Repositorio Institucional de Documentos

Master Thesis FABRICATION AND CHARACTERIZATION OF NANOPARTICLES/PMMA ELECTROSPUN NANOFIBER MEMBRANES Erasmus Mundus Master in Membrane Engineering Magdalena Malankowska Supervisor: Silvia Irusta Zaragoza, 18.06.2013 The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 2 Abstract Currently, one of the most serious problems is to maintain the water balance in terms of quality and quantity. In recent times, membrane technology is considered to be essential as it ensures high water quality with low cost and maintenance of sustainable water resources. In this work different membranes were fabricated by electrospinning technique. PMMA (polymethyl methacrylate) was used as a base polymer material and the silver nanoparticles, silver nanowires or titanium dioxide were incorporated into the matrix. The aim was to produce polymeric membranes containing nanoparticles, fabricated by electrospinning in order to obtain the highest bactericidal effect for water treatment. The bactericidal effect was observed for three kinds of membranes however, the best results were obtained by the silver nanoparticles membrane with the in situ manufacturing method. The distribution of the nanoparticles in this membrane resulted more uniform, the size of the nanoparticles was smaller and the texture was the most mechanically resistant. The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 3 Acknowledgments: I would like to thank the EM3E master and Reyes Mallada as a coordinator for the opportunity of developing my scientific skills and participating in the program, University of Zaragoza, Jesus Santamaria and all the INA group for the possibility of developing the project, supplying all of the required devices, Silvia Irusta for supervising me and giving essential advices and Ivan Moreno for help with the equipments and scientific support. The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 4 EN: The EM3E education programme has been funded with support from the European Commission. This publication reflects the views only of the author, and the Commission cannot be held responsible for any use which may be made of the information contained therein. Translation of this phrase in all EU languages. The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 5 List of tables: Table 1. CFU/100mL of water USEPA recommendations for E. coli Table 2. Advantages and disadvantages of different membranes. (Where, CA: cellulose acetate membrane, PES: polyethersulfone membrane, PVDF: polyvinylidene fluoride membrane, PE: phosphatidylethanolamine membrane and PP: polypropylene membrane.) Table 3. General comparison of different membrane processes Table 4. Final amounts of all the constituents of AgNW synthesis Table 5. Final amounts of all the constituents of AgNW synthesis Table 6. Final amounts of all the constituents of AgNPs in situ synthesis Table 7. Final amounts of all the constituents of TiO 2 synthesis Table 8. Concentration values of different nanoparticles Table 9. Zeta potential values for NPs at their original pH Table 10. Colony forming units values of the three suspensions examined against E.coli and S. aureus Table 11. Measurement parameters for different concentrations of PMMA fibers; *pump diameter for 10ml Norm Ject syringe Table 12. Contact angle values for electrospun membranes Table 13. List of chemicals used in the AgNP, AgNW, TiO 2 membranes synthesis with prices and amounts Table 14. Total price of AgNW + PMMA membrane production. Amounts of all the reagents required for the synthesis were taken into account. The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 6 List of figures: Figure 1. Various antimicrobial mechanisms of nanomaterials Figure 2. The crystalline structure of titanium dioxide anatase Figure 3. Agar well diffusion method Figure 4. Dilution bacteria test method Figure 5. Cellulose Acetate Membrane (Type ST 68, 0.8 µm) Figure 6. CHMLAB GROUP and Prat Dumas cellulose acetate filter papers Figure 7. Diagram showing fibre formation by electrospinning Figure 8. Electrospinning/electrospraying schematic with variations for different processing outcomes Figure 9. Microwave apparatus, CEM Discover, Institute of Nanomaterials Aragon, Zaragoza, Spain Figure 10. Representative scheme of the bactericidal test for suspensions Figure 11. Representative scheme of the pipetting method for Petri plates Figure 12. Ultraviolet lamp VL-4.LC, 365 and 254 nm tubes Figure 13. Ultraviolet lamp VL-4.LC, 365 and 254 nm tubes Figure 14. Flow nanotechnology solutions, Electrospinner 2.2.D-500, INA, Zaragoza, Spain Figure 15. Flow nanotechnology solutions, Electrospinner 2.2.D-500, INA, Zaragoza, Spain Figure 16. Representative scheme of the solids bacteria test Figure 17. Titanium dioxide membrane bacteria test under UV illumination Figure 18. SEM image of Silver nanowires Figure 19. TEM image of Silver nanowires Figure 20. Particle size distribution of Ag nanowires Figure 21. TEM image of silver nanospheres Figure 22. TEM image of silver nanospheres Figure 23. Particle size distribution of Ag nanoparticles Figure 24. TEM image of TiO 2 The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 7 Figure 25. TEM image of TiO 2 Figure 26. Particle size distribution of TiO 2 Figure 27. UV-Vis absorption spectrum of silver nanowires Figure 28. UV-Vis absorption spectrum of silver nanoparticles Figure 29. UV-Vis absorption spectrum of titanium dioxide Figure 30. Linear representation of nanoparticles suspensions used for producing a composite membranes Figure 31. Zeta potential figure representing values for stable and unstable solutions Figure 32. Raman spectroscopy for AgNPs Figure 33. Raman spectroscopy for TiO 2 Figure 34. Bacteria test of AgNWs and AgNPs against Staphylococcus aureus Figure 35. Bacteria test of AgNWs and AgNPs against E.coli Figure 36. Bacteria test of titanium dioxide suspension against S. aureus Figure 37. Bacteria test of titanium dioxide suspension against E.coli Figure 38. SEM image of 16%wt PMMA fibers Figure 39. SEM image of 18%wt PMMA fibers Figure 40. SEM image of 20%wt PMMA fibers Figure 41. Fiber size distribution of 16% wt ofPMMA fibers Figure 42. Fiber size distribution of 18% wt ofPMMA fibers Figure 43. Fiber size distribution of 20% wt ofPMMA fibers Figure 44. SEM images of silver nanowires incorporated into PMMA fibers Figure 45. SEM images of silver nanowires incorporated into PMMA fibers Figure 46. Fiber size distribution of PMMA fibers with AgNWs Figure 47. Particle size distribution of AgNWs incorporated into PMMA fibers Figure 48. SEM images of silver nanoparticles incorporated into PMMA fibers Figure 49. SEM images of silver nanoparticles incorporated into PMMA fibers Figure 50. Fiber size distribution of PMMA fibers with AgNPs The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 8 Figure 51. Particle size distribution of AgNPs incorporated into PMMA fibers Figure 52. SEM images of titanium dioxide incorporated into PMMA fibers Figure 53. SEM images of titanium dioxide incorporated into PMMA fibers Figure 54. Fiber size distribution of PMMA fibers with TiO 2 Figure 55. Particle size distribution of TiO 2 incorporated into PMMA fibers Figure 56. UV-Vis absorption spectrum of AgNWs/PMMA membrane Figure 57. UV-Vis absorption spectrum of AgNPs/PMMA membrane Figure 58. UV-Vis absorption spectrum of TiO 2 /PMMA membrane Figure 59. Percentage loss of a AgNP + PMMA membrane Figure 60. Percentage loss of a AgNW + PMMA membrane Figure 61. Percentage loss of a TiO 2 + PMMA membrane Figure 62. Raman spectroscopy for silver nanoparticles incorporated into PMMA fibers Figures 63. Raman spectroscopy for TiO 2 incorporated into PMMA fibers Figure 64. Bacteria test of silver nanoparticles membrane against S. aureus Figure 65. Visual representation of a AgNWs + PMMA electrospun membrane and setup filtration with the membrane inside the filter holder Figure 66. Visual representation of a AgNWs + PMMA electrospun membrane and setup filtration with the membrane inside the filter holder Figure 67. Graphic representation of flux of AgNW+PMMA membrane with the maximum flux equal to 3,74 [ml/sec] Figure 68. Graphic representation of flux of AgNP+PMMA membrane with the maximum flux equal to 10 [ml/sec] Figure 69. Graphic representation of flux of TiO 2 +PMMA membrane with the maximum flux equal to 10 [ml/sec] Figure 70. Graphic representation of flux of commercial chmlab group membrane with the maximum flux equal to 1,88 [ml/sec] Figure 71. UV-VIS absorption spectrum of the membrane in the water solution Figure 72. An example of a contact angles measurements of water drop on a lotus leaf The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 9 Tableofcontent: List of tables……………………………………………………………………………5 List of figures…………………………………………………………………………..6 1. Introduction…………………………………………………………………………...11 1.1. Silver nanoparticles…………………………………………………….…..…….12 1.2. TiO 2 nanoparticles……………………………………………………………..…13 1.3. Nanoparticles synthesis-literature review………………………………………..14 1.4. Bacteria…………………………………………………………………………..16 1.5. Commercial membranes comparison…………………………………………….19 1.5.1. Cellulose acetate membrane………………………………………………20 1.6. Electrospinning…………………………………………………………………..21 2. Experimental section………………………………………………………………….23 2.1. Nanoparticles synthesis…………………………………………………………..23 2.1.1. Silver nanowires (AgNWs) synthesis…………………………………….23 2.1.2. Silver nanoparticles (AgNPs) synthesis…………………………………..24 2.1.2.1. In situ silver nanoparticles synthesis…………………………………25 2.1.3. Titanium dioxide (TiO 2 ) nanoparticles synthesis…………………………25 2.2. Bacteria test for suspensions……………………………………………………..26 2.3. Electrospinning.………………………………………………………………….28 2.3.1. Control sample preparation……………………………………………….28 2.3.2. AgNWs/PMMA solution preparation…………………………………….30 2.3.3. AgNPs/PMMA solution preparation……………………………………..31 2.3.4. TiO 2 /PMMA solution preparation………………………………………..32 2.4. Membrane bacteria test…………………………………………………………..32 3. Results and discussion………………………………………………………………..33 3.1. Characterization of nanoparticles………………………………………………..34 3.1.1. Microscopy and diameter distribution examination………………………35 3.1.2. UV-visible spectroscopy………………………………………………….37 3.1.3. Concentration measurement by microbalance……………………………38 The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 16 charge (M 0 ) is generated either by the direct reduction of the metal source, salt or another complex, or by the reduction of metal ions by generated intermediates, such as radicals [15]. This method was used in the electrospun membrane fabrication due to the overcoming of the common problems connected with agglomeration of AgNPs, high viscosity of PMMA which made it difficult to disperse NPs in the uniform way. In the following experiment metal nanoparticles were reduced inside the polymer matrix. N,N-dimethyl formamide (DMF) was chosen as a reducing agent. The polymerization of MMA monomers and reduction of silver ions occurred simultaneously which lead to the formation of Ag/PMMA nanocomposites [16]. TiO 2 might be synthesized by variety of different methods such as: chloride process, sulfate process, coprecipitation, impregnation, hydrothermal method, sol-gel method etc. Sol-gel method became one of the most suitable way for synthesizing variety of different metal oxides due to the low processing temperatures, low cost and ease of fabrication. This process involves the transformation from a liquid state “sol” into a solid phase “gel”. The homogeneity of the solid phase depends on a couple of factors such as: the solubility of reagents in the solvent, the temperature, pH and the sequence of addition of reactants [11]. In the case of anatase titanium dioxide photocatalytic process, which was described in the previous sections, switching to more ecological friendly and sustainable methods and technologies, became of high importance in recent years. However, the main limitation of this photocatalyst is that under the visible light condition it does no longer have photocatalytic activity [17]. In order to avoid this problem the solution might be doping titanium by Nd (Neodymium) or other element with absorption in the visible range. Materials of Nd-doped titania which are prepared by mild microwave-assisted protocol possess excellent activities in the photodegradation of water impurity [17]. 1.4. Bacteria The main application of the fabricated membrane will be purifying fresh or drinking water from bacteria, mainly Staphylococcus aureus and Escherichia coli. Bacteria are a large group of prokaryotic microorganisms, usually with few micrometers length. The main bacteria division is connected with retaining the crystal violet dye in the Gram staining protocol. Gram positive bacteria retain crystal violet dye while the Gram negative do not [18]. Staphylococcus The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 17 aureus is the example of Gram positive bacteria and might be found in the soil and other environmental areas and can simply contaminate the wound. It can also be taken into account that S. aureus in drinking water may be a source for colonizing residents exposed to contaminated water [19]. While the Escherichia coli is the example of Gram negative bacteria and can be found in the gastrointestinal system of the human body [10]. There are many sources of E. coli contamination, mainly intestinal tracts of human and other warm-blooded animals such as livestock and wildlife. If the concentration of bacteria in a fresh water is higher than the standard numbers there is a high risk of being affected with diarrhea, fewer, chest pain or hepatitis. According to USEPA (United States Environmental Protection Agency) recommendations, some set of agreements connected with the E. coli CFU must be obeyed in the fresh water reservoirs [20]. Designated swimming Moderate swimming area Light swimming area Infrequent swimming area E. coli (CFU/100mL of water) 235 298 410 576 Table. 1 CFU/100mL of water USEPA recommendations for E. coli [20] E. coli is a major drinking water indicator proving that water is contaminated by for example human or animal wastes. According to European Union’s drinking water standards with the Council Directive 98/83/EC on the quality of water intended for human consumption the amount of E. coli in the 250 mL of water should be equal to 0 [21]. Antibacterial effect of water filtering membranes against Gram negative and Gram positive bacteria became of high importance. In order to prove the bactericidal properties of nanoparticles, various bactericidal tests might be taken into account. The most popular method for determination the antibacterial activity of various suspensions is agar well diffusion method. The zone of inhibition is examined, which should appear as a clear area around the wells [2]. It was reported that the greater the zone of inhibition the greater the bactericidal effect of the suspension. Moreover, silver nanoparticles have higher influence on Gram-negative than on Gram-positive bacteria. During the treatment by silver ions, DNA loses its replication ability, expression of ribosomal subunit proteins and some other cellular The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 18 proteins and enzymes which are crucial to the production of ATP (Adenosine triphosphate) [2]. The inhibition of bacterial growth is dependent on the concentration of AgNPs in the medium [2]. Generally, larger zones are connected with the smaller Minimum Inhibitory concentration (MIC) of antibiotic for that bacterium. MIC is the lowest concentration of an antimicrobial that will stop the growth of bacteria which is visible for a naked eye after 24 hours incubation [18]. Another important bacteria test parameter is Colony-forming unit (CFU) which is an estimate number of viable bacteria or fungi. The units of this number are CFU/ml (colony-forming units per milliliter) or CFU/g (colony forming units per gram) in the case of solids [18]. Fig. 3 Agar well diffusion method [22-23] Fig. 4. Dilution bacteria test method However, more important, accurate and reliable method is dilution bacteria test. The procedure consists of cultivating the bacteria and keeping them in the oven for 24 hours and next, serial dilutions of the suspension starting from 10 8 CFU/ml are prepared. The last step is transferring the solution into Petri plates filled with Agar solution and incubating for 24 hours in the oven once more. The results are examined by visual counting the grown of colony on a Petri plate [24]. The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 19 1.5. Commercial membranes comparison Nowadays, population explosion and industrial activities became responsible for large water resources consumption and water pollution. As a consequence, one of the most serious problem and issue is to sustain water balance, in terms of quantity and quality. Membrane technology is recently regarded as essential since it ensures plenty of high grade water with low cost and the maintenance of sustainable water resources [25]. The figure below presents advantages and disadvantages of different membranes according to the material they are made of. Table 3 presents major membrane module designs. It is clearly visible that some characteristic features of membrane, like cost, resistance to fouling, packing density etc., highly depends on the membrane module. In the designed test the experimentally obtained electrospun membranes were compared to the commercial cellulose acetate membranes in terms of and mechanical resistance. Table 2. Advantages and disadvantages of different membranes. (Where, CA: cellulose acetate membrane, PES: polyethersulfone membrane, PVDF: polyvinylidene fluoride membrane, PE: phosphatidylethanolamine membrane and PP: polypropylene membrane.) [25] The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 20 Table 3. General comparison of different membrane processes [26] 1.5.1. Cellulose acetate membrane Among polymer membranes which can be used for separation, cellulose acetate (CA) was one of the foremost. CA, as hydrophilic membrane, possesses a good fouling resistance, its price is low, it has moderate chlorine resistance and good biocompatibility, but at the same time its disadvantages are: poor mechanical strength, low oxidation, thermal and chemical resistances. Another obstacle of using CA membranes is crucial for applications in chemical and pharmaceutical industries when organic solvents are part of the feed or in process operating at temperatures higher than 50 o C and pH lower than 3 or higher than 7 [27]. Figure 5 presents CA membrane of type ST 68. Fig. 5.Cellulose Acetate Membrane (Type ST 68, 0.8 µm) [27] The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 21 Two commercial filters were examined: • Cellulose acetate filter by CHMLAB GROUP These filters with 150 nm diameter possess excellent properties for quantitative and qualitative analysis. They are free of possible residual acids which might be used in some production methods. What is more, they contain extremely low amount of ash [28] • Cellulose acetate filter by Prat Dumas, France Prat Dumas filters with the diameter of 0,2 µm, possess an extremely high thermal stability. For this reason they can be autoclaved which is a great property in the bacteria environment. Perfect for biological and clinical analysis [29] Fig. 6. CHMLAB GROUP and Prat Dumas cellulose acetate filter papers [28-29] 1.6. Electrospinning Electrospinning process is a simple and low cost technology for producing fibers in the range of few microns by using an electrical charge. In this method the sufficiently high voltage must be applied to a droplet of a liquid, an electrostatic repulsion counteracts the surface tension and the droplet is stretched due to the charge process of the body liquid. At a critical point a stream of liquid explode from the surface. It is called Taylor cone [18]. If the molar cohesion of the liquid is high enough, there is no occurrence of a stream breakup and a formation of charged liquid jet is observed [18]. The current flow alteration from Ohmic to convective mode occurs due to the jet drying in flow. The next step is elongation of the jet by whipping process caused by electrostatic repulsion, until the final deposition on the grounded The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 22 collector. The elongation and thinning of the fiber is the result of bending instability which leads to the production of uniform fibers with nanometer-scale diameters [18]. Fig. 7.Diagram showing fibre formation by electrospinning [18].Fig. 8. Electrospinning/electrospraying schematic with variations for different processing outcomes [18]. The typical laboratory electrospinning apparatus consists of a spinneret, which is typically a hypodermic syringe needle, connected to a high voltage (5-50kV), syringe pump, direct current power supply and a grounded collector. A solution is placed in the syringe and the liquid is extruded from the needle tip by a syringe pump at a constant rate. The electrospun non-woven mats provide extremely high porosity and surface area-to-volume ratio. They are light and mechanically flexible which emphasizes their attractive properties for potential applications, for example in biomedical engineering or as filter membranes. Electrospun mats could exhibit higher bactericidal effect than conventional microfibers due to their high surface area-to-volume ratio mentioned above [4]. 2. Experimental section The experimental measurements were taken in laboratories of the Institute of Nanoscience, Aragon (INA) and in the Centro de Investigacion Biomedica de Aragon (CIBA) in Zaragoza, Spain. The first part of the project was synthesizing and characterizing silver and titanium dioxide nanoparticles, testing the bactericidal effect of the suspensions and afterwards the composite membranes were fabricated by electrospinning process made of The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 23 Poly(methyl methacrylate) and already synthesized nanoparticles. The last step was verifying the bactericidal effect of created membranes and compare their mechanical resistance with commercial membrane filters for the water treatment. 2.1. Nanoparticles synthesis Three synthesizing methods were used to produce three different nanoparticles: silver nanowires, silver nanospheres and titanium oxide. Most of the chemicals were purchased from Sigma-Aldrich Co. Spruce Street, St. Louis, USA and PRS Panreac Quimica Sau, Castellar del Valles, Barcelona, Spain. 2.1.1. Silver nanowires (AgNWs) synthesis Silver nanowires have been used in a broad variety of applications such as: photonic crystals, optical polarizers, microelectronics, catalysts and SERS (Surface Enhanced Raman Scattering). Particularly, great effort was made to obtain nanowires with controllable size [30]. The solvothermal method, developed by the NFP (Nanoporous films and particles) group [30] was used for fabrication of silver nanowires by reducing silver nitrate (AgNO 3 , 99.9% ),with ethylene glycol (EG, 99.8%), and using Polyvinyl pyrrolidone (PVP, molecular weight = 55000), as an adsorption agent with all of the chemicals purchased from Sigma Aldrich. Two solutions have been prepared in 50 ml beakers: 1. 10 mL of EG and 169,87 mg of AgNO 3 (0,1 M) 2. 10 mL of EG and 166,71 mg of PVP (0,15 M) The first solution was mixed using a magnetic stirrer and the second was added slowly to the first one drop by drop. The second solution was added only after the first one was mixed correctly. The final mixture was placed in the autoclave and closed in the oven for 2,5h in 160 o C. Afterwards, the suspension was divided into two plastic centrifugation tubes and thoroughly washed with acetone (10 mL of a suspension and 30 mL of acetone). Tubes were placed in the centrifugation machine (Jouan B4 number 1995/3777a) at 3000 rpm (revolutions The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 24 per minute) for 20 min. The procedure was repeated 3 times. After each centrifugation supernatant was removed and replaced again by acetone [31]. The solutions were placed in the ultrasonic bath for about 5 minutes in order to avoid agglomeration of particles after each centrifugation during all of the nanoparticles synthesis. 2.1.2. Silver nanoparticles (AgNPs) synthesis The synthesis of silver nanospheres was carried out also by a polyol method. It is commonly used for preparation of metals which are easily reducible. The polyols, such as: diethylene or ethylene glycol are able to act as a reducing agent but also as a solvent in which the metal salts might be dissolved or suspended. The capping agents are used to protect nanoparticles from sintering with each other and from forming large particles. PVP is one of the commonly used capping agent [6]. Most of the chemical reducing reactions require elevated temperatures to increase the reaction rate. The energy which is used to heat up the media can be laser irradiation, conventional thermal heating, ultrasonic, UV irradiation etc. Microwave radiation however, has the fastest heating rate and is used to synthesize platinum and silver nanoparticles. It heats up the material through its dielectric loss, and then it converts the radiation energy into a thermal one [6]. The method used for preparation of silver nanospheres was chemical reduction of silver salt in the presence of a stabilizing agent and MW heating [32]. Fig. 9 Microwave apparatus, CEM Discover, Institute of Nanomaterials Aragon, Zaragoza, Spain 2,4 g of PVP (Mw = 10000) was added slowly to 20 mL of EG and mixed by magnetic stirrer for 2h in the 50 mL beaker. The consecutive step was adding 0,158 g of AgNO 3 and mixing for exactly 20 min. The chemicals in this synthesis were purchased from Sigma Aldrich. The solution was placed in the microwave (CEM Discover) for 16 sec in 200 o C with The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 25 SintNPsAg16s program which produces the smallest nanoparticles in terms of diameter. After cooling the solution down, the tube with the suspension was placed in the centrifugation machine (Beckman Coulter Allegra 64R) for 1h and with the speed of 21000 rpm. The same procedure was repeated 6 times, each time supernatant was taken out only from the top and then filled with water due to the slow precipitation of silver nanospheres [33]. 2.1.2.1. In situ silver nanoparticles synthesis Due to the difficulties connected with obtaining high concentration of AgNPs another synthesis method was applied. In order to fabricate a silver nanoparticles – polymeric membrane by electrospin a particular concentration was required due to the limitations connected with the size of the syringe used by the device. Taking these limitations into account and after many attempts with the solution of silver nanoparticles and different solvents, it was decided that in the case of AgNPs, “in situ” synthesis will be applied. As it was described above, in situ method possess only one step. Silver nitrate (Sigma Aldrich) was added directly to the polymer solution of Poly (methyl) methacrylate and its solvents and mixed for around 12 hours. DMF as one of the PMMA’s solvent worked as a reducing agent. The amount of AgNO 3 added was calculated and it was taken into account that the amount of pure silver in the silver nitrate mixture is lower than in the AgNPs solution. What is more, 2%wt of silver was chosen to be in the solution due to its best solubility. 2.1.3. Titanium dioxide (TiO 2 ) nanoparticles synthesis TiO 2 nanoparticles were synthesized by a sol-gel method. 2 mL of Titanium Isopropoxide (TIPO - Sigma Aldrich, 97%) was added drop-wise to 30 mL of absolute ethanol (PRS Panreac) in the 50 mL beaker. The solution was mixed by a magnetic stirrer and 3 ml of acetic acid (HAC – Sigma Aldrich, 99-100%) was added. The following step was addition of 5 ml of deionized water and stirring for 5 min again. The solution was placed in the autoclave that was sealed and heated up in the microwave oven ( Milestone – microwave laboratory systems, Ethos Plus, High performance microwave labstation) up to 120 o C (measured by IR) for 15 min. The next step was isolation of TiO 2 by centrifugation (10 min at 9000 rpm), thoroughly The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 32 However, AgNO 3 is not pure silver which means that some recalculation had to be done: M AgNO3 - 169,87 [g/mol] M Ag – 107,87 [g/mol] m = 87,12 ∙  , ,  m = 137 [mg] of AgNO 3 had to be added Substance Amount PMMA [g] 4,269 AgNO 3 [mg] 137 Acetone [ml] 12 DMF [ml] 8 Table 6. Final amounts of all the constituents of AgNPs in situ synthesis 2.3.4. TiO 2 /PMMA solution preparation The last fabricated film was the titanium dioxide membrane. Solution with 7,8 mg/mL concentration was treated in the same way as the previous ones. TiO 2 solution exhibited one of the best dispersion in the polymeric fibers which was visible in the SEM characterization technique. The amounts of all the constituents added to the mixture are presented in table 7. Substance Amount PMMA [g] 6,4 TiO 2 [ml] 8,32 Acetone [ml] 9,67 DMF [ml] 12 Table 7. Final amounts of all the constituents of TiO 2 synthesis 2.4. Membrane bacteria test A bacteria colony had to be suspended in TSB solution and incubated for 24 h at 37 o C in order to prepare the bacteria test for solids. 1 ml of the suspended bacteria was transferred to the 1 L Agar solution, which was autoclaved before and which had exactly 37 o C. The proper temperature was crucial due to the Agar solution and bacteria properties. Above 37 o C bacteria would be killed and below this temperature Agar solution would start to solidify. After stirring the Agar and bacteria mixture, 1 ml of the solution was placed on the membrane by the micropipete, as it is shown in the Figure 16. Small (3cm x 3cm) piece of membrane was placed in a plastic Petri plate which was situated inside the big glass Petri plate filled with The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 33 water. The water was required in order to sustain the aqueous environment for bacteria. Petri plates together with the membrane and bacteria were inserted into the oven for 24 h at 37 o C. The last step was transferring the membrane into the 50 mL beaker filled with 10 mL of TSB, mixing it and sonicating for 1 min. Then, the standard dilution and bacteria test was prepared [34]. Fig. 16 Representative scheme of the solids bacteria test In order to prepare titanium dioxide bacteria test for solids different procedure must be taken into account. The different part was connected with the treatment of the Petri plates with the membrane. The membrane was illuminated with the UV lamp for 5 h with 365 nm wavelength at room temperature instead of placing it into the oven. This particular treatment is connected with the TiO 2 different properties under ultraviolet light. Fig. 17 Titanium dioxide membrane bacteria test under UV illumination 3. Results and discussion The characterization was divided into two parts: characterization of nanoparticles suspensions and characterization of composite membranes obtained by electrospinning technique. High amount of trials was done in case of nanoparticles suspensions and The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 34 electrospun membranes. The best results have been chosen and presented below. What is more, the biggest problem was the choice of a proper solvent because PMMA solvents are acetone and DMF as it was mentioned above. The idea was to use one of these solvents in the nanoparticle synthesis. 3.1. Characterization of nanoparticles Silver and titanium dioxide nanoparticles were synthesized by methods described in the previous sections. The silver nanowires solution became brownish/grey, silver nanoparticles turned to pale brown and titanium dioxide solution was white. 3.1.1. Microscopy and diameter distribution examination Microscopy is one of the best technique for characterization of nanoparticles in terms of morphology. Transmission electron Microscopy (FEI TECNAI T20) and Scanning Electron Microscopy (INSPECT S) were used in order to characterize obtained suspensions (TEM) and membranes (SEM). TEM sample preparation did not required special treatment, diluted suspension was poured on a grid and dried, while in the case of SEM the sample needed to be conductive, hence the drop of a solution was placed on a carbon tape. What is more, the microscopy examination was combined together with the particle size histograms. The diameter distribution histograms were obtained in the computer software IMAQ Vision Builder from at least 50 measurements of different TEM images. Figures 18-26 represent TEM and SEM (in the case of nanowires) and diameter size distribution histograms images of silver nanowires, silver nanoparticles and titanium dioxide respectively. Silver nanoparticle suspensions which finally were replaced by in situ method in the membrane fabrication are presented in the results below as well in order to prove the reproducibility and to show the working mechanism of the method. Silver nanowires presented in the figures below show satisfactory particle size distribution. One of the images is a SEM photo which proves that AgNWs are nanoparticles with the biggest size. The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 35 Fig. 18. SEM image of Silver nanowires Fig. 19. TEM image of silver nanowires 100 200 300 400 0,0 0,2 0,4 Relative Frequency mean diam eter [nm] Frequency Counts of A Fig. 20. Particle size distribution of Ag nanowires The silver nanowires histogram shows that the wire diameter distribution range from 63,3 to 439 nm with the mean diameter equal to 148 nm. It is very difficult to obtain uniform distribution in case of AgNWs due to their agglomeration even after sonication of the suspension used for sample preparation. Silver nanoparticles presented in the figures below exhibit one of the smallest particles size and better diameter distribution in comparison to the silver nanowires. Fig. 21. TEM image of silver nanospheres Fig. 22. TEM image of silver nanospheres The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 36 6 12 18 0,0 0,1 0,2 0,3 0,4 Relative Frequency mean diam eter [nm] Frequency Counts of A Fig. 23. Particle size distribution of Ag nanoparticles The silver nanoparticles histogram exhibits the mean particles size between 4,06 and 19 nm with the mean diameter equal to 13, 9 nm. Titanium dioxide anatase nanoparticles exhibit a tendency of agglomeration, however their particles size distribution is good and the size is also small. Nevertheless, it was a big challenge to keep TiO 2 nanoparticles not agglomerated with each other due to the high concentration of the suspension with acetone which caused the particles agglomerate at the bottom of the sample. Fig. 24. TEM image of TiO 2 Fig. 25. TEM image of TiO 2 3 4 5 6 7 8 9 10 11 12 13 0,0 0,1 0,2 0,3 Relative Frequency Mean diameter [nm] Frequency Counts of A Fig. 26. Particle size distribution of TiO 2 The TiO 2 particle size histogram shows the particles range between 3,56 and 12,4 nm with the mean diameter 6,8 nm. The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 37 From the histograms above it is clear that the biggest nanoparticles are silver nanowires and the smallest titanium dioxide nanoparticles. 3.1.2. UV-visible spectroscopy UV-visible spectroscopy is a commonly used technique for structural characterization of nanoparticles. The measurements were carried out in Jasco V-670 Spectrophotometer in the Institute of Nanoscience of Aragon in Zaragoza, Spain. The range of the measurement was between 800 and 300 nm. Three suspensions were examined: silver nanowires, silver nanoparticles and titanium dioxide respectively. The results are presented in the figures below. 400 600 800 0,3 0,4 0,5 0,6 B absorbance wavelength [nm] The absorption spectrum of silver nanowires presented in the Figure 27 shows a surface Plasmon absorption band with the value of 429 nm demonstrating the presence of silver nanowires. The colour of the solution was brownish/grey with a high turbidity which had an influence on the sharpness of the peak [36]. Fig. 27 UV-Vis absorption spectrum of silver nanowires 400 600 800 0,0 0,1 0,2 0,3 B absorbance wavelength [nm] The absorption spectrum of silver nanoparticles presented in the Figure 28 is equal to 424,5 nm and the peak is sharper than in the case of nanowires. It is connected with the pale brown colour and no turbidity present in the solution [36]. Fig. 28 UV-Vis absorption spectrum of silver nanoparticles The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 38 400 600 800 0,0 0,4 0,8 1,2 B absorbance wavelength [nm] Fig. 29 UV-Vis absorption spectrum of titanium dioxide The last figure shows the absorption spectrum of TiO 2 nanoparticles in the form of anatase. The surface Plasmon absorption band exhibits its maximum at 363 nm which indicates titanium dioxide particles [37]. What is more, the reason of the peak getting sharper is the decrease of the size of nanoparticles which was indicated by the particle size distribution in the previous section [37]. All of the presented above spectrums indicated the presence of AgNWs, AgNPs and TiO 2 nanoparticles respectively. 3.1.3. Concentration measurement by microbalance The measurements were carried out using RAWDAG, Wagi Elektroniczne, MYA 5/2Y, Poland microbalance in order to check the concentration of the solutions which was a crucial characterization technique for the usage by electrospinning. The small aluminum vessels were used in order to weight the solution in the microbalance. First, empty vessels were measured, and after they were filled with the solution and placed into the oven in order to evaporate the solvent. The vessels were weighted once more and the difference was used to plot the graph: mass versus volume. The resulting graph was a linear representation of the amount of the nanoparticles in the suspension. The linear function with the formula: y = ax + b demonstrates the values of intersection (y) and the slope (a) of the curve. The value of the concentration is equal to 1/a. The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 39 Fig. 30 Linear representation of nanoparticles suspensions used for producing a composite membranes The graph above represents the silver nanowires (blue colour), silver nanoparticles (purple colour) and titanium dioxide (green colour) linear representations. The values of concentrations are presented in the table below. Many attempts were done in order to check concentrations of many solutions. Only three of them are presented in this section. Nanoparticles solution Concentration [mg/ml] AgNWs 4,78 AgNPs 1,02 TiO 2 7,8 Table 8. Concentration values of different nanoparticles 3.1.4. Zeta potential Zeta potential measurements were important in terms of the stability of the solution. The measurement was taken by PALSPhase Analysis Light Scattering, which is an extension of laser, Electrophoretic Light Scattering (ELS). ELS is used to the measurement of the velocity of moving particles that scatter laser light, to the measurement of electrophoretic mobility (EPM) and the calculation of zeta potential. y = 0,2098x + 0,0173 R² = 0,9994 y = 0,9724x + 0,0027 R² = 0,9433 y = 0,1281x + 0,0002 R² = 0,8701 0 0,01 0,02 0,03 0,04 0,05 0,06 0,07 0,08 0,09 0 0,1 0,2 0,3 0,4 vol [ml] mass [mg] The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 40 Fig. 31 Zeta potential figure representing values for stable and unstable solutions [38]. According to the literature, solutions with high negative or high positive values of zeta potential are stable, while those with low values, close to zero are not stable. Particles are in the constant movement (Brownian motion), if they exhibit high zeta potential values it means that they have big charges and they repulse from each other, which means no attraction occurs and the solution is stable [18]. Zeta potential measurements of three nanoparticle suspensions indicated their stability at their natural pH. Results are presented below: NPs pH Zeta potential AgNW 6,65 -16,51 AgNP 5,94 -14,86 TiO 2 5,17 +21,6 Table 9. Zeta potential values for NPs at their original pH None of the suspension exhibit value of zeta potential close to zero, which means suspensions are stable because the more negative or positive value of zeta potential the highest the stability. The most stable should be titanium dioxide suspension with the highest zeta potential value. But in the electrospinning process titanium dioxide was added to the acetone suspension where the agglomeration tendency increases. 3.1.5. Raman spectroscopy Raman spectroscopy technique takes into consideration inelastic scattering (Raman scattering) of monochromatic light which usually comes from laser in the visible or ultraviolet range. The light of the laser interacts with molecular vibrations which results as an energy of the laser photons being shifted up or down. That particular shift is different for different compounds so it gives information about the vibrational modes in the system [18]. Two samples were carried out in order to examine the spectroscopy of the system: suspensions The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 41 containing silver and titanium dioxide nanoparticles. The suspensions were dried in order to obtain the pure nanoparticles without the solvent and were placed on the optical microscopy glass. 2000 4000 0 8000 intensity Raman shift [cm -1 ] B The Raman spectroscopy figure shows variety of bands: 1596 cm -1 for glassy carbon [39], 1319cm -1 lonsdaleite (hexagonal diamond) [40], 2847 cm -1 for CH 2 stretching [41] coming from carbon contamination, while the most important is silver bonding: 245 cm -1 for Ag-O [42]. Fig. 32. Raman spectroscopy for AgNPs 0 2000 4000 0 10000 20000 intensity Raman shift [cm -1 ] B The Raman spectroscopy figure shows the dominating bands of 639, 502 and 406 cm -1 which are the typical peaks for titanium dioxide anatase [43-44] Fig. 33. Raman spectroscopy for TiO 2 3.1.6. Bacteria testing The bacteria testing measurements were held in CIBA (Centro de Investigacion Biomedica de Aragon). Tests with two kinds of bacteria strains were examined: S. aureus and E. coli. Two different bacteria testing were carried out: suspension bacteria test and bacteria test for solids in order to test the fabricated electrospun membranes. The results are presented in the figures below. The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 48 Fig. 52-53 SEM images of titanium dioxide incorporated into PMMA fibers TiO 2 nanoparticles present good distribution with the most uniform diameter distribution as it is shown in next sections. 1,4 2,1 2,8 0,00 0,07 0,14 0,21 Relative Frequency mean diameter [ µ m] Frequency Counts of A 0,3 0,6 0,9 0,0 0,2 0,4 0,6 Relative Frequency mean diameter [ µ m] Frequency Counts of A Fig. 54. Fiber size distribution of PMMA fibers with TiO 2 Fig. 55. Particle size distribution of TiO 2 incorporated into PMMA fibers The histograms show the fiber size distribution of PMMA in the sample of TiO 2 + PMMA with the mean diameter equal to 2,51 µm and the particle size distribution of TiO 2 in the same sample with the mean diameter equal to 0,59 µm showing the agglomeration of the primary nanoparticles. As it is visible from the results above, the PMMA fibers with the highest diameter are present in the case of TiO 2 + PMMA membrane, and the lowest for AgNW + PMMA membrane. What is more, the most uniform nanoparticles diameter distribution is observed for the AgNP + PMMA in situ membrane due to the excellent distribution characteristic of the method which was described in the previous sections. The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 49 3.2.3. UV-VIS 400 600 0,20 0,22 0,24 B wavelength [nm] B The absorption spectrum of silver nanowires + PMMA membrane presented in the figure shows a surface Plasmon absorption band with the value of 373 nm demonstrating the presence of silver nanowires [36]. Presence of two peaks might be a reason of a nonhomogenous silver nanowires distribution. Fig. 56. UV-Vis absorption spectrum of AgNWs/PMMA membrane 400 600 800 0,2 0,4 absorbance wavelength [nm] B The absorption spectrum of silver nanoparticles + PMMA membrane presented in the figure is equal to 410 nm and the peak is sharper than in the case of nanowires. It is connected with better in situ silver nanoparticles distribution inside polymer fibers than in the case of nanowires [36]. Fig. 57. UV-Vis absorption spectrum of AgNPs/PMMA membrane 400 600 800 0,2 0,4 0,6 absorbance wavelength [nm] B The last figure shows the absorption spectrum of TiO 2 nanoparticles in the form of anatase + PMMA membrane. The surface Plasmon absorption band exhibits its maximum at 288 nm which indicates titanium dioxide nanoparticles [37]. Fig. 58. UV-Vis absorption spectrum of TiO 2 /PMMA membrane The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 50 3.2.4. TGA Thermogravimetric analysis is a thermal analysis method where changes of the chemical and physical properties of an examined material are measured as a function of an increasing temperature. TGA can provide information about physical phenomena, for example decomposition [18]. This method was used in order to find out what was the concentration of the nanoparticles inside the polymeric membrane matrix. Proper heating temperature was required in order to decompose the polymer but not the nanoparticles. The process finished when the whole amount of polymer evaporated and after simple calculations the concentration of the nanoparticles could be estimated. 0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 Loss (%) Temperature ( o C) C 0 50 100 150 200 250 300 350 400 450 0 20 40 60 80 100 loss [%] temperature [ o C] B Fig. 59. Percentage loss of a AgNP + PMMA membrane Fig. 60. Percentage loss of a AgNWs + PMMA membrane 0 100 200 300 400 500 0 20 40 60 80 100 loss [%] temperature [ o C] B Fig. 61. Percentage loss of a TiO 2 + PMMA membrane Calculations of concentration for AgNPs + PMMA membrane were based on the TGA measurements, taking into account initial and final % weight. The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 51 99,97 %- initial percentage of a membrane 0,205 %- final percentage of a membrane x =   ,   ,   ∗ 100% x = 0,205% The same calculation procedure was done for TiO 2 + PMMA and AgNWs + PMMA membranes with result equal to 0,010941% and 0,065% respectively. TGA measurements indicate the highest nanoparticle concentration in the AgNPs + PMMA membrane and the lowest in the titanium dioxide membrane. This is connected with the agglomeration tendency and coagulation at the bottom of the electrospin syringe of silver nanowires and titanium dioxide. 3.2.5. Raman spectroscopy Raman spectroscopy technique was used in order to define the electrospun membrane components. Figures presented below show the dominating bands connected with PMMA as a membrane base. However, there are also representative peaks for silver and titanium respectively. Peak 2948 cm -1 corresponds to (C-H) of α-CH3 and α-CH2, peak 1720 cm -1 is responsible for (C=O) of (C-OO) bonds, peak 1446 cm -1 corresponds to (C-H) of α-CH3 and (C-H) of (O-CH3), 984 cm -1 is connected with the (O-CH3) bonds and 595 cm -1 for (C-COO) and (C-C-O) bonds [45]. Peak 2415 cm -1 is connected with the diamond vibration [46]. 0 2000 0 20 40 60 80 intensity Raman shift [cm -1 ] B Figure 61 shows variety of peaks connected with PMMA. All the carbon related peaks were presented above nevertheless, the crucial ones are those of silver nanoparticles: 239 cm -1 (AgO) and 1057 cm -1 (NO 3 ) [42]. Fig. 62. Raman spectroscopy for silver nanoparticles incorporated into PMMA fibers The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 52 0 2000 0 intensity Raman shift [cm -1 ] B The Raman spectroscopy figure shows the dominating bands connected with poly methyl methacrylate bonds described above. What is more peaks: 370, 476 and 595 cm-1 possess typical values for titanium dioxide anatase nanoparticles [43-44] Fig. 63. Raman spectroscopy for TiO 2 nanoparticles incorporated into PMMA fibers 3.2.6. Bacteria test Bacteria tests of membranes were held in CIBA as in the case of nanoparticles suspensions. Figures below present the results obtained. Fig. 64. Bacteria test of silver nanoparticles membrane against S. aureus Three different membranes were examined: AgNW + PMMA, AgNP + PMMA and TiO 2 + PMMA. PMMA membrane was also examined as a control sample with no killing bacteria process being observed. Moreover, TiO 2 membrane without UV illumination exhibits little bactericidal effect. The AgNP membrane has the highest bactericidal activity due, not only to the higher activity of the filler, but also due to its higher concentration in the fibers. 3.2.7. Mechanical resistance Several measurements were carried out in order to prove the mechanical resistance of a membrane. The experimental setup was placed in the hood as it is demonstrated in the figure below. Distilled water was poured to the glass vessel and transferred through the Pall 1 100 10000 1000000 100000000 1E+10 PMMA AgNW AgNP TiO2 TiO2+UV CFU/ml The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 53 Corporation filter holder by the CHMLAB group filter with the membrane inside. Pressure, which is an external driving force was required in order to increase the flow velocity due to high hydrophobicity of the membranes, discussed in the next section. Fig. 65-66 Visual representation of a AgNWs + PMMA electrospun membrane and setup filtration with the membrane inside the filter holder. Pressure and the time needed to fill 10 ml glass beaker with water were measured hence, the flow rate of a membrane was calculated. . The pressure was increased until the destruction of the membranes. AgNP + PMMA membrane was the only one which withstood until 40 kPa. Moreover, mechanical resistant of chmlab commercial membrane was measured as well and the pressure values were kept constant in order to have comparable results even if the commercial membranes was not destroyed after that pressure. 20 22 24 26 28 30 2,4 2,7 3,0 3,3 3,6 Q [ml/sec] pressure [kPa] A 24 32 40 3 6 9 Q [ml/sec] pressure [kPa] A Fig. 67. Graphic representation of flux of AgNW+PMMA membrane with the maximum flux equal to 3,74 [ml/sec] Fig. 68. Graphic representation of flux of AgNP+PMMA membrane with the maximum flux equal to 10 [ml/sec] The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 54 20 22 24 26 28 30 6 7 8 9 10 Q [ml/sec] pressure [kPa] A 21 24 27 30 1,2 1,5 1,8 Q [ml/sec] pressure [kPa] A Fig. 69. Graphic representation of flux of TiO 2 +PMMA membrane with the maximum flux equal to 10 [ml/sec] Fig. 70. Graphic representation of flux of commercial chmlab group membrane with the maximum flux equal to 1,88 [ml/sec] The value of the flux was measured at the last available pressure, before the membrane destruction. Differences in values might be connected with high hydrophobicity of the membrane, worse particles distribution characteristic inside the polymer fibers that affect the mechanical properties. The highest flow rate was exhibited by AgNP in situ and titanium dioxide membranes probably due to the best and the most uniform particle distribution which could lead to most homogenous porosity. In the case of silver nanowires membrane where the particle distribution was not uniform, there could be a problem with agglomeration in some places so the flux was lower. 3.2.7.1.Nanoparticles stability in the membrane During the water transfer through the membrane there was a probability of eluting nanoparticles from the polymer fibers. That is why ultraviolet visible light spectrophotometer was used in order to ensure that no nanoparticles are present in the permeate after the filtration. Results are presented in the figure below. The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 55 200 300 400 500 600 700 800 -20000 0 20000 40000 Absorbance Wavelength [nm] B Fig. 71 UV-VIS absorption spectrum of the membrane in the water solution Nine samples were prepared, for three kinds of membranes and at three different pressures. All the results were the same, so only one is presented in figure 70. Any characteristic peak for nanoparticles is visible, which indicates the absence of any nanoparticles in the permeate. The same results were obtained by more accurate method called Inductively coupled plasma atomic emission spectroscopy (ICP-AES). This technique is used in order to detect trace metals. It uses inductively coupled plasma to produce excited ions and atoms that emit electromagnetic radiation with the characteristic wavelengths for a particular element. The intensity of this emission indicates the concentration of the element in the sample [18]. 3.2.8. Contact angle The contact angle measurements indicate the hydrophobicity or hydrophilicity of the material. It is an angle where a liquid/vapor interface meets a solid surface. It describes the wettability of a solid surface [18]. Fig. 72 An example of a contact angles measurements of water drop on a lotus leaf [18]. Values of the contact angle directly represent the properties of the material. 0 o -90 o - hydrophilic material 90 o -120 o -hydrophobic material >120 o -super hydrophibic material [18] The measurements were carried out in the Faculty of SCIENCE in the University of Zaragoza, Spain with the optical tensiometer Theta Lite 101 by Attension. The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 56 Four membranes were tested: AgNP + PMMA, AgNW + PMMA, TiO 2 + PMMA and PMMA without nanoparticles with two measurements for each. Results are presented in the table below: AgNP+PMMA 118,47 o AgNW+PMMA 120,05 o TiO 2 +PMMA 124,05 o PMMA 92,88 o Table. 12. Contact angle values for electrospun membranes All three membranes exhibit hydrophobic or super hydrophobic properties. Control sample of PMMA without nanoparticles is hydrophobic as well but possesses lower values of contact angle. The addition of inorganic filler was already known as a method to incresase the hydrophobicity of membranes [48]. 4. Conclusion The silver nanoparticles and silver nanowires were synthesized by the polyol method. The titanium dioxide nanoparticles were synthesized by the sol-gel method with the use of microwaves. Three different kinds of electrospun membranes were fabricated. Variety of characterization methods were used in order to check well distribution of nanoparticles, diameter size, bactericidal effect and mechanical resistance of membranes. The comparison to the commercial membranes were taken into account as well. Results presented in the previous sections prove the AgNP in situ method fabricated membrane being the best in terms of the most uniform nanoparticles distribution, the strongest bactericidal effect and the most mechanically resistant with the highest water flux. Titanium dioxide membrane possesses similar properties however illuminating it by UV lamp each time might be time consuming and complicated from industrial point of view and it is no effective for Gram negative bacteria. What is more, the production of silver nanoparticles in situ membrane is the cheapest among all three membranes. The production of such membranes is fast and simple. All of the three membranes are hydrophobic thanks to the membrane polymeric support and the fabrication technique. Moreover, hydrophobicity is improved by the presence of inorganic fillerssx. Thanks to the hydrophobic properties the possible fouling, swelling or fast damage of a membrane might be avoided. Water passing through the membrane will not stay inside for a long time but will elute very fast thanks to an external driving force. The comparison of the chmlab and prat dumas commercial filters were executed. Results of prat dumas The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities | 57 commercial filter are not presented due to improper surface behavior of the filter in the water flow with an external driving force. The future work of the project would be improving the mechanical resistance of a membrane and finding the convenient and commercial fabrication process. 5. Bibliography: [1] Springer Handbook for Nanotechnology, Bharat Bhushan Edition, USA, 2004 [2] Silver nanoparticles: Antibacterial activity against wound isolates and invitro cytotoxic activity on Human Caucasian colon adenocarcinoma, J. S. Devi, B. V. Bhimba, Asian Pacific Journal of Tropical Disease, Chennai, 2012 [3] Synthesis, characterization, and evaluation of antimicrobial and cytotoxic effect of silver and titanium nanoparticles, F. Martinez-Gutierrez, P. L. Olive, A. Banuelos, E. Orrantia, N. Nino, E. Morales Sanchez, F. Ruiz, H. Bach, Y. Av-Gay, Nanomedicine: Nanotechnology, Biology, and Medicine, 2010 [4] Ag nanoparticle-embedded one-dimenshional β-CD/PVP composite nanofibers prepared via electrospinning for use in antibacterial material, S. Wang, J. Bai, C. Li, Y. Zhang, J. Zhang, China, 2012 [5] Immobilization of silver nanoparticles synthesized using Curcuma longa tuber powder and extract on cotton cloth for bactericidal activity, M. Sathishkumar, K. Sneha, Yeoung-Sang Yun, Bioresource Technology, Korea, 2010 [6] Variable frequency microwave synthesis of silver nanoparticles, Journal of Nanoparticle Research, Hongjin Jiang, Kyoung-sik Moon, Zhuqing Zhang, Suresh Pothukuchi, C.P. Wong, 2005 [7] Biofunctionalized silver nanoparticles: Advances and prospects, Colloids and Surfaces B: Biointerfaces, A.Ravindran, P.Chandran, S.Sudheer Khan, 2012 [8] Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gramnegative bacteria, I. Sondi, B. Salopek-Sondi, Journal of Colloid and Interface Science, Croatia, 2004