Chapter 3 SYNTHESIS AND CHARACTERIZATION OF SILVER NANOPARTICLES 3.1 Introduction The important properties of nanoparticles that could affect nanoparticle behaviour and toxicity comprises of particle size, shape, surface properties, aggregation state, solubility, structure and chemical make-up. Therefore, when analysing nanoparticles in different matrices, it is not only the composition and concentration that will need to be determined but also the physical and chemical properties of the engineered nanoparticles within the sample and the chemical characteristics of any capping/functional layer on the particle surface. Methods are available that have been developed for natural nanomaterials or engineered nanomaterials in simple matrices which could be optimized to provide the necessary information. These include microscopy, chromatography, spectroscopy, centrifugation as well as filtration and related techniques. A combination of these is often required (Tiede et al., 2008). This chapter describes the synthesis of silver nanoparticles in yeast growth media (YM media). The verification of the synthesis process and particle characterization was done by the techniques of UV-Vis absorption spectroscopy, transmission electron microscopy (TEM) and X-ray diffraction (XRD). A general discussion of the experimental findings, in view of previous reports, concludes the chapter. A wide range of spectroscopic methods is available for nanoparticle analysis and characterization. UV-Vis and infrared spectroscopy offer the possibility to characterise nanoparticles, especially quantum dots and organic based nanoaprticles like fullerenes and carbon nanotubes. Fourier transformation infrared (FTIR) and UV-Vis spectroscopy have been used to compare aqueous
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Chapter 3
SYNTHESIS AND CHARACTERIZATION OF SILVER
NANOPARTICLES
3.1 Introduction
The important properties of nanoparticles that could affect nanoparticle behaviour
and toxicity comprises of particle size, shape, surface properties, aggregation
state, solubility, structure and chemical make-up. Therefore, when analysing
nanoparticles in different matrices, it is not only the composition and
concentration that will need to be determined but also the physical and chemical
properties of the engineered nanoparticles within the sample and the chemical
characteristics of any capping/functional layer on the particle surface. Methods
are available that have been developed for natural nanomaterials or engineered
nanomaterials in simple matrices which could be optimized to provide the
necessary information. These include microscopy, chromatography,
spectroscopy, centrifugation as well as filtration and related techniques. A
combination of these is often required (Tiede et al., 2008).
This chapter describes the synthesis of silver nanoparticles in yeast growth media
(YM media). The verification of the synthesis process and particle
characterization was done by the techniques of UV-Vis absorption spectroscopy,
transmission electron microscopy (TEM) and X-ray diffraction (XRD). A general
discussion of the experimental findings, in view of previous reports, concludes
the chapter.
A wide range of spectroscopic methods is available for nanoparticle analysis and
characterization. UV-Vis and infrared spectroscopy offer the possibility to
characterise nanoparticles, especially quantum dots and organic based
nanoaprticles like fullerenes and carbon nanotubes. Fourier transformation
infrared (FTIR) and UV-Vis spectroscopy have been used to compare aqueous
Synthesis and Characterization of Silver Nanoparticles 53
colloidal suspensions of C60 (Andrievsky et al., 2002). Pesika et al. (2003) also
used UV spectroscopy to study the relationship between absorbance spectra and
particle size distributions for quantum-sized nanocrystals. Particularly in the case
of silver nanoparticles, the UV-Vis absorption spectra have proved to be quite
sensitive as the plasmon peak and full width at half maximum (fwhm) depends on
the extent of colloid aggregation (Yamamoto et al., 2001).
X-ray diffraction is a non-destructive technique and can reveal information about
the crystallographic structure, size and elemental composition of natural and
manufactured materials. It has been widely applied in the characterization of
nanoparticles including those synthesized using biological agents. The diffraction
spectra can be conveniently compared with powder diffraction files (PDF)
available from the International Centre for Diffraction Data, USA for verification
of nanoparticle composition.
Microscopic techniques can be applied not only to visualize nanoparticles but
also to generate useful data on the size, size distribution and other measurable
properties (Rabinski and Thomas, 2004). Electron microscopy is one of the
techniques that have played a major role on studding nanoparticles. Since the
early conventional bright field images and the intermediate resolution dark field
techniques, to the high-resolution atomic images of nanoparticles the results have
shown that indeed the nanoparticles, in the range of a few nanometers, can have
well-defined crystal structures (Jose-Yacaman et al., 2001).
3.2 Materials and Methods
A list of all buffers and reagents used in the following protocols are described in
Appendix I.
3.2.1 Synthesis of Silver Nanoparticles
Silver nanoparticles used throughout this study were synthesized in the lab by the
Creighton method which involves reduction of silver nitrate (AgNO3) by sodium
Synthesis and Characterization of Silver Nanoparticles 54
borohydride (NaBH4). Reactions were carried out in de-ionized water as well as
in liquid Yeast Malt (YM) growth media.
Requirements
§ AgNO3 solution: 1.0 x 10-3
M
§ NaBH4 solution: 2.0 x 10-3
M
§ Magnetic stirrer & bar
§ Burette and glasswares
Procedure
(i) In a 100 ml Erlenmeyer flask 0.84 g of Yeast Malt media (HiMedia Labs)
was dissolved in 15 ml of de-ionized water.
(ii) 15 ml of 2.0 x 10-3
M NaBH4 (Merck) was then added and the flask was
cooled at 4ºC for 30 min.
(iii) A stir bar was placed in the Erlenmeyer and the assembly was centered on a
magnetic stir plate.
(iv) Under constant stirring, 10 ml of 1 x 10-3
M AgNO3 (SRL) was added drop-
wise using a burette supported with a clamp. The stirring was stopped
immediately after all of the silver nitrate solution had been added.
Note: All glasswares were first cleaned with chromic acid and then with
detergent. A final rinse was done in alcoholic KOH and then in de-ionized water
before use.
If stirring is continued once all the silver nitrate has been added, aggregation is
likely to occur.
The method described above was used for the synthesis of silver nanoparticles of
concentration 26.95 µg/ml in YM media. Other concentrations of Ag NPs were
also synthesized which are detailed in Table 3.1.
Synthesis and Characterization of Silver Nanoparticles 55
Stock Solutions
(I) Silver Nitrate Solution (AgNO3)
(a) 2.0 x 10-3
M (b) 2.6 x 10-3
M
(II) Sodium Borohydride Solution (NaBH4)
(c) 4.0 x 10-3
M (d) 4.0 x 10-2
M (e) 5.2 x 10-2
M
Table 3.1: Cocktail for nanoparticle synthesis
CONCENTRATION
(µg/ml)
AgNO3 (M) * NaBH4 (M) #
YM media (g)
5.39
2.0x10-4
(1ml stock a)
4.0x10-4
(3ml stock c)
0.84
16.17
6.0x10-4
(3ml stock a)
1.2x10-3
(9ml stock c)
0.84
26.95
1.0x10-3
(5ml stock a)
2.0x10-3
(15ml stock c)
0.84
37.73
1.4x10-3
(7ml stock a)
2.8x10-2
(21ml stock d)
0.84
48.51
1.8x10-3
(9ml stock a)
3.6x10-2
(27ml stock c)
0.84
53.90
2.0x10-3
(10ml stock a)
4.0x10-2
(30ml stock c)
0.84
70.07
2.6x10-3
(10ml stock b)
5.2x10-2
(30ml stock e)
0.84
* Final volume made up to 10 ml with de-ionized water. # Final volume made up
to 30 ml with de-ionized water.
Synthesis and Characterization of Silver Nanoparticles 56
3.2.2 Characterization of Nanoparticles
The synthesized particles were characterized by absorption spectroscopy, TEM
and XRD. This section describes how the analyses were made.
3.2.2.1 Analysis by Absorption Spectroscopy
The absorbance spectrum of the nanoparticle solutions – both in de-ionized water
and in YM media – were recorded immediately (within 1h) after the synthesis
was done. Before recording the absorbance spectrum a reference of de-ionized
water or pure YM media was recorded, as appropriate.
To assess the stability of the nanoparticle solutions, the absorbance spectrum was
recorded at 1 day, 15 days and 30 days after synthesis.
The spectrophotometer used was a Chemito UV Scan 2600 (Thermo Fisher) and
the software was Spectrum™ Version 6.87. Absorbance spectra were recorded
over the range of 300 – 700 nm. Wavelength of peak absorbance, λmax was noted
and the fwhm (full width at half-maximum) was calculated according to He et al.
(2001).
3.2.2.2 Analysis by Transmission Electron Microscopy
Particle size and shape were determined with a JEOL JSM 100 CX II
transmission electron microscope operating at a maximum of 100 kV.
100 µl of nanoparticle solutions were deposited on carbon coated copper grids
(400 mesh) and dried at 30ºC before image capture.
Particle size distributions were determined from TEM images and results were
plotted using GraphPad Prism®
Version 5.03.
Synthesis and Characterization of Silver Nanoparticles 57
3.2.2.3 Analysis by X-Ray Diffractometer
The crystal structure and also the particle size were studied by X-ray Diffraction.
1 ml of the nanoparticle solutions were spread on a glass slide and dried at 40ºC
in an oven. The process was repeated 3-4 times to obtain a thin film.
The spectra were recorded in a Phillips Xpert Pro Diffractometer (Cu Kα
radiation, λ1 = 1.54056; λ2 = 1.54439) running at 40 kV and 30 mA. The
diffracted intensities were recorded from 35.01 degrees to 79.99 degrees 2θ
angles.
The crystalline size was calculated from the half-height width of the diffraction
peak of XRD pattern using the Debye-Scherrer equation:
Where,
D = crystalline size, Ǻ
K = crystalline-shape factor
λ = X-ray wavelength
θ = observed peak angle, degree
β = X-ray diffraction broadening, radian
The crystallinity of the particles was evaluated through a comparison of
crystallite size from XRD and TEM particle size determination by the following
equation:
Where,
Icry is the crystallinity index
Dp is the particle size (obtained from either TEM or SEM morphological
analysis)
Dcry is the particle size (calculated from the Scherrer equation)
Synthesis and Characterization of Silver Nanoparticles 58
3.3 Experimental Findings
3.3.1 Particle synthesis and concentrations
Nanoparticles were synthesized in de-ionized water as well as in YM media
without the use of any stabilizing agent.
Figure 3.1 Silver nanoparticles synthesized in YM media at various
concentrations. (a) YM media (w/o Ag NPs) and YM media with 5.39, 26.95,
43.12, 48.51 and 53.90 µg/ml Ag NPs (left – right) and (b) Pure YM media (w/o
Ag NPs) and YM media with 58.29, 64.68 and 70.07 µg/ml Ag NPs (left – right).
The colour of the colloidal solutions varied from light yellow to dark brown
(Figure 3.1) depending on the concentration of nanoparticles. There was no
obvious aggregation of particles as the colour remained stable even after
Synthesis and Characterization of Silver Nanoparticles 59
autoclaving at 15 psi for 20 min. Aggregation is generally visualized by a change
in colour as the yellow darkens, turns violet then grayish as the particles settle
out. Finally a colourless solution with black precipitates of silver is seen. The
formation process and the optical properties of the silver nanoparticles can also
be identified from both the colour change and the UV-Vis spectra of the solutions
(He et al., 2002).
An important aspect of the synthesis process was the determination of particle
concentration, as the effect on cells would likely be dose dependent. We followed
a theoretical method based on the assumption that there is a compete reduction of
the AgNO3 used in the synthesis process. This is likely to be the case as the
reducing agent, NaBH4, was used in excess (up to 2 x 102 times) to that of the
silver salt. The UV-vis spectra of the synthesized nanoparticle solutions described
in Section 3.3.2 indicate that indeed there is total reduction of the silver salt as
absorption peaks attributable to silver ions (Ag+) were not detected.
In a typical synthesis reaction the silver salt was reduced to metallic silver
according to the following reaction:
The concentration of silver particles in the solution would then depend on the
molarity of the initial silver nitrate solution and the total reaction volume.
For example, in the synthesis of 26.95 µg/ml nanoparticle concentration:
10 ml of 10-3
M AgNO3 was reduced with 30 ml of 2 x 10-3
M NaBH4. Total
silver (At. wt. 107.8) content of the reaction volume would be 1.078 x 10-3
g in
40 ml which is equivalent to 26.95 µg/ml.
Likewise, nanoparticle solutions of different concentrations were prepared by
varying the strength of the starting silver nitrate solution albeit with concomitant
changes in the NaBH4 concentration to ensure complete reduction. A similar
method to calculate nanoparticle concentration was described by Xu et al. (2004).
Synthesis and Characterization of Silver Nanoparticles 60
3.3.2 Optical properties
The optical properties of nanoparticle colloidal solutions of various
concentrations were determined in a UV-Vis spectrophotometer. The spectra
recorded are presented below:
Figure 3.2 UV-Vis absorption spectra of silver nanoparticle solutions of
concentrations 5.39 µg/ml (I) and 16.17 µg/ml (II).
Synthesis and Characterization of Silver Nanoparticles 61
Figure 3.3 UV-Vis absorption spectra of silver nanoparticle solutions of
concentrations 26.95 µg/ml (III) and 37.73 µg/ml (IV).
Synthesis and Characterization of Silver Nanoparticles 62
Figure 3.4 UV-Vis absorption spectra of silver nanoparticle solutions of
concentrations 48.51 µg/ml (V) and 53.90 µg/ml (VI).
Synthesis and Characterization of Silver Nanoparticles 63
Figure 3.5 UV-Vis absorption spectra of silver nanoparticle solutions of
concentrations 64.68 µg/ml (VII) and 70.07 µg/ml (VIII).
Synthesis and Characterization of Silver Nanoparticles 64
Figure 3.6: Super-imposed UV-Vis spectra of the various nanoparticles
concentrations presented in Figures 3.2 – 3.5 above. Peak absorption can be
observed between 380 nm to 420 nm. Time: 1 h after synthesis.
Table 3.2: Optical characteristics of synthesized nanoparticle solutions: Peak
absorbance (λmax) & full width at half maximum (FWHM).
S. No Nanoparticle Concentration
(µg/ml)
λmax
(nm)
FWHM
(nm)
1 5.39 416.5 77.50
2 16.17 416.5 97.50
3 26.95 407.0 92.50
4 37.73 396.5 96.25
5 48.51 392.0 108.75
6 53.90 391.0 82.50
7 64.68 390.5 93.75
8 70.07 393.5 85.00
Synthesis and Characterization of Silver Nanoparticles 65
Figure 3.7 UV-Vis spectra of representative silver nanoparticle solution recorded
at different time intervals: 1 day (a); 15 days (b) and 30 days (c).
Table 3.3: Optical characteristics of a representative nanoparticle solution
recorded at different time intervals.
Time after Synthesis
(days)
λmax
(nm)
FWHM
(nm)
1 398.5 97.50
15 398.2 97.75
30 408.2 101.25
The absorption spectrum of the samples (Table 3.2) show a well-defined plasmon
band between 416 – 390 nm with a fwhm range of 77 – 109 nm, which are
characteristic of nanosized silver. Theoretical and experimental studies
(Chakraborty, 1998; Otter, 1961), in which the optical properties of silver
particles have been discussed report the appearance in the electronic absorption
spectrum of a band located at 396 nm, associated with the presence of small
spherical silver nanoparticles. If the particles would not be spherical (or
equiaxial), the absorption band would appear at longer wavelengths and would
Synthesis and Characterization of Silver Nanoparticles 66
gradually shift to shorter wavelengths as the particles become more spherical
(Chakraborty, 1998; Baia et al., 2006). Because the position of the electronic
absorption bands shown in Figure 3.6 and Table 3.2 are close to the above-
mentioned value we assume that the particles synthesized in YM media have a
roughly spherical shape. Particle size and shape were further confirmed by
electron microscopy as described below in Section 3.3.4.
The individual UV-VIS absorption spectra of the Ag NP samples, with particle
concentrations of 5.39 µg/ml to 70.07 µg/ml, are displayed in Figures 3.2 to 3.5.
Absorption bands are smooth with a single pronounced plasmon resonance that
appear around 400 nm. Lu et al. (2005) have reported that the electronic
transitions involving the Ag+ ion give rise to absorption bands located between
200 and 230 nm, whereas the electronic transitions of metallic Ag0 appear in the
250-330 nm spectral range. The UV-VIS spectra of all samples analyzed in this
study did not show distinct absorption signals around 230 nm due to the
electronic transitions involving Ag+ ions. The presence of silver ions in the
synthesized nanoparticle solutions can so be safely assumed to be either non-
existent or of infinitesimal concentration. As has been mentioned in Section
3.3.1, a large excess of the reducing agent was used during the synthesis process,
possibly leading to a complete reduction of the silver salt.
The stability of synthesized silver nanoparticle solutions was assessed by
recording the UV-vis spectra at intervals of 1, 15 and 30 days after storage at
ambient temperature. The evolution of UV-VIS spectra is shown in Figure 3.7 (b
and c curves). There was no obvious change in peak position for two weeks,
except for the increase of absorbance. Increase of absorption indicates that
amount of silver nanoparticles increases. The stable position of absorbance peak
indicates that new particles do not aggregate. After the fourth week (curve c) the
fwhm of the spectrum starts to become wider than before, and the position of the
peak has a slight red shift, implying the onset of nanoparticle aggregation. These
spectra demonstrate that the silver nanoparticle colloidal solution can remain
stable for about 1 month.
Synthesis and Characterization of Silver Nanoparticles 67
3.3.3 Crystalline structure
The X-ray diffraction spectrum of the synthesized nanoparticles is shown in
Figure 3.8.
Figure 3.8 XRD spectra of representative nanoparticle concentration (26.95
µg/ml): Peak indices and 2θ positions.
A number of Bragg reflections in the (111), (200), (220) and (311) set of lattice
planes were observed. The high intensity for fcc materials is generally (111)
reflection, which is observed in the samples. This confirmed the lattice structure
to be fcc (face centered cubic). The crystalline size was calculated from the half-
height width of diffraction peaks using the Debye-Scherrer equation. Data are
presented in Table 3.4.
Table 3.4: Particle size derived from XRD spectra.
Particle
concentration
2θ
(degree)
FWHM
(radian)
Particle size
(nm)
Crystal
lattice
26.95 µg/ml 38.31 0.014 10.3 fcc
70.07 µg/ml 38.35 0.013 10.9 fcc
Synthesis and Characterization of Silver Nanoparticles 68
Particle size of around 10 nm was derived from the XRD spectra. These values
represent a rough estimate as the total broadening of the diffraction peak is due to
the sample and the instrument.
Table 3.5: Crystallinity index of silver nanoparticles.