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

2.1 Materials

Gold (III) chloride hydrate (HAuCl₄⋅3H₂0, 99.99%) and IL[EMIM][OAc] (1-ethyl-3-methylimidazolium 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 of analytical grade.

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, 800 W) at power outputs ranging between 10% and 100%. 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 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 were 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 particle size distributions.

2.4 Characterization of AuNPs

Perkin Elmer Lambda 25 UV-visible spectrophotometer was used to record SPR absorbance and peak wavelength (λ_max) 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 15,000 rpm for 10 min and deposited on zero back ground plate followed by drying at 60 °C for 90 min.

3.1 UV-vis analysis

Preliminary assessment for AuNPs synthesis can be observed from color change occurred in reaction mixture (Fig. 1 inset). Initial color of aqueous extract of OPK was translucent white. 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 color using OPK and OPK-EMIM extracts, respectively due to their SPR (Dzimitrowicz et al., 2019a). Parallel reactions were performed using OPK and OPK-EMIM extracts and sharp SPR peaks were observed. For OPK, the value of λ_max 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., 2019c; Mostafa et al., 2012) as shown in Fig. 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., 2019b). No other SPR peaks were found near infrared region indicated the absence of any other shape particles such as trigonal and hexagonal (Shankar et al., 2005). This observation was confirmed by TEM image analysis.

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Figure 1 Comparison of λ_max for AuNPs using OPK-EMIM and OPK. Inset; physical color appearance of (a) OPK extract, (b) AuNPs using OPK and (c) AuNPs using OPK-EMIM extract.

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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 Fig. 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 OPK-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 (λ_max) from 527 nm to 524 nm when power output was increased from 30% to 70% (Fig. 2 inset).

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Figure 2 UV-vis analysis: change of SPR absorbance peak intensities with changing total microwave power output efficiency, Inset; variation of λ_max with total microwave power output efficiency.

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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. A clear solution was visible in the vial showing close to zero absorbance as shown in Fig. 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 Fig. 3. AuNPs mixtures retained its red color for 120 days but became faint, as compared to day 22 (Fig. 3 inset) due to less absorbance value at day 120. Using OPK-EMIM extract, SPR band was found to be sharp and narrow even after 120 days. However, 20.83% reduction in absorbance was detected in comparison with maximum absorbance as measured on day 22.

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

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Initial absorbance value (day 1) of AuNPs synthesized using OPK-EMIM extract was 0.82 A with λ_max 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%, and 20.83% was recorded after 2, 3 and 4 months, respectively as shown in Fig. 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 λ_max (527 nm) was shifted toward 524 nm in first 30 days but remained firm in this position up to day 120 indicating the absence of any aggregation of particles as shown in Fig. 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 h using ipomoea carnea extract indicating agglomeration and settling of particles (Abbasi et al., 2015).

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

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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 520–530 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)

High value of R₁ (>1) indicates isolated character of AuNPs. On the contrary, R₁ ≤ 1 when both absorbance values in numerator and denominator tend to become equal showing a wide distribution of SPR band and the presence of agglomerated nanoparticles (Ivanov et al., 2009). R₁ values for AuNPs-OPK-EMIM were observed high as indicated in Fig. 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.

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Figure 5 Absorbance ratios of AuNPs as a function of time.

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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 to be maximum on day 22 as compared to the initial absorbance recorded on day 1 as shown in Fig. 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.

Data given above confirm 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.

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 (Fig. 6 inset) due to the formation of small nanoparticles (Mostafa et al., 2014). When 0.3 mL of OPK-EMIM extract was used, SPR peak sharply decreased in the region of 530–541 nm and significantly increased in higher wavelength region 600–700 nm indicating strong aggregation of particles after 15 days as shown in Fig. 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 (Fig. S2(B)).

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Figure 6 UV-vis spectra: reduction in absorbance (%) with time using different volumes of extract, Inset; change in λ_max with increase in volume of extract.

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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 Fig. S2(E). The reduction in absorbance after 120 days was 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 (Fig. 6). Higher volume of extract contained higher mass fraction of IL and bio-compounds such as 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 structures were observed while using both OPK and OPK-EMIM extracts. Data collected from more than 200 particles revealed that average particle diameter using OPK-EMIM was 8.72 nm which was marginally lower than the average particle diameter 9.64 nm recorded using OPK. The presence of IL [EMIM][OAc] reduced particle diameter and the particles were well-dispersed as indicated by TEM images shown in Fig. 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 extracts. 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 present 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 Figs. 7 and 8.

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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.

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TEM analysis of AuNPs prepared using OPK-EMIM extract was carried out periodically after day 30, day 60, day 90 and day 120 to observe size variation and morphological characteristics as shown in Fig. 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.

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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.

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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 Fig. 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 the 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, and −19.9 mV after day 1, day 30, day 60, day 90 and day 120, respectively, indicating good colloidal stability as shown in Fig. 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 is shown in Fig. 3.

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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.

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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 background plate. Characteristics peaks were appeared at 38.24°, 44.03°, 64.55° and 77.60° as shown in Fig. 11. By comparing these peaks positions with standard JCPDS data, peaks can be assigned to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) reflections of face centered cubic AuNPs (Kanchi et al., 2018; Nadaf and Kanase, 2019). Predominant orientation was observed at 1 1 1 due to the most dominant peak raised at 38.24°. Intensity ratio for 2 0 0/1 1 1 was measured to be 0.38, which was quite low as compared to bulk conventional bulk intensity ratio (0.52). This result indicates that plane 1 1 1 is considered as primary one. The Debye-Scherer equation was used to estimate average crystalline diameter based on 1 1 1 peak and observed diameter was 7.91 nm. XRD diffraction pattern indisputably exhibits the crystalline nature of synthesized AuNPs.

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Figure 11 XRD pattern of AuNPs.

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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 such as 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 assists in synthesis and control of the size of metal nanoparticles (Kareem and Kaliani, 2019).

Electrostatic stabilization originates from double layer electric charges presented around the surface of nanoparticles although maximum stability cannot be achieved by the 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 Fig. 12. Derjaguin-Landau-Verwey-Overbeek (DLVO) theory explains that IL can provide electrosteric protection by covering the metal surface 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).

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

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TEM analysis of AuNPs reveals that particle sizes for most of the gold particles were in range of 7–10 nm. As the particle size increased, height of primary peaks formed at 524 nm became less and rose up in the region of 600–700 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 (Fig. 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)

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)

Considering 4.5 nm mean radius for AuNPs as confirmed by TEM analysis, settling velocity of the AuNPs was measured as 9 × 10⁻¹⁰ m/s or 1 cm/4.26 months showing a relatively stable suspension. For vial length of 6 cm, it requires more than 24 months for particles  to be completely settled 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).

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, a 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 with 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 1.80 × 10⁻⁹ 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 dispersed AuNPs which endorsed the validity of data and stability of colloidal gold solution.