aqueous solutions onto kaolinite has been studied at different temperatures. The adsorption isotherm follows the Langmuir model: the thermodynamic parameters Delta G(ads)(0), Delta H-ads(0) and Delta S-ads(0) as were calculated and found to be consistent with an interaction model involving the formation of an inner-sphere complex between HMP and aluminol groups. Also, the dependence of the adsorption behavior on the kaolinite volume fraction has been studied and discussed in term of association processes between the clay particles.

2004 - The role of sodium hexametaphosphate in the dissolution process of kaolinite and kaolin [Articolo su rivista]
Andreola, Nora Maria; Castellini, Elena; Manfredini, Tiziano; Romagnoli, Marcello

Suspensions of kaolinite and ceramic grade kaolin in sodium hexametaphosphate solutions at different concentrations were prepared and kept at constant temperature for 4 h: the solutions obtained after centrifugation and filtration were analysed using a ICP technique to determine the concentration of significant cations. The results allowed to stress the role of NaHMP in the dissolution process of kaolinite and ceramic grade kaolin. The role of accessory minerals in ceramic grade kaolin dissolution was discussed in the light of the obtained data.

2003 - Delamination of italian kaolin: effect of reaction temperature and cristallinity on the urea rate intercalation [Abstract in Atti di Convegno]
L., Garrido; Andreola, Nora Maria; Castellini, Elena

Urea is effective in kaolin delamination and cristallinity. The role of temperature in delamination has been investigated in different conditions.

2003 - Effect of ionic strength and temperature on the interaction between kaolin suspensions and sodium hexametaphosphate [Abstract in Atti di Convegno]
Andreola, Nora Maria; Castellini, Elena; Manfredini, Tiziano; Romagnoli, Marcello

The effect of ionic strength on the interaction between kaolin and sodium hexametaphosphate has been investigated at different temperatures.

2002 - Chemical additives for pressure tape casting [Articolo su rivista]
Andreola, Nora Maria; Castellini, Elena; Romagnoli, Marcello

Suspensions of ceramic raw material and different deflocculant polyelectrolytes were prepared with a solid content of about 78% in industrial water. The rheological behavior of these suspensions was tested by evaluation of the shear dependence in a 4-step cycle and by a modified off-on procedure that reproduced the typical times involved in tape casting. The best deflocculants were found to be Na hexametaphosphate and polyacrylate, but their capability differed a little from mixtures of hexametaphosphate and Na disilicate or from ternary mixtures. The deflocculants made the suspensions slightly dilatant, had a low viscosity and yield stress values close to zero, that permitted an easy degasification of the suspensions. The additives, alone or in mixture, had adequate characteristics for use in pressure tape casting.

2002 - Zeta potential and rheological behaviour of aqueous kaolin suspensions dispersed with sodium hexametaphosphate [Capitolo/Saggio]
Andreola, F.; Castellini, E.; Romagnoli, M.; Olhero, S. M.; Ferreria, J. M. F.

Deflocculants adsorb on particles' surfaces, increase the repulsive forces among them, reducing viscosity, while the soluble salts have an opposite effect on viscosity. This work studies the influence of deflocculant and salt concentration on zeta potential and correlates the results with rheological measurements.

2000 - Effetto di Additivi Chimici sulle Caratteristiche Reologiche di Sospensioni Ceramiche Tradizionali per Tape-Casting. [Relazione in Atti di Convegno]
Andreola, Nora Maria; Castellini, E.; Romagnoli, Marcello

Sono presentati i risultati di una sperimentazione che ha visto l'utilizzo di tre diverse tipologie di additivi su un impasto di tipo industriale e avente come intento l'individuazione di additivi che permettano, a parità di contenuto solido, di ridurre la viscosità della sospensione, attraverso una razionale serie di prove. Viene mostrato quali additivi o miscele permettono di ottenere i comportamenti reologici piu adeguati a tale tecnica di formatura. Parametri reologici quali la viscosità e la tissotropia delle sospensioni sono messi in relazionealla composizione degli additivi.

2000 - Optimization of fritting parameters of ceramic frits: effect on the rheological behavior of the glaze suspensions. [Articolo su rivista]
Andreola, Nora Maria; Castellini, E.; Romagnoli, Marcello

Studies to optimize the prepn. parameters used in industrial frits are reported. Two frit compns. widely used for the prodn. of single-fired ceramic-glazes were chosen: an alk. boric and an earth-alk. boric compn. The chem. durability and rheol. behavior of the frit suspensions were studied in relation to changes in the fritting temp. and time. The results confirm that the two frits studied react differently to changes in fritting parameters due to the dependence on their chem. durability and compn. Suspensions prepd. with the alk.-boric frit displayed no significant change in their rheol. properties; the earth-alk.-boric frit suspensions were more markedly affected.

2000 - Optimization of fritting parameters of ceramic frits: effect on the rheological behaviour of the glaze suspensions [Articolo su rivista]
Andreola, Nora Maria; Castellini, Elena; Romagnoli, Marcello

Studies to optimize the preparation parameters used in industrial frits are reported. Two frit compositions widely used for the production of single-fired ceramic-glazes were chosen: an alkaline boric and an earth-alkaline boric composition. The chemical durability and rheological behavior of the frit suspensions were studied in relation to changes in the fritting temperature and time. The results confirm that the two frits studied react differently to changes in fritting parameters due to the dependence on their chemical durability and composition. Suspensions prepared with the alkaline-boric frit displayed no significant change in their rheological properties; the earth-alkaline-boric frit suspensions were more markedly affected.

1999 - Rheology of ceramics slurries with mixtures of deflocculanting agents. [Relazione in Atti di Convegno]
Andreola, Nora Maria; Castellini, Elena; Romagnoli, Marcello

In whiteware processing, as in most other technical processes involving concentrated suspensions, additives are used to decrease viscosity and control stability. Silicate, phosphate and polyacrylate sodium salts are the most used additives in the production of tiles, tablewares, sanitarywares etc. They are commercially available products with good deflocculant abilities and performance/costs ratio. The chemical action of each single component was studied1,2,3 but the authors are not aware of any studies on mixtures. The systems are complex because they are multi-phases and constituted by natural raw material. In the industrial field, liquid mixture of additives are generally used. They take an inferior amount of time before being active with respect to when they are used as solid salts and permit a better management in the plant.