Synthesis of Silver Nanoparticles from Silver Nitrate Solution Using Green Tea Extract (Camelia sinensis) as Bioreductor

Wara Dyah Pita Rengga, Arie Yufitasari, Wismoyo Adi

DOI 10.15294/jbat.v6i1.6628

Chemical Engineering Department, Fakultas Teknik, Universitas Negeri Semarang, Kampus Sekaran Gunung Pati, Semarang 50229, Indonesia

Article Info Abstract Article history: The synthesis of silver nanoparticles with micro size is highly required in antibacterial fields. Accepted The biorefinery material is highly potential as a bioreductor which is applied in the synthesis July 2016 of nanoparticles. The bioreductor is made from green tea leaves extraction using aquadest to Approved extract its active substance, the catechin which is derived from polyphenol. The polyphenol March 2017 can reduce the synthesis of silver nanoparticles naturally. The result of FTIR analysis from Published green tea leaves extract containing polyphenol shown in the uptake functional groups is -OH June 2017 group located in 3425 cm-1, C=O group located in 1635 cm-1, C=C group located in 1527, -1 -1 Keywords : and 1442 cm , and C-O group located in 1234 cm . The precursors of AgNO3 was used as Bio-reductor, green the main synthetic material. The synthetic condition was resulted from the reaction between tea leaves, the extraction of green tea extract and AgNO3 as the precursors in the variation of nanoparticles, silver synthetizing time. The heating process during synthesizing is done in 50 ˚C along with stirring to foster the creation of silver nanoparticles. The analysis result of XRD shows that silver nanoparticles has the diffraction peaks in the angle of 2 theta that are 44.08, 64.40, and 77.51. The types of silver nanoparticles is Ag0 nanoparticles with face-centered cubic crystal structure. Based on TEM analysis, the size and particle size distribution can be determined using image J. The distribution shows that the longer synthesizing time, the bigger nanoparticles produced. With synthesizing times at 24 hours, 6 hours, 3 hours, and 2 hours produce average particle size of 26.4 nm; 9.2 nm; 8.4 nm; and 7.4 nm respectively.

INTRODUCTION The colloid of silver has been known as an antimicrobial. The antimicrobial ability of silver Nanotechnology is an interesting inno- can kill all pathogenic microorganism and there has vation of technology in research area which is not been any report of microbes which is resistant related to production, size, and form in industry to silver (Ariyanta et al., 2014). Silver nanoparticles (Amiruddin et al., 2013). Researches in have wider surface allowing it improving its contact nanotechnology produces numerous invention of to bacterial or fungus and able to improve the products with better performance. It motivates the effectiveness as bactericides and fungicides research of chemistry to synthesize nano size (Montazer et al., 2012). The silver nanoparticle is particle from precious metal. One of the metal slowly able to release silver ion which can break the which is mostly investigated is silver which is RNA and DNA of bacterial making them unable to commonly used in the application as antibacterial replicate (Blaker et al., 2004). and antifungal. (Dwandaru et al., 2016; Rengga et Several methods used in producing al., 2017). nanoparticles are chemical reduction, photo- © 2017 Semarang State University Corresponding author: ISSN 2303-0623 Chemical Engineering Department, Fakultas Teknik, Universitas Negeri Semarang, e-ISSN 2407-2370 Kampus Sekaran Gunung Pati, Semarang 50229, Indonesia E-mail: [email protected]

Wara Dyah Pita Rengga, Arie Yufitasari, Wismoyo Adi / JBAT 6 (1) (2017) 32-38

chemistry and sonochemistry. Nevertheless, the that, the heated green tea solution is filtered using most used method is chemical reduction method. whatman filter paper number 1 in chemical glass. The reduction method is mostly chosen since it is The extract of green tea is cooled to room simple and easy. The synthesis of silver nano- temperature. The extract of green tea was then particles with eco-friendly chemical reduction analyzed using FTIR to find the catechin method is using the extract of plants as the reductor composition in the green tea. agents (Sathishkumar et al., 2009). Several plants containing these chemical compounds can be used Preparation of 0,1 N AgNO3 SOLUTION as reducing agents. The antioxidant compounds Solid AgNO3 solution is weighed as 1.7 contained inside plant have been known to be metal grams and taken into the 100 mL chemical glass. ion reducing agents in the biosynthetic process AgNO3 is dissolved with 100 mL aquadest. First,

(Vidhu et al., 2011). AgNO3 was dissolved with 50 mL aquadest in the The use of plants in the biosynthesis of chemical glass. The mixtures then was stirred until nanoparticle is related to the composition of it was perfectly dissolved perfectly. AgNO3 solution secondary metabolite compounds with antioxidant was then moved into a 100mL volumetric flask and activities. The antioxidant is the alternative to added with aquadest to the size of 100mL. The produce green chemistry nanoparticles, because it is mixtures were shaken until they were blended able to deduce the use of dangerous chemical perfectly. substance along with its waste (Dyduch-Sieminska et al., 2015). The Synthesis of Nanoparticles Using Reduction Tea can be used as a bioreductor for Method reducing silver to become nanoparticle. It is because The solution of green tea leaves was taken tea possess many antioxidant material which can be in 1000 mL. The solution of 100 mL AgNO3 and a reductor of metal in the process of biosynthesis 1000 mL the extract of green tea leaves is mixed in (Begum et al., 2009). One of the plants with high 2000 mL chemical glass. The mixture of green tea antioxidants is green tea. The composition of leaves and AgNO3 solution is stirred at 50°C for 1 chemical substance in green tea leaves are including hour. Then, bit by bit, it was added with 0.1 N caffeine, catechin, flavonol, theanin, glutamate NaOH solution until the pH surpass the level of 8. acid, aspartate acid, arginine, glucose, and The mixture was respectively heated for an hour potassium. The biggest composition in the bud of until the Ag nanoparticles were deposited. The green tea leaf is catechin or epigallocatechin-3- deposition of Ag nanoparticles was separated from gallate (EGCG) in the level of 20-29%. The activity the filtrate using a filtration. The deposition of Ag of antioxidant from catechin is 200 times stronger nanoparticles which had been separated then was in fighting free radicals comparing to vitamin E consequently roasted to dispel the existing water. (Widyaningrum, 2013). This deposition was then analyzed using XRD and TEM. The variation of samples used in different RESEARCH METHODOLOGY time allocations, which were 24 hours, 6 hours, 3 hours, and 2 hours. The material used in this research were green tea leaves, AgNO3 (Merck), aquadest, and RESULTS AND DISCUSSIONS NaOH (Merck). The tools used in this research were chemical glasses, measuring pipette, glass Results of Green Tea Characterization Using stirrer, watch glass, hot plate, spatula, analytical FTIR balance, oven, whatman filter paper number 42, Figure 1 shows the comparison between Fourier Transform InfraRed (FTIR), Transmission FT-IR spectra analysis result for catechin Electron Microscopy (TEM), X-Ray Diffraction compounds from the extraction of green tea leaves. (XRD), and Image-J application. The absorbance peak at 3425 cm-1 show -OH bond indicating the existence of hydroxyl groups. The Preparation of Green Tea Extract Solution absorbance peak at 1635 cm-1 shows C=C bond in Green tea of 2 g and water of 1000 mL are a aromatic compounds. The absorbance peak at placed into 2000 mL of beaker. It is stirred and 1527 cm-1 and 1447 cm-1 show the C=O bond heated using hot plate at 60 ° C for 20 minutes. After indicating presence of a carbonyl group. The

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(%) , ,

Ramos-Tejada et al. (2002)

Experimental result Transmittance

Wave Number, cm-1

Figure 1. The comparison of spectra result from FTIR analysis to catechin compounds.

Table 1. The Comparison of Functional Group in Catechin Compounds based on FTIR Analysis Results. Absorption Area (cm-1) Types of Bond Researches (Ramos-Tejada et al., 2002) 3425 3400 –OH (hydroxyl group) 1635 1628 C=O (carbonyl group) 1527, 1442 1519, 1464 C=C (aromatic) 1234 1240 C-O (polyol group) absorbance peak at 1234 cm-1 shows the C-O bonds indicating the existence of ether and polyol groups. The bonds of several functional groups were the functional group found in catechin compounds. The researches of Ramos-Tejada et al. (2002), explain that the result of analysis from catechin compound analysis with the existence of –OH, C=C, C=O, and C-O groups. Thus, the result of green tea extract supporting the research of Begum Figure 2. The extract of green tea after mixed et al. (2009) that specifically, the absorbance peak at with AgNO3 solution. 1226 cm-1 containing C-O group indicating polyol, like hydroxy flavonoid or catechin. This result is light brown. In the 10th minutes, the color shown in Table 1. In summary, the samples of green will change again to be darker brown, and after 60 tea extract is proven containing catechin. minutes, the solution will be darker as shown in Figure 3. The changes of color will show the Silver Nanoparticles Synthesis reaction of reduction from the mixtures. The Catechin compounds contained in green changes of color from white turbid into dark brown tea extracts can be used to reduce AgNO3 become is caused by the oxidation of catechin to create Ag nanoparticles, or reducing Ag+ into Ag0. The another compound of theaflavin and theaburigin. color of extraction is light brown and the color of After the washing process, the result of the synthesis

0,1 N AgNO3 solution is turbid white, when both of became dark grey in color and shone like crystal them is mixed, it resulted light white color mixture indicating that the deposition is a silver as shown in Figure 2. nanoparticle, as can be seen in Figure 4. The reduction reaction occurs between the Synthesis of nanoparticles was treated with bioreductor solution and AgNO3 0.1 N which heating at 50 ˚C for 1 hour aimed at accelerating undergoes rapid color change. This color change oxidation of catechin and reduction of Ag+ become indicates a reduction reaction. After 5 minutes of Ag0. During the process, the pH was set until pH = mixing, the mixture will change from light white to 8 with the addition of 0.1 N NaOH. The function of

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Wara Dyah Pita Rengga, Arie Yufitasari, Wismoyo Adi / JBAT 6 (1) (2017) 32-38

a b c

Figure 3. The change of color after mixing bioreductor and AgNO3 solution in (a) 5 minutes, (b) 10 minutes, and (c) 60 minutes.

Raghavendra et al. (2016) Experimental result

Figure 4. Silver Nanoparticles from the reaction of reduction.

NaOH addition was to speed up silver nanoparticle Intensity formation. The increment of pH can increase the 2 Theta - - concentration of OH , so that OH will be more Figure 5. The result of XRD analysis of silver effective in producing negative surface charge in the nanoparticles base pH environment (El Badawy et al., 2010). The electron in bioreductor reduce the Ag+ become Ag0, Table 2. The Comparison of Peak Diffraction in so, it became nanoparticle, whilst bioreductor the Research and Reference. experiences the oxidation process where this Research Raghavendra et al. (2016) antioxidant is the compound which produces 37.83o 38.06o electron (Silalahi, 2000). 44.08o 44.14o 64.40o 64.38o Result of Silver Nanoparticles Analysis Using 77.51o 77.26o XRD Based on Figure 5, it can be inferred that Results of Silver Nanoparticles Analysis Using the sample of deposition reached its peak position Transmission Electron Microscopy (TEM) and intensity. In details, the peak of diffraction The deposition of nanoparticles were appears at 2 angle of 44.08, 64.40, and 77.51 each analyzed using TEM to predict the size of the in the reflection area of (2 0 0), (3 0 0), (3 1 1) with nanoparticles. The TEM results presented are a crystal structure휃 of face centered cubic (FCC) for form of nanoparticles based on their synthesis silver nanoparticle. Meanwhile, based on the reaction time of 24 hours, 6 hours, 3 hours, and 2 research of Raghavendra et al., (2016), the peak hours. Based on these figures, the nanoparticles positions are in the angles of 38.06, 44.14, 64.38, were shown in the figure with black color. The time and 77.26 where each of them is in the reflection of synthesis between precursor and bioreductor area of (1 1 1), (2 0 0), (3 0 0), and (3 1 1). The influence the formed nanoparticles. The longer the comparison between XRD results of silver time of synthesis contact results the bigger size of nanoparticles from the research conducted by nanoparticle size, since there is an aggregation of Raghavendra, et al., (2016) can be seen in Table 2. particle formation which is shown in Figure 6 (a)

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Wara Dyah Pita Rengga, Arie Yufitasari, Wismoyo Adi / JBAT 6 (1) (2017) 32-38

(a) (b)

(c) (d) Figure 6. The analysis of TEM from silver nanoparticles in the synthesis time of (a) 24 hours; (b) 6 hours; (c) 3 hours (d) 2 hours where the size of the nanoparticles is bigger than the size than nanoparticle size for 24 hours synthesis. other figures. The aggregation is resulted from the The distribution of nanoparticle diameter size for 6 force between particles, thus the particles will hours synthesis was mostly in the diameter size of approach to each other and gather creating a cluster 10 nm (64.2%), followed by 20 nm (32.1%), and 30 of particle which gets bigger each time. The analysis nm (3.8%). The nanoparticle with 10 nm size is of TEM in the nanoparticle synthesis in 6 hours can increasing in number comparing to the 24 hours be seen in Figure 6 (b) where the particle is smaller synthesizing process. The whole nanoparticle in size and not similar to particle which is average size was 9.2 nm. According to Figure 7(c), synthesized in 24 and 2 hours. The particles in silver nanoparticles was distributed in the diameter synthesis time of 3 hours in Figure 6 (c) and 2 hours size of 10-60 nm. The distribution of silver in Figure 6 (d) has smaller size than the synthesis of nanoparticles for 3 hours synthesizing process was 6 hours. The particle size during the reduction more variative than the 6 hours process of process appears on the TEM results of varying size synthesizing, but the size of 10 nm nanoparticles is indicating the collision process between the increasing until become 71.3% than the 60 nm reactants has not been maximized. Nonetheless, particles in 1.2%, the other size were 20-40 nm. The many crystals seeds have slowly appeared though it average size of silver nanoparticles was 8.4 nm. The have not reached in the process where the crystal distribution of nanoparticle size in 2 hours seed can become bigger crystal. synthesizing process can be seen in Figure 7(d) The analysis of TEM can determine the shown that the diameter of nanoparticles was about distribution of particles size as pictured in Figure 7 10 nm in the highest proportion (79.4%) comparing (a) where the results of synthesis of 24 hours were to 20 nm nanoparticles and the lowest diameter (30 still bigger comparing to other figures with the nm (5.56%). The size of silver nanoparticle in the biggest size about 80 nm. In the figure it appears synthesis time of 2 hours resulting a narrower size that the dominant diameter of nanoparticles were of particle, 10-30 nm, with the average diameter of 20 and 40 whereas the particle with a size of 10 nm 7.4 nm. The synthesis time of 2 hours resulting was only 18%. The average size of silver smallest nanoparticles. The process of synthesizing nanoparticles in 24 hours synthesis was 26.4 nm. of silver nanoparticles with reaction of reduction Figure 7 (b) showed the TEM analysis results in will result less nanoparticles until the limit where synthesis time of 6 hour with smaller nanoparticle the nanoparticles is formed. Natural bioreductor,

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Distribution (%) Distribution(%) Distribution (%)

Nanoparticle diameter (nm) Nanoparticle diameter (nm)

(a) (b)

Distribution (%) Distribution(%) Distribution (%)

Nanoparticle diameter (nm) Nanoparticle diameter (nm) (c) (d) Figure 7. The Distribution of Silver Nanoparticle Size in Syntheses (a) 24 hours; (b) 6 hours; (c) 3 hours; (d) 2 hours the extract of green tea is impossibly possessing nanoparticles. Based on TEM analysis, the time of catechin, so, even when the size of nanoparticles is synthesizing process influences the formed silver less than 10 nm, the research of Rengga et al. (2017) nanoparticles. The longer the synthesizing time, the was proven able to produce silver nanoparticle with bigger size of nanoparticles formed, since there is a the average size of 5.44 nm which is attached to particle aggregation, so the creation of initial crystal active carbon because the used bioreductor is a pure and crystal formation should be concerned more. starch. The results of the research was better than the extraction of tea by Nestor et al. (2008). ACKOWLEDGEMENT Camiellia sinensis can result silver nanoparticles in The author would like to deliver the the size of 40 nm. The extract of other leaves is gratitude to the Direktorat Riset dan Pengabdian Cytrus sinensis which results silver nanoparticles of Kepada Masyarakat, Kementerian Ristek dan 22.3 nm (Logeswari et al., 2015). pendidikan Tinggi Indonesia for the funding of this research according to Fundamental Research CONCLUSION Funding in 2016.

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