Size and stability modulation of ionic liquid functionalized gold nanoparticles synthesized using Elaeis guineensis (oil palm) kernel extract

Accepted Manuscript Original article Size and Stability Modulation of Ionic Liquid Functionalized Gold Nanoparticles Synthesized Using Elaeis guineensis (Oil Palm) Kernel Extract Muhammad Irfan, Tausif Ahmad, Muhammad Moniruzzaman, Sekhar Bhattacharjee, Bawadi B Abdullah PII: DOI: Reference:

S1878-5352(17)30043-6 http://dx.doi.org/10.1016/j.arabjc.2017.02.001 ARABJC 2058

To appear in:

Arabian Journal of Chemistry

Received Date: Revised Date: Accepted Date:

8 November 2016 1 February 2017 1 February 2017

Please cite this article as: M. Irfan, T. Ahmad, M. Moniruzzaman, S. Bhattacharjee, B.B. Abdullah, Size and Stability Modulation of Ionic Liquid Functionalized Gold Nanoparticles Synthesized Using Elaeis guineensis (Oil Palm) Kernel Extract, Arabian Journal of Chemistry (2017), doi: http://dx.doi.org/10.1016/j.arabjc.2017.02.001

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Size and Stability Modulation of Ionic Liquid Functionalized Gold Nanoparticles Synthesized Using Elaeis guineensis (Oil Palm) Kernel Extract Muhammad Irfana, Tausif Ahmada, Muhammad Moniruzzamana,b*, Sekhar Bhattacharjeea, Bawadi B Abdullaha, a

Chemical Engineering Department, Universiti Teknologi PETRONAS, Bandar Seri Iskandar,

32610, Perak, Malaysia b

Centre of Researches in Ionic liquids, Universiti Teknologi PETRONAS, Bandar Seri

Iskandar, 32610, Perak, Malaysia

* To whom correspondence should be addressed

Dr. Muhammad Moniruzzaman Office: +605 - 368 7572 Fax: +605 - 368 6176 Email: [email protected]

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Abstract Bio-synthesis approach for gold nanoparticles (AuNPs) has received tremendous attention as an efficient and eco-friendly process. However, kinetic growth and colloidal stability of AuNPs synthesized by this process remained challenging. In this study, Elaeis guineensis (oil palm) kernel

(OPK)

extract

prepared

in

an

ionic

liquid

(IL)[EMIM][OAc]

(1-ethyl-3-

methylimidazolium acetate) was employed to control and tune the size and morphology of AuNPs. Synthesized AuNPs were characterized using UV-vis spectrophotometer, dynamic light scattering (DLS) and transmission electron microscopy (TEM) to observe any changes in absorbance, surface charge and particle size, respectively. IL mediated AuNPs were examined for 120 days and found well disperse and stable at room temperature. UV-vis analysis demonstrated that volume of extract played an important role to control the stability of AuNPs. After 120 days, only 8.86% reduction from maximum absorbance was observed using 2 mL of volume of extract, which was elevated to 47.64% in case of 0.3 mL.

TEM analysis was

performed periodically after day 1, day 30, day 60, day 90 and day 120 and minor increase in the size was observed.

Insignificant change in zeta potential value after 120 days supported

enhanced stability of IL mediated AuNPs. Crystalline nature of AuNPs was confirmed by X-ray diffraction (XRD) pattern. Absence of [EMIM][OAc] from OPK extract resulted into larger particles size (9.64 nm), low zeta potential value (-13.9 mV) and enhanced aggregation of particles. Finally, experimental data was used to predict the theoretical and the experimental settling time for AuNPs to evaluate colloidal stability. Keywords — Gold nanoparticles; Elaeis guineensis; Ionic liquids; Particle size; Particle growth; Stability

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1 Introduction Synthesis of gold nanoparticles (AuNPs) has received immense research attention due to their unique applications in catalysis, optics, sensing, imaging and medical diagnostic kits. AuNPs have also been used to increase electroluminescent and quantum efficiency in organic light emitting diodes (Islam et al., 2015a). These have been used significantly as carriers for drug delivery due to their excellent biocompatibility (Kanchi et al., 2014, Daniel and Astruc, 2004). Suitability of AuNPs for biomedical applications is highly dependent on their size, shape and stability. AuNPs must possess high surface area for effective conjugation with biomolecules, well dispersed in an aqueous medium with good stability at room temperature for an extended period. In fact, AuNPs possess large surface area that tends to aggregate smaller particles. This agglomeration process has been well explained by the Ostwald ripening model (Janiak, 2013). To prevent agglomeration, various stabilizers and capping agents, such as bio-surfactants, polymers, starch and lipids are added to colloidal gold (Dzimitrowicz et al., 2016b). Additives cover a protective shield on AuNPs surfaces to improve the stability of the AuNPs (Das et al., 2014). Surface charge on nanoparticles offset Van der Waals attractive forces by steric and electrostatic repulsions (Jiang et al., 2009). Currently, various methods including physical, chemical and biological have been used to synthesize AuNPs (Chakraborty et al., 2014; Islam et al., 2015b). However, toxicity issues of chemicals and stabilizers used in physical and chemical methods are major impediments to biomedical applications (Islam et al., 2016). Green synthesis of AuNPs is an attractive option due its simplicity, eco-friendliness and biocompatibility (Islam et al., 2015b; Kanchi et al., 2014).

For green synthesis, aqueous extracts of leaves, seeds, barks and fruits of various

medicinal plants have been used as they contain different bioactive phytochemicals including 3

phenolics and flavonoids, which are able to reduce trivalent gold ions to zero valent gold atom (Gan et al., 2012; Islam et al., 2015b). Among the different process parameters, Precursor (Au3+) concentration, selection of an appropriate solvent for phytochemicals extraction, reaction temperature and nature of stabilizing agents play important roles in defining morphology, size distribution and stability of synthesized AuNPs (An et al., 2012). Ionic liquids (ILs) are environmentally friendly materials with melting points below 100 oC (Li et al., 2008). ILs have been used as a potential alternative of volatile organic solvents (VOSs) due to their attractive and unique properties including non-volatility, multiple solvation interaction with many organic and inorganic compounds, superior thermal and chemical stabilities and low surface energy (Messali, 2014; Moniruzzaman et al., 2010). In the past decade, ILs have been used significantly in a wide range of applications including biological application (Adawiyah et al., 2016; Sivapragasam et al., 2016; Financie et al., 2016, Moniruzzaman et. al., 2012). ILs have been used for extraction of phytochemicals and biopolymers (Ibrahim et. al., 2015; Moniruzzaman and Ono, 2013; Yang and Dionysiou, 2004) with high extraction efficiency as compared to many conventional solvents (Cláudio et al., 2013; Ressmann et al., 2012). In microwave induced AuNPs synthesis, ILs were used as an excellent solvent for rapid synthesis of AuNPs (Lin et al., 2006). ILs has also been used as a stabilizer in microwave, electron beam, gamma irradiation and sonochemical and photochemical reduction methods of AuNPs synthesis (Wang et al., 2011). Intrinsic polarity of ILs provides electrostatic protection and colloidal stability to nanoparticles (Safavi and Zeinali, 2010b). Anions adsorb on the surface of nanoparticles creates a negative protective layer whereas hydrocarbon moiety of IL extends outward to produce stable AuNPs dispersions in an aqueous medium (Safavi and Zeinali, 2010a; Scheeren et al., 2006). Anionic 4

part of an IL is responsible for morphological characteristics of nanoparticles (Ren et al., 2008). ILs with an imidazolium cation has been used as a solvent as well as stabilizer for synthesis of AuNPs (Safavi et al., 2012; Zhang et al., 2014). However, none of the reported studies has been carried out to investigate ILs’ dual roles as solvent in phytoextraction and as stabilizers after synthesis of AuNPs. In this study, our aim was to develop a single step method to synthesize IL mediated AuNPs using extract of oil palm kernel (OPK), which is abundantly available as bio waste materials in Malaysia (Jamil et al., 2016). IL[EMIM][OAc] (1-ethyl-3-methylimidazolium acetate) was used for synthesis of AuNPs for its triple role: (i) as a solvent for extraction of phytochemicals from oil palm kernels (OPK); (ii) as a reaction medium where bioactive compounds present in OPK[EMIM][OAc] extract reduce Au (III) ions to colloidal gold and (iii) as a stabilizer to impart long term stability to the synthesized AuNPs. Progress of the synthesis reaction was monitored using UV-vis spectrophotometer, whereas size distribution and particles’ morphology were monitored using TEM.

2 Materials and Methods 2.1 Materials Gold (III) chloride hydrate (HAuCl4.3H20, 99.99%) and IL[EMIM][OAc] (1-ethyl-3methylimidazolium acetate) (97%) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO) and used as received. Oil palm kernels were procured from Felcra Berhad Nasaruddin Oil Palm Plantation located in Bota, Perak, Malaysia. All other reagents used in the experiments were analytical grade.

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2.2 Preparation of oil palm kernel (OPK) extract Fresh oil palm kernels were collected and washed with distilled water to remove dust and other impurities. These kernels were dried in an oven for 12 h at 70 °C and grinded to powder form using IKA® grinder with a 0.25 mm sieve. The powder was kept in an airtight container for further use. 0.12 M aqueous solution of IL [EMIM][OAc] was prepared by adding 2 g of the [EMIM][OAc] into 100 mL of distilled water. Then 10 g finely grinded kernel powder was added to IL solution and heated at 90 °C for 20 min. The supernatant liquid was filtered by gravity filtration using Whatman No. 1 filter paper. The filtrate (OPK-EMIM) extract was collected in a 100 mL glass vial and stored at 4 °C for further use. For a control experiment, another batch of OPK aqueous extract was prepared using identical procedure as described above without IL. Both extracts were used for AuNPs synthesis within seven days of their preparations as efficacy of extracts drops substantially over an extended period of time (Anuradha et al., 2014). 2.3 AuNPs synthesis Au (III) (12.96 mM) stock solution was prepared by dissolving 500 mg of chloroauric acid into 100 mL distilled water. This stock solution was further diluted to 2.28 mM used in all experiments. AuNPs synthesis was performed in a domestic microwave oven (Sharp – R 268R(S)-M, 2450 MHz, 800W) at power outputs ranging between 10% and100%. 1 mL of 2.28 mM gold precursor solution diluted with 5 mL distilled water was preheated for 10 s in microwave followed by addition of 1 mL of OPK extracts. Formation of AuNPs was indicated by changing the color of the reaction medium from cloudy white to ruby red that began to appear after 52 s of microwave irradiation. Aliquot samples collected from the reaction medium at different time intervals were monitored for surface plasmon resonance (SPR) intensity by UV- vis 6

spectrophotometer whereas TEM was used to study AuNPs morphology. DLS analysis was performed to record zeta potential of the synthesized nanoparticles. Effects of volume of extract (0.30-2 mL) added to the gold precursor was studied on SPR absorbance, stability and particle size. For stability study, the final gold sols were preserved in 10 mL vials at room temperature and monitored periodically for 4 months using UV- vis to observe any change in the SPR peak intensities after 1, 3, 5, 8, 15, 22, 30, 60, 90 and 120 days. TEM analysis was also performed after each month to record changes in the morphology and particles size distributions. 2.4. Characterization of AuNPs Perkin Elmer Lambda 25 UV-visible spectrophotometer was used to record SPR absorbance and peak wavelength (λmax) at 1 nm resolution and a scan speed of 480 nm/min. Zeiss Libra 200 TEM was employed to observe size and shape of synthesized AuNPs by putting one drop of reaction mixture on a copper grid that was allowed to dry prior to TEM imaging and size measurement. Malvern, Zetasizer Nano ZSP (Malvern Instruments Ltd., Malvern, Worcester, U.K.) was used to measure the particle size distribution and surface zeta potential of AuNPs by using dynamic light scattering (DLS) analysis technique. Crystalline structure of AuNPs was evaluated using XRD, X’Pert3 powder, PANalytical. To prepare XRD sample, colloidal solution of AuNPs was centrifuged at 15000 rpm for 10 min and deposited on zero back ground plate followed by drying at 60 °C for 90 min. 3 Results and Discussion 3.1 UV-vis analysis Preliminary assessment for AuNPs synthesis can be observed from color change occurred in reaction mixture (Figure 1 inset). Initial color of aqueous extract of OPK was translucent white.

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Addition of chloroauric acid into the extract resulted in the reduction of trivalent gold ions and the colloidal solution exhibited intense purple and ruby red colour using OPK and OPK-EMIM extracts, respectively due to their SPR (Dzimitrowicz et al., 2016a). Parallel reactions were performed using OPK and OPK-EMIM extracts and sharp SPR peaks were observed. For OPK, the value of λmax was observed at 548 nm, which was blue shifted toward lower wavelength region i.e. 527 nm using OPK-EMIM extract indicating formation of smaller particles (Islam et al., 2016; Mostafa et al., 2012) as shown by Figure 1. Any changes in absorption band originated due to the surface plasmon oscillation of gold electrons provided sufficient information about variation of nanoparticles size (Islam et al., 2015b). No other SPR peaks were found near infrared region indicated the absence of any other shape particles like trigonal and hexagonal (Shankar et al., 2005). This observation was confirmed by TEM image analysis. Figure 1 here 3.2 Effect of microwave irradiation High polarity and conductivity of ILs make them excellent absorbents of microwave (MW) irradiation (Lin et al., 2006). MW induced AuNPs synthesis in an IL facilitated the enhanced heating rate with fast nucleation to complete the reaction in less time (Richter et al., 2013). AuNPs synthesis was performed by mixing 1 mL of 2.28 mM gold precursor that was diluted with 5 mL distilled water and 1 mL OPK-EMIM extract. The resulting mixture was subjected to MW irradiation for 90 s at various power outputs ranging from 10 % to 100% of total power capacity. At 10% power output no SPR peak was observed indicating that temperature rise was not enough to initiate the synthesis reaction as shown in Figure 2. At 30% power output, SPR peaks began to appear with low absorbance but with broad peak. There was substantial increase in SPR peak intensity at 50%, 70% and 100% power outputs. Initial cloudy white color of the OPK8

EMIM extract turned pale yellow on the addition of gold chloride and finally to deep red color after 90 s of MW exposure. Increase in absorbance at higher MW power output might be due to higher temperature rise, which resulted in higher reduction rate and formation of AuNPs. There was minor blue shifting change in the maximum wavelength (λmax) from 527 nm to 524 nm when power output was increased from 30 % to 70 % (Figure 2 inset). Figure 2 here 3.3 Stability of AuNPs Solutions of AuNPs mixture prepared using OPK and OPK-EMIM were preserved in a sealed vial at room temperature to observe any changes of SPR peak positions over time. AuNPs prepared with OPK extract were found to be stable for two weeks, and after which almost all gold particles were settled down to the bottom and a clear solution was visible in the vial showing close to zero absorbance as shown in Figure S1. At this moment, it is not clear why AuNPs prepared using OPK extract showed such a poor stability. Various process parameters including solution pH, temperature and volume of extract are needed to optimize to enhance the colloidal stability. However, colloidal solution of AuNPs prepared using OPK-EMIM was remained stable. For more investigation, a stability evaluation of AuNPs was performed by monitoring the changes of SPR absorbance and particle size for 120 days using UV-vis and TEM analysis (Mechler et al., 2010) as shown in Figure 3. AuNPs mixtures retained its red color for 120 days but became faint, as compared to day 22 (Figure 3 inset) due to less absorbance value at day 120. Using OPKEMIM extract, SPR band was found sharp and narrow even after 120 days. However, 20.83 % reduction in absorbance was detected in comparison to maximum absorbance as measured on day 22. Figure 3 here 9

Initial absorbance value (day 1) of AuNPs synthesized using OPK-EMIM extract was 0.82A with λmax of 527 nm. The value was increased up to 1.85 A after 22 days, and then a slight reduction (~4.9% ) was observed after passing 30 days. The reduction in absorbance values of 8.66 %, 14.94 %, 20.83 % were recorded after 2, 3 and 4 months, respectively as shown in Figure 4 inset, although absorbance value was still quite high as compared to day 1. A linear reduction in absorbance values up to 120 days with R2 > 95% was observed. SPR bands for AuNPs using OPK-EMIM extract were still sharp after 120 days. Initial value for λmax (527 nm) was shifted toward 524 nm in first 30 days but remained firm in this position up to day 120 indicating absence of any aggregation of particles as shown in Figure 4. Reduction in absorbance of AuNPs was also reported after one month of synthesis using grape waste extract (Krishnaswamy et al., 2014) and even decreased after 48 hours using ipomoea carnea extract indicating agglomeration and settling of particles (Abbasi et al., 2015). Figure 4 Position of SPR band of an AuNPs hydrosol is dependent on its morphological characteristics, such as size and shape of the nanoparticles, surrounding medium and interparticle distance (Ghosh and Pal, 2007). Agglomeration of nanoparticles leads to changes in band position or evolution of a new SPR peak at higher wavelength (Richter et al., 2010). Freshly prepared AuNPs sol contained isolated particles and showed high absorbance value around 520 nm-530 nm and a minor peak at longer wavelength region (600 -700 nm). Ratio of absorbance values (R1) was used to predict the isolated character of synthesized nanoparticles (Ivanov et al., 2009)

(1)

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High value of R1 (>1) indicates isolated character of AuNPs. On contrary, R1  1 when both absorbance values in numerator and denominator tend to become equal showing a wide distribution of SPR band and presence of agglomerated nanoparticles (Ivanov et al., 2009). R1 values for AuNPs- OPK-EMIM were observed high as indicated in Figure 5. It increased from 2.73 to 3.39 from day 1 to day 15 respectively, followed by minor reduction up to day 30 i.e. 3.03 and reached up to 2.16 on day 120. However, its value was still higher than 1 indicated isolated and disperse nature of particles emphasizing an estimable sign for enhanced colloidal stability. Maximum value of R1 was observed between day 8 and day 15 where absorbance peak at 527 nm was still growing. If ratio of maximum absorbance over initial absorbance is taken as signal toward time dependent stability of AuNPs (R2) as indicated by Eq. 2. Using OPK-EMIM extract this ratio gradually increased up to day 22 (3.04) as value of absorbance was found maximum on day 22 as compared to the initial absorbance recorded on day 1 as shown in Figure 5. R2 value reduced after day 22 and moved down to 1.79 on days 120 that was still high and indicative of colloidal stability of AuNPs solution. (2) Data given above confirms that AuNPs synthesized using OPK-EMIM extract were significantly stable possibly due to the surface capping of particles by the negatively charged acetate ions of IL. Figure 5

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3.4 Effect of volume of extract on AuNPs stability Effect of volume of extract on the stability of AuNPs nanoparticles was conducted by reacting 1 mL of 2.28 mM gold precursor with different volumes of OPK-EMIM extract (0.3 mL, 0.6 mL, 1 mL, 1.5 mL and 2 mL). UV-vis spectrometer analysis showed a blue shift of SPR band position from 541 nm to 525 nm with an increase in the volume of extract from 0.3 mL to 2 mL (Figure 6 inset) due to the formation of small nanoparticles (Khalil et al., 2014). When 0.3 mL of OPKEMIM extract was used, SPR peak sharply decreased in the region of 530-541 nm and significantly increased in higher wavelength region 600 nm – 700 nm indicating strong aggregation of particles after 15 days as shown in Figure S2 (A). With an increase in extract volume to 0.6 mL, similar behavior was observed but peak height at higher wavelength region was comparatively lower as compared to that observed for 0.3 mL (Figure S2 (B)). Figure 6 here Using higher volume of extract i.e., 2 mL, absorbance value almost remained constant for 60 days, and there was only 3.01 % reduction in absorbance from maximum absorbance as shown in Figure S2 (E). The reduction in absorbance after 120 days were found to be 47.64%, 41.35%, 20.83%, 17.5% and 8.86% using 0.3 mL, 0.6 mL, 1 mL, 1.5 mL and 2 mL volume of extract, respectively (Figure 6). Higher volume of extract contained higher mass fraction of IL and biocompounds like phenols and flavonoids (Gan et al., 2012) that capped the nanoparticles resulting the stability of particles in suspended form. 3.5 TEM analysis TEM analysis was performed to measure the size and shape of the synthesized nanoparticles. Predominately, spherical shapes of nanoparticles along with few triangular and multiple twinned

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structure were observed while using both OPK and OPK-EMIM extracts. Data collected from more than 200 particles revealed that average particles diameter using OPK-EMIM was 8.72 nm which was marginally lower than the average particle diameter 9.64 nm recorded using OPK. Presence of IL [EMIM][OAc] reduced particle diameter and the particles were well-dispersed as indicated by TEM images shown in Figure 7. Using OPK extract, particles’ size was broad ranged with sign of little agglomeration and few larger particles even more than 20 nm diameters were observed. It is believed that phytochemicals presented in the plant species are acted as a stabilizers and capping agents. It was observed a minor difference in the AuNPs size using both extract. However, use of IL not only increased the extraction efficiency of phytochemicals (Passos et al., 2014) but slowed down the rate of aggregation of particles. Addition of IL coupled with bio-compounds presents in kernel extract was supportive to get higher uniformity and narrow size distribution. TEM histogram of day 1 revealed narrow particle size distribution for EMIM extract as compared to OPK extract as shown in Figure 7 and Figure 8. Figure 7 here Figure 8 here TEM analysis of AuNPs prepared using OPK-EMIM extract were carried out periodically after day 30, day 60, day 90 and day 120 to observe size variation and morphological characteristics as shown in Figure 9. TEM image showed a minor increase in mean particle size with time as 9.39 nm, 10.21 nm, 11.30 nm and 12.67 nm after day 30, day 60, day 90 and day 120, respectively. TEM image clearly demonstrated that nanoparticles remain dispersed and scattered throughout this period. Smaller size, highly disperse and minor variation in size exhibited that AuNPs particles were stabilized by capping action of imidazolium acetate ions. Figure 9 here 13

3.6 Surface zeta potential study Surface zeta potential was measured to record the surface charge on AuNPs. Using 1 mL of extract, zeta values of AuNPs using OPK and OPK-EMIM extract were observed -13.9 mV and 18.7 mV, respectively as shown in Figure 10 inset. Nanoparticles are considered to be stable if their surface potential values are between -30 mV and +30 mV (Anand et al., 2015). Many studies have proposed that surface active molecules or stabilizers in the reaction mixture create electrostatic interactions providing more stable nanoparticles. Hence, it is suggested that presence of IL can produce electrostatic and electrosteric forces to improve stability of AuNPs that was quite evident from higher zeta value from aqueous extract. Zeta values for IL mediated AuNPs were almost remained firm around its initial value and were measured as -18.7 mV, -21.4 mV, 19.6 mV, -20.1 mV, -19.9 mV after day 1, day 30, day 60, day 90 and day 120, respectively, indicating good colloidal stability as shown in Figure 10. Increase in zeta potential value up to day 30 possibly due to the formation of new particles as evident by higher absorbance intensities peaks up to day 22 as shown in Figure 3. Figure 10 here 3.7 XRD analysis XRD analysis was performed to evaluate crystallographic structure for AuNPs synthesized using OPK-EMIM extract after making a thin film on zero back ground plate. Characteristics peaks were appeared at 38.24°, 44.03°, 64.55° and 77.60° as shown in Figure 11. By comparing these peaks positions with standard JCPDS data, peaks can be assigned to (111), (200), (220) and (311) reflections of face centered cubic AuNPs (Kanchi et al., 2014; Nadaf and Kanase, 2016). Predominant orientation was observed at 111 due to the most dominant peak raised at 38.24°. Intensity ratio for 200/111 was measured to be 0.38, which was quite low as compared to bulk 14

conventional bulk intensity ratio (0.52). This result indicates that plane 111 is considered as primary one. The Debye-Scherer equation was used to estimate average crystalline diameter based on 111 peak and observed diameter was 7.91 nm. XRD diffraction pattern indisputably exhibits the crystalline nature of synthesized AuNPs. Figure 11 here 3.8 Estimation of settling time for AuNPs aggregates Nanoparticles have natural tendency to minimize their surface energy to acquire thermodynamic stability. In order to become stable, these nanoparticles either adsorb surrounding molecules or reduce their surface area to grow into larger particles as explained by Ostwald ripening growth (Dare et al., 2015). Addition of macromolecules like surfactants or polymers disrupts the hydrogen network resulting in a change of surface properties. When IL is used, ions cover the particles’ surface to produce electrosteric repulsion force. Accordingly, these nanoparticles keep away from each other to remain stable in the solution and form a three dimensional hydrogen bonding network that assist in synthesis and control of the size of metal nanoparticles (Kareem and Kaliani, 2015). Electrostatic stabilization originates from double layer electric charges presented around the surface of nanoparticles although maximum stability cannot be achieved by presence of only induced charges (Ott and Finke, 2007). IL[EMIM][OAc] can provide electrostatic stabilization and enable the generation of strong electrosteric stabilization due to the presence of acetate ion and alkyl chain. The small negative CH3COO- ions are adsorbed on the surface of AuNPs while imidazolium cations orientated outwardly create a bulky geometry for steric repulsion (Kraynov and Müller, 2011) as described in Figure 12. Derjaugin-Landau-Verwey-Overbeek (DLVO) theory explains that IL can provide electrosteric protection by covering the metal surface 15

generating a protective shell around the metal nanoparticles (Janiak, 2013). This approach predicts that first capping of nanoparticles is performed by anions that are the primary source for stabilization. In addition, these ions form loose coordination bonds with the surface of nanoparticles (Kraynov and Müller, 2011). Figure 12 here TEM analysis of AuNPs reveals that particle sizes for most of the gold particles were in range of 7-10 nm. As the particles size increased, height of primary peaks formed at 524 nm became less and rose up in the region of 600 nm -700 nm. Reduction in absorbance as a function of time can be illustrated by Lambert Beer’s law (Vanecht, 2012). It states that absorbance is directly proportional to concentration of absorbing species. Peaks moving down slowly and periodically with continuous decrease in absorbance value (Figure 3) indicated removal of nanoparticles from its colloidal form due to the increase in size, aggregate formation and settling down in the bottom as sediment. The number of collisions of each particle with other particles per unit time is expressed by Einstein–Smoluchowski equation for Brownian motion of colloids (Kraynov and Müller, 2011)

(3) Particle settling rate is dependent on particle density, fluid density, temperature of solution, degree of turbulence, size and shape of particles. All these factors are nearly the same for both extracts i.e. OPK-EMIM and OPK. However, they mainly differ in size of particles with the time as observed by UV-vis and TEM analysis. Stoke’s equation can be applied to predict the settling velocity of particles in stagnant fluid (Vanecht, 2012)

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(4) where ρp is density of particles (for AuNPs it is 19.3x103 kg/m3), ρf is density of fluid and that is 1000 kg/m3 for water and 1027 kg/m3 for [EMIM][OAc]. g is the gravitational force (9.8 m/s2), η is dynamic viscosity and that is 0.89 mPa.s for water. Dynamic viscosity for OPK-EMIM is considered as the same as OPK due to the small concentration of IL present (2%) in the mixture (Fendt et al., 2010). Stoke’s equation was initially designed for small micro size particles with low Reynolds number (Re ≤ 0.3) and it is not suitable for application on small size nanoparticle where surface forces are high as compared to gravitational forces. Intermolecular forces including Van der Waals gold ions interaction, induced dipole, dipole - dipole interactions, dispersion forces and Brownian motion dominate over Newtonian forces which means that gravitation forces have no impact over settling velocity of nanoparticles (Liyanage et al., 2016). Under these conditions, AuNPs are not settled until these form aggregates. Physical observation, UV absorbance and TEM images confirmed about minor presence of aggregates that become reasons to dominate the Newtonian force over intermolecular forces. Considering 4.5 nm mean radius for AuNPs as confirmed by TEM analysis, settling velocity of the AuNPs was measured as 9x10-10 m/s or 1 cm/4.26 months showing a relatively stable suspension. For vial length of 6 cm, it requires more than 24 nm particle diameter to completely settle down under these conditions. Average number of gold atoms per nanoparticles may be derived from Eq. 5 assuming that all particles are spherical in shape (Liu et al., 2007). (5) ρ is density of AuNPs and that is 19.3g/cm3 and atomic weight (M) of gold is 197 g/mole. Substituting these values in eq. 5, it becomes:

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(6) This equation explained that more numbers of atoms are occupied by a particle with larger diameter and hence tend to settle down rapidly. TEM image analysis after 120 days confirmed the increase in particles size and consequently it contains more number of gold atoms. Ultimately, particles tend to settle down rapidly showing less absorbance value as confirmed by UV-vis spectra. However, using higher volume of extract such as 2 mL, higher mass fraction of bio-compounds and imidazolium acetate ions were present in the reaction mixture. These were able to create strong repulsive forces and only 8.86 % reduction in absorbance was observed which was significant less in comparison to 20.83 % using 1 mL of extract. Moreover, with increase in average particle size (12.67 nm) after 120 days; mean settling velocity was changed to 1.80x10-9 m/s or 1 cm/2.15 months. This increase in settling velocity from initial one depicting that these particles will settle down rapidly. However, colloidal stability of solution was still quite enough to exist for 12 months. TEM image analyses after 120 days showed well disperse AuNPs which endorsed the validity of data and stability of colloidal gold solution. 4 Conclusions A single step bio-synthesis method was developed to synthesize stable AuNPs using oil palm kernels (OPK) extracts in the presence of IL[EMIM][OAc]. The use of IL facilitated to produce small, dispersed and stable AuNPs. Presence of IL resulted into blue shifting of SPR band position from 548 nm to 524 nm, smaller particle size formation from 9.64 nm to 8.72 nm and elevation of zeta potential value from -13.9 mV to -18.7 mV, which certainly indicated its supremacy to synthesize stable AuNPs in comparison to aqueous extract. IL mediated AuNPs were examined for 120 days and found to be stable with only 8.86% reduction in absorbance value. Application of ILs in synthesis of inorganic nanoparticles through bio-synthesis is still on

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its roots and required further research and development to investigate its effects on size, morphology, mechanism, composition, growth and functionality. We believe that the results obtained in this study will be beneficial for nanoscience community to design and develop ILassisted inorganic/organic nano-materials. Acknowledgement The present work is supported by STIRF fund (0153AA-C77) from Universiti Teknologi PETRONAS and FRGS fund (0153AB-I96) from Ministry of Higher Education, Malaysia. Authors gratefully acknowledge the financial assistance through Graduate Assistance (GA) scheme by Universiti Teknologi PETRONAS. References Abbasi, T., Anuradha, J., Ganaie, S., Abbasi, S. 2015. Gainful utilization of the highly intransigent weed ipomoea in the synthesis of gold nanoparticles. JKSUES, 27(1), 15-22. Adawiyah, N., Moniruzzaman, M. Hawatulaila, S., Goto M. 2016. Ionic liquids as a potential tool for drug delivery systems. MedChemComm, 7(10).1881-1897. An, K., Alayoglu, S., Ewers, T., Somorjai, G.A. 2012. Colloid chemistry of nanocatalysts: A molecular view. J. Colloid Interface Sci., 373(1), 1-13. Anand, K., Gengan, R., Phulukdaree, A., Chuturgoon, A. 2015. Agroforestry waste Moringa oleifera petals mediated green synthesis of gold nanoparticles and their anti-cancer and catalytic activity. J Ind Eng Chem., 21, 1105-1111. Anuradha, J., Abbasi, T., Abbasi, S. 2014. An eco-friendly method of synthesizing gold nanoparticles using an otherwise worthless weed pistia (Pistia stratiotes L.). JAR. 6(5), 711-720

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Figure Captions: Figure 1: Comparison of λmax for AuNPs using OPK-EMIM and OPK Inset; physical colour appearance of (a) OPK extract, (b) AuNPs using OPK (c) AuNPs using OPK-EMIM extract Figure 2: UV-vis analysis: change of SPR absorbance peak intensities with changing total microwave power output efficiency, Inset; variation of λmax with total microwave power output efficiency Figure 3: UV-vis spectra analysis; variation of absorbance intensities for AuNPs with time using OPK-EMIM extract, Inset; Colour of colloidal gold on (a) day 1, (b) day 22, (c) day 120 Figure 4: UV-vis spectra analysis; variation in absorbance value and shifting of λmax with time, Inset; Reduction of absorbance (%) with time based on periodic UV-vis analysis Figure 5: Absorbance ratios of AuNPs as a function of time Figure 6: UV-vis spectra: Reduction of absorbance (%) with time using different volume of extract, Inset; Change in λmax with increase in volume of extract Figure 7: TEM image and Histogram for AuNPs synthesized using OPK-EMIM extract, predominantly spherical and dispersed particles

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Figure 8: TEM image and Histogram for AuNPs synthesized using OPK extract, predominantly spherical particles with minor aggregation Figure 9: TEM images for AuNPs synthesized using OPK-EMIM extract with time on (A) day 1, (B) day 30, (C) day 60, (D) day 90, (E) day 120 Figure 10: Change of surface zeta potential for AuNPs with time using OPK-EMIM extract, Inset; surface zeta potential value for AuNPs using (a) OPK-EMIM extract, (b) OPK extract Figure 11: XRD pattern of AuNPs Figure 12: Schematic layout to stabilize AuNPs using OPK-EMIM extract Figure S1: UV-vis spectra: change of absorption intensities with settling of AuNPs prepared using OPK extract Figure S2: UV-vis spectra: absorbance variation using different volume of OPK-EMIM extract; A (0.3 mL), B (0.6 mL), C (1 mL), D (1.5 mL), E (2 mL)

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All Figure revised

Figure 1: Comparison of λmax for AuNPs using OPK-EMIM and OPK, Inset; physical colour appearance of (a) OPK extract, (b) AuNPs using OPK extract (c) AuNPs using OPK-EMIM extract

Figure 2: UV – vis analysis: change of SPR absorbance peak intensities with total microwave power output efficiency, Inset; variation of λmax with total microwave power output efficiency

Figure 3: UV-vis spectra analysis; variation of absorbance intensities for AuNPs with time using OPK-EMIM extract, Inset; Colour of colloidal gold on (a) day 1, (b) day 22, (c) day 120

Figure 4: UV-vis spectra analysis; variation in absorbance value and shifting of λmax with time, Inset; Reduction of absorbance (%) with time based on periodic UV-vis analysis

Figure 5: Absorbance ratio of AuNPs as a function of time

Figure 6: UV –vis spectra: Reduction of absorbance (%) with time using different volume of extract, Inset; Change in λmax with increase in volume of extract

Figure 7: TEM image and Histogram for AuNPs synthesized using OPK-EMIM extract, spherical and dispersed particles

Figure 8: TEM image and Histogram for AuNPs synthesized using OPK-Aq. extract, spherical particles with minor aggregation

Figure 9: TEM images for AuNPs synthesized using OPK-EMIM extract with time on (A) day 1, (B) day 30, (C) day 60, (D) day 90, (E) day 120

Figure 10: Change of surface zeta potential for AuNPs with time using OPK-EMIM extract, Inset; surface zeta potential value for AuNPs using (a) OPK-EMIM extract, (b) OPK-Aq. extract

Figure 11: XRD pattern of AuNPs

Figure 12: Schematic layout to stabilize AuNPs using OPK-EMIM extract