This work is an investigation of the effects of the processing parameters (E.G., reaction temperatures and reactant concentrations) on the characteristics of copper-based particles synthesized using chemical reduction technique, a simple chemical method that involves the use of reducing agents to trigger the formation of copper metal.
Journal of Science: Advanced Materials and Devices (2019) 66e71 Contents lists available at ScienceDirect Journal of Science: Advanced Materials and Devices journal homepage: www.elsevier.com/locate/jsamd Original Article Effects of reaction temperatures and reactant concentrations on the antimicrobial characteristics of copper precipitates synthesized using L-ascorbic acid as reducing agent ~ ol a, Nacita B Lantican b, Ana Rose Ramos a, Alvin Karlo G Tapia a, Chrysline Margus N Pin b c Ma Lourdes F del Mundo , Ronniel D Manalo , Marvin U Herrera a, * a b c ~ os College, Laguna, 4031, Philippines Institute of Mathematical Sciences and Physics, University of the Philippines Los Ban ~ os College, Laguna, 4031, Philippines Institute of Biological Sciences, University of the Philippines Los Ban ~ os College, Laguna, 4031, Philippines Department of Forest Products and Paper Science, University of the Philippines Los Ban a r t i c l e i n f o a b s t r a c t Article history: Received 18 October 2018 Received in revised form 23 December 2018 Accepted 29 December 2018 Available online January 2019 Copper-based precipitates were synthesized using a straightforward wet chemical reduction method with copper sulfate salts as precursor and L-ascorbic acid as reducing agent Scanning electron micrographs of the precipitates reveal particle-like structures with edges The percent age yield was observed to increase with the reaction temperature and the reductant-to-precursor concentration ratio Comparison of samples prepared at different times showed that the bulk of the precipitates was formed during the first 24 hours New yield added to the bulk decreased over time Here, the ascorbic acid served as a capping agent, which impeded the oxidation This capping was not visible in the electron micrographs but may be inferred from the infrared spectra Faster capping reduced the amount of oxides in the precipitates This was seen in samples prepared at a higher temperature and with a lower reductant-toprecursor concentration ratio Antimicrobial testing under dynamic contact conditions showed that the copper-based precipitates were more effective against Staphylococcus aureus (a gram-positive bacteria) than Escherichia coli (a gram-negative bacteria) Copper-based precipitates collected after the first 24 hours were more potent than those collected after eight days Furthermore, samples prepared at room temperature and lower reductant-to-precursor concentration ratios were found to be more effective in reducing the number of S aureus after one hour of contact The synthesized copper-based precipitates was observed to reduce the population of S aureus (CFU/mL) by up to 98% after one hour of contact © 2019 The Authors Publishing services by Elsevier B.V on behalf of Vietnam National University, Hanoi This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Keywords: Wet-chemical method Capping Antimicrobial Staphylococcus aureus Escherichia coli Introduction Copper is a popular cheaper alternative to silver Often, it is used as a conductor of heat and electricity Like silver, antimicrobial properties of copper [1e6] are well known Knowledge of its sanitizing ability dates back to Ancient Egypt, when it was used to clean drinking water but it was not until the 19th and early 20th centuries when its applications in the field of medicine became prevalent [5] Antimicrobial properties may be attributed to the ability of charges (e.g., ions, oxidation states, free electrons, radicals) to directly or indirectly disrupt the vital charge transports within the * Corresponding author E-mail address: muherrera@up.edu.ph (M.U Herrera) Peer review under responsibility of Vietnam National University, Hanoi cells Thus, when placed on different materials and surfaces, antimicrobial agents can minimize, inhibit, or stop the spread of infectious diseases Antimicrobial agents may be in the form of liquids, gels, sheets, and powders Unlike liquids and gels, powders not evaporate and, thus, have longer effective lifetime Compared to sheets, powders have the advantage of being easily integrated, mixed, and incorporated in different materials Copper particles or powder may be produced using physical, electrochemical, and chemical means [7e21] Physical methods (e.g., pulse laser ablation or deposition, ball milling method, and pulsed wire discharge method) and electrochemical methods (e.g., chemical reduction) may be faster However, these methods usually require more expensive equipment and/or higher energy cost Chemical methods (e.g., chemical reduction) may offer cheaper alternatives But these, at times, use reagents and/or produce byproducts which are harmful to the environment https://doi.org/10.1016/j.jsamd.2018.12.009 2468-2179/© 2019 The Authors Publishing services by Elsevier B.V on behalf of Vietnam National University, Hanoi This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) A.R Ramos et al / Journal of Science: Advanced Materials and Devices (2019) 66e71 67 growth, the aggregation, and the interaction of the synthesized particles with the external environment [26] Methodology Fig X-ray diffraction patterns of precipitates produced at room temperature (~25 C) after 24 hours using 0.2M copper sulfate solution and 1.2M ascorbic acid solution This work is an investigation of the effects of the processing parameters (e.g., reaction temperatures and reactant concentrations) on the characteristics of copper-based particles synthesized using chemical reduction technique, a simple chemical method that involves the use of reducing agents to trigger the formation of copper metal One non-toxic, abundant, and cheap reducing agent used in the synthesis of copper-based particles is the ascorbic acid [22e26] Ascorbic acid, like copper, is known for its antimicrobial properties But, aside from being a reductant, ascorbic acid also acts as a capping agent, which affects the Copper particles were synthesized via straightforward wetchemical reduction with copper sulfate pentahydrate (Technical grade) and L-ascorbic acid powder (US Pharmacopoeia grade) as the precursor and reducing agent, respectively Different concentrations of the copper sulfate solution were prepared by dissolving copper sulfate pentahydrate salt in water The copper sulfate solution was added dropwise to a 1.2M ascorbic acid solution, which was made by dissolving an appropriate amount of L-ascorbic acid powder in water Equal volumes of the two solutions were mixed at room temperature (~25 C) Another sample was prepared at a higher temperature (85 C) The precipitates were gathered by suction filtration 24 hours after the first drop of the copper sulfate solution was added to the ascorbic acid solution and air-dried immediately After eight days, precipitates were collected again using suction filtration and likewise air-dried The percentage yield (%yield) was computed using the equation: %yield ¼ output  100; input where the ‘output’ is the mass of the precipitates while the ‘input’ is the total mass of copper element in the precursor The phases present in the samples were identified using an X-ray Diffractometer The infrared spectra of the precipitates were also determined using Fourier-Transform Infrared (FTIR) spectroscopy Fig Scanning electron micrographs of the precipitates produced at room temperature (~25 C) after 24 hours using 0.2 M copper sulfate solution and 1.2 M ascorbic acid solution The magnifications were (a) 1000Â, (b) 2000Â, (c) 5000 and (d) 8000 68 A.R Ramos et al / Journal of Science: Advanced Materials and Devices (2019) 66e71 Table Amount of precipitate collected after 24 hours at different temperatures using 0.2M copper sulfate solution and 1.2M ascorbic acid solution Fig Infrared spectrum of precipitates produced at 25 C after 24 hours using 0.2M copper sulfate solution and 1.2M ascorbic acid solution Finally, antimicrobial properties of the precipitates against one gram-positive (Staphylococcus aureus) and one gram-negative (Escherichia coli) bacteria were tested under dynamic contact conditions (ASTM E2149-01) Results and discussion 3.1 Copper-based precipitates X-ray diffraction patterns confirm the presence of copper in the samples Fig shows the pattern obtained from precipitates processed at room temperature (25 C) using the 0.2 M copper sulfate solution and the 1.2 M ascorbic acid solution The diffraction peaks at 2q ¼ 43.28 and 50.41 reveal that the precipitates are mainly composed of copper metal The peaks at 2q ¼ 36.4 and 42.3 indicate small amounts of cupric oxide (CuO) and cuprous oxide (Cu2O) in the samples The majority of the structures seen in the scanning electron micrographs of the samples (Fig 2) are particlelike This means that there is no excessive elongation in any of the dimensions The particles are not perfect spheres Instead, they have edges This implies that the particle growth may have some degree of preference in certain planes The average size of the precipitates was 10 mm Table Peaks of the infrared spectra of precipitates produced at 25 C after 24 hours using 0.2M copper sulfate solution and 1.2M ascorbic acid solution and their corresponding interpretation Wavenumber, cmÀ1 Assignment 3568e3211 O-H stretching of free alcohol group/water O-H stretching of intermolecular bonded alcohol group O-H stretching of intramolecular bonded alcohol group C-H asymmetrical stretching in CH2 group not identified but attributed to the presence of complex between the Cu atoms and polyhydrated ascorbic acid molecules C-O stretching C-C stretching O-H bending C-OH stretching O-H in-plane bending C-O stretch of 1 alcohol C-H in-plane bending C-C in-plane bending O-H out-of-plane bending 3198 2893 2830 2569e1980 1623 1562 1398 1303 1220 1080 1018 804 631 Temperature, C Yield, g Percent Yield, % 25 85 0.5072 2.9092 15.96 91.56 The sample's infrared spectrum presented in Fig shows the vibrational modes associated with the presence of the polyhydrated ascorbic acid molecules The peaks of the infrared spectrum and their corresponding interpretations are detailed in Table An L-ascorbic acid molecule contains two hydroxyl (OH) groups attached to double bonded carbon atoms nfrared spectrum of the synthesized particle, however, does not show vibration modes associated with the C¼C bond The ascorbic acid may be oxidized into dehydroascorbic acid Dehydroascorbic acids in the aqueous solution may have undergone hydration, thus forming polyhydrated ascorbic acid molecules The broad peak at 3198 cmÀ1 associated with O-H stretching modes found in the intermolecular bonded alcohol groups suggests the formation of networks of the ascorbic acid molecules The infrared spectrum in Fig also shows unidentified peaks in the range from 2569 to 1980 cmÀ1 These peaks are attributed to a possible bond or a complex formed between the Cu atoms and the polyhydrated ascorbic acid molecules The ascorbic acid did not merely coat the copper metal Otherwise, it would have dissolved in water Instead, it formed a complex, which capped the molecules This capping may be only a few molecules thick and is too thin to be visible in the electron micrographs 3.2 Variation of the reaction temperature Table presents a comparison of the yield obtained at different reaction temperatures This reflects the amount of synthesized copper particles per unit of available copper ions The precipitates were collected 24 hours after mixing 0.2M copper sulfate and 1.2M ascorbic acid solutions Samples prepared at 85 C generated a percentage yield of 91.56% while those produced at room temperature (~25 C) gave only a yield of 15.96% The energy needed to trigger the reduction of copper ions into copper metal decreases with the temperature Moreover, at higher temperature, the probability of one molecule colliding and reacting with another increases With the increased probability of collision and reaction among molecules in the mixture, precipitates are also more likely to be capped before they interact with oxygen molecules in the Fig X-ray diffraction patterns of precipitates produced at (a) 85 C and (b) ~25 C using 0.2M copper sulfate solution and 1.2M ascorbic acid solution A.R Ramos et al / Journal of Science: Advanced Materials and Devices (2019) 66e71 69 Table Antimicrobial properties of the precipitates against S aureus and E.coli under dynamic contact conditions The samples were synthesized at different reaction temperatures using 0.2M copper sulfate solution and 1.2M ascorbic acid solution Temperature, C Reduction, % (after one hour) 25 85 S aureus E coli 98 83 33 32 atmosphere The effect of faster capping at higher temperatures is seen in the XRD patterns of the samples (Fig 4) Both show strong diffraction peaks associated with the presence of copper metal However, the peaks associated with CuO differ in intensity The sample synthesized at higher temperature has a weaker diffraction peak at 36.4 Samples prepared at room temperature contain more oxides It also has a peak at 42.3 , which is associated with presence of Cu2O Table shows the antimicrobial properties of the different precipitates against S aureus and E coli under dynamic contact conditions The copper-based particles synthesized at 85 C, has reduced the population of S aureus (CFU/mL) by 83% after one hour of contact This value is lower compared to the effect of copperbased particles that was prepared at 25 C (98%) At higher temperatures, the increase in probabilistic collisions between ascorbic acid molecules and copper-based particles may lead to a more extensive capping of precipitates Capping minimizes interaction between the precipitates and S aureus, thus, reducing its antimicrobial activity against the gram-positive bacteria The same was observed when the samples prepared at different temperatures were tested against E.coli However, in general, the precipitates were more effective in reducing the number of S aureus (grampositive) than that of E.coli (gram-negative) after one hour of contact 3.3 Variation of the reaction concentrations Table summarizes the effect of different reductant-toprecursor concentration ratios on the yield At higher copper sulfate concentrations, more copper ions are available for reduction This enhances the yield However, in terms of percentage, the yield becomes less This downward trend in the percentage yield may be attributed to the decrease in the number of available ascorbic acid molecules for every unit of copper ions In the experiment, the L-ascorbic acid solution was maintained at 1.2M while the copper sulfate concentration was varied to achieve 12:1, 6:1, and 3:1 reductant-to-precursor concentration ratios As the concentration of copper sulfate was increased, the ratio of the reductants to the precursors decreased A greater percentage yield was obtained by limiting the precursor., thus, reducing the amount of copper sulfate in the solution Fig presents a comparison of the XRD patterns All show strong diffraction peaks confirming the presence of copper metal in the precipitates Traces of oxides are also seen in the samples Table Amount of precipitate produced at ~25 C using different concentrations of copper sulfate solution and an ascorbic acid solution of 1.2 M Copper sulfate concentration (M) Reductant-toprecursor concentration ratio Yield, g 0.1 0.2 0.4 12:1 6:1 3:1 0.2777 0.5072 0.7271 Percent Yield, % 17.48 15.96 11.44 Fig X-ray diffraction patterns of precipitates produced at ~25 C reaction temperature using 1.2 M ascorbic acid solution and varying copper sulfate concentration (a) 0.4M, (b) 0.2 M and (c) 0.1 M However, the associated peaks are very low for the precipitates produced using 0.4M copper sulfate concentration or 3:1 reductant-to-precursor ratio Capping impedes oxidation Based on the results, capping may have proceeded faster when the disproportion between the concentrations of ascorbic acid (the capping agent) and copper sulfate became less The antimicrobial properties of these samples were likewise tested against the S aureus and E coli under dynamic contact conditions The precipitates were found to be more effective against S aureus The data in Table also show the effect of varying reactant concentrations on the antimicrobial properties of the samples The precipitates prepared using high reductant-to-precursor concentration ratio (12:1) demonstrated the least activity against S aureus, with only 68% reduction after one hour of contact In the previous section, the antimicrobial activity was related to the degree of capping In this case, while it may have proceeded faster at 3:1 reductant-to-precursor ratio, capping may not be as extensive So far, the data presented correspond to precipitates gathered after the first 24 hours (or one day) The yield is proportional to the reaction time The amount of precipitates yielded more than doubled after eight days However, the reaction rates usually slow down with time due to the consumption of reactants From Table 6, it can be inferred that around 32% of the total product was formed on the first day and, on the average, only about 0.1939 g or 12.5% was added each day after The precipitates were again found to be more effective in reducing the number of S aureus compared to that of E.coli after one hour of contact (Table 7) The samples collected after days, however, showed poor antimicrobial activity against both bacteria This decrease in the antimicrobial activity may still be attributed to extensive capping Samples collected after days were found to have significantly lower oxide content compared to those collected after the first 24 hours The diffraction peaks associated with oxides Table Antimicrobial properties of the precipitates synthesized at ~25 C using different reductant-to-precursor concentration ratios against S aureus and E.coli under dynamic contact conditions Copper sulfate concentration (M) 0.1 0.2 0.4 Reductant-to-precursor concentration ratio 12:1 6:1 3:1 Reduction, % (after one hour) S aureus E.coli 68 98 93 37 33 31 70 A.R Ramos et al / Journal of Science: Advanced Materials and Devices (2019) 66e71 Table Amount of precipitate produced at ~25 C using 3:1 reductant-to-precursor concentration ratio at different collection times Collection Amount of precipitate (g) 1st (after day) 2nd (after days) 0.7271 1.5512 have become very small and almost indistinguishable (Fig 6) After the first 24 hours, conditions may have favored capping over further aggregation and improving the yield Capping may have continued and become more extensive lessening oxidation and lowering the antimicrobial property of the precipitates In general, the synthesized copper-based particles have better antimicrobial properties against S aureus than against E coli The higher percentage reduction observed in S aureus as compared to the percentage reduction of E coli may be attributed to the structural difference in their cell wall Gram-positive bacteria such as S aureus have abundant pores, which may facilitate foreign particles to pass through On the other hand, gram-negative bacteria such as E coli have lipopolysaccharide, lipoproteins and phospholipids, which make up a better barrier against foreign particles The higher antimicrobial activity of different materials against S Aureus than against E.coli is also observed in other works [27e30] Conclusion A straightforward wet chemical reduction technique was employed to produce copper-based particles Copper sulfate salts and L-ascorbic acid were used as precursor and reducing agent, respectively Synthesis of copper-based precipitates was confirmed through an X-ray Diffractometer Scanning electron micrographs of the precipitates showed particle-like structures with edges The amount of synthesized copper particles per unit of available copper ions varied with the reaction temperature and the reactant concentration Greater percent age yield was obtained when the sample preparation was performed above room temperature and a higher reductant-to-precursor concentration ratio was used Infrared spectra of the samples suggest formation of networks of ascorbic acid molecules and complex between the Cu atoms and the polyhydrated ascorbic acid molecules Ascorbic acid served as a capping agent Faster capping reduced the amount of oxides in the precipitates, particularly in samples prepared at a higher temperature and with a lower reductant-toprecursor concentration ratio The rate of capping, however, may not be directly related to its extent Antimicrobial testing under dynamic contact conditions showed that the copper-based precipitates were more effective against S aureus (a gram-positive bacteria) than against E coli (a gram-negative bacteria) Samples prepared at room temperature and lower reductant-to-precursor concentration ratio were more successful in reducing the number of S aureus after one hour of contact Capping may limit the interaction between the precipitates and the bacteria Therefore, capping may have been less extensive in the aforementioned cases Table Antimicrobial properties of the precipitates synthesized at ~25 C using 3:1 reductant-to-precursor ratio, against S aureus and E.coli under dynamic contact conditions Time Collection After first 24 hours After eight days Reduction, % (after one hour) S aureus E coli 93 45 31 27 Fig X-ray diffraction patterns of precipitates that were taken from the second collection of the samples The samples were produced at 25 C reaction temperature using 1.2 M of ascorbic acid solution and (a) 0.4M, (b) 0.2M and (c) 0.1M copper sulfate solutions The corresponding reductant-to-precursor ratios are (a) 3:1, (b) 6:1 and (c) 12:1, respectively than in the samples prepared at a higher temperature and using a greater reductant-to-precursor concentration ratio Comparison of precipitates 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phase: synthesis, DFT calculations, and enhanced antibacterial activities, ACS Sustain Chem Eng (2017) 6585e6596 ... involves the use of reducing agents to trigger the formation of copper metal One non-toxic, abundant, and cheap reducing agent used in the synthesis of copper- based particles is the ascorbic acid. .. the effects of the processing parameters (e.g., reaction temperatures and reactant concentrations) on the characteristics of copper- based particles synthesized using chemical reduction technique,... employed to produce copper- based particles Copper sulfate salts and L-ascorbic acid were used as precursor and reducing agent, respectively Synthesis of copper- based precipitates was confirmed through