J Nanopart Res (2010) 12:237–246

DOI 10.1007/s11051-009-9602-5

RESEARCH PAPER

Process variables in biomimetic synthesis of silver

nanoparticles by aqueous extract of Azadirachta indica
(Neem) leaves
A. Tripathy Æ Ashok M. Raichur Æ
N. Chandrasekaran Æ T. C. Prathna Æ
Amitava Mukherjee

Received: 10 June 2008 / Accepted: 6 February 2009 / Published online: 20 February 2009
Ó Springer Science+Business Media B.V. 2009

Abstract Owing to widespread applications, syn- size with nearly spherical shape were produced. On
thesis and characterization of silver nanoparticles is increasing interaction time (ageing) to 66 days, both
recently attracting considerable attention. Increasing aggregation and shape anisotropy (ellipsoidal, poly-
environmental concerns over chemical synthesis hedral and capsular) of the particles increased. In
routes have resulted in attempts to develop biomi- alkaline pH range, the stability of cluster distribution
metic approaches. One of them is synthesis using increased with a declined tendency for aggregation of
plant parts, which eliminates the elaborate process of the particles. It can be inferred from the study that
maintaining the microbial culture and often found to fine tuning the bioprocess parameters will enhance
be kinetically favourable than other bioprocesses. possibilities of desired nano-product tailor made for
The present study deals with investigating the effect particular applications.
of process variables like reductant concentrations,
reaction pH, mixing ratio of the reactants and Keywords Biomimetic process

interaction time on the morphology and size of silver Azadirachta indica
Silver nanoparticles

nanoparticles synthesized using aqueous extract of Process variability
Shape anisotropy

Azadirachta indica (Neem) leaves. The formation of Size aggregation
Synthesis
Plant biotechnology
crystalline silver nanoparticles was confirmed using
X-ray diffraction analysis. By means of UV spec-
troscopy, Scanning and Transmission Electron Introduction
Microscopy techniques, it was observed that the
morphology and size of the nanoparticles were Recently, synthesis of silver nanoparticles has
strongly dependent on the process parameters. Within attracted considerable attention owing to their diverse
4 h interaction period, nanoparticles below 20-nm- properties like catalysis (Shiraishi and Toshima
2000), magnetic and optical polarizability (Shiraishi
and Toshima 2000), electrical conductivity (Chang
A. Tripathy
N. Chandrasekaran
T. C. Prathna

and Yen 1995), antimicrobial activity (Sharverdi
A. Mukherjee (&)
School of Biotechnology, Chemical and Biomedical et al. 2007) and Surface Enhanced Raman Scattering
Engineering, VIT University, Vellore, India (SERS) (Matejka et al. 1992).
e-mail: [email protected] A number of synthesis techniques have been
developed including chemical reduction of silver ions
A. M. Raichur
Department of Materials Engineering, Indian Institute of in aqueous solutions, with or without stabilizing
Science, Bangalore, India agents (Liz-Marzan and Lado-Tourino 1996), thermal

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decomposition in organic solvents (Esumi et al. 1990) like reductant concentrations, reaction pH, mixing
and chemical and photo reduction in reverse micelles ratio of the reactants and interaction time on the
(Pileni 2000; Sun et al. 2001), to name a few. Most of biomimetic synthesis of silver nanoparticles by
these methods are extremely expensive and they also aqueous extract of Azadirachta indica (Neem) leaves.
involve the use of toxic, hazardous chemicals which
may pose potential environmental and biological risks.
A quest for an environmentally sustainable synthe- Experimental
sis process has led to few biomimetic approaches.
Biomimetics refers to applying biological principles in Materials
materials formation. One of the fundamental processes
in the biomimetic synthesis involves bioreduction. Fresh Neem leaves were finely cut and washed with
Recently, a number of inorganic nanomaterials distilled water. A known amount of leaves was added
have been synthesized by bioreduction processes to 100 mL of deionized water and boiled for 15 min
employing different microorganisms. Nanocrystals in a water bath. The mixture was then filtered to
of gold, silver and their alloys have been synthesized obtain aqueous extract of definite concentrations. For
within cells of lactic acid bacteria (Nair and Pradeep synthesis of silver nanoparticles, a known concentra-
2002). The bacterium Pseudomonas stutzeri AG259, tion of Neem leaf broth was interacted with 0.01 M
an isolate from silver mine, reduced the Ag? ions to AgNO3 solution at a definite mixing ratio to make up
form nanoparticles of well-defined size and distinct 100 mL volume in 250 mL Erlenmeyer flasks. The
morphology within the periplasmic space of the flasks were incubated in a rotary shaker at 120 rpm
bacteria (Joerger et al. 2000; Klaus et al. 2001). In speed for a desired time at 28 °C.
addition, eukaryotic organisms such as fungi have also
been used to grow nanoparticles of different chemical Methods
composition and sizes like Verticillum sp. (Mukherjee
et al. 2001), Fusarium oxysporum (Ahmad et al. 2003) To enhance the reproducibility of the experimental
and Aspergillus flavus (Vigneshwaran et al. 2007). results and to optimize the biosynthesis process as a
Sometimes, the synthesis of nanoparticles utilizing whole, the factorial Design of Experiments, the ‘‘one-
plants/parts of plants could prove advantageous over factor-at-a time’’ method, was employed in this study.
other biological processes by eliminating the elaborate Here, the experimental factors are varied one at a
process of maintaining the microbial culture. Recently, time with the remaining factors held constant.
Jose-Yacaman and co-workers biosynthesized gold Different concentrations (5, 10, 15, 20, 40, 50 and
nanoparticles within live alfalfa plants (Gardea- 70%) of aqueous Neem leaf extract were prepared by
Torresdey et al. 2007). Gold nanoparticles have also boiling different amounts of freshly cut leaves in
been successfully synthesized using tamarind leaves 100 mL of deionized water for 2 min. They were
extract (Ankamwar et al. 2005). Pelargonium graveo- added to 0.01 M AgNO3 solution at 1:4 ratio and
lens (geranium plant) leaves extract and its endophytic incubated for 4 h in rotary shaker at 120 rpm.
fungus Colletotrichum sp. have been identified as The silver reduction reaction was also carried out at
exciting candidates for the synthesis of gold nanopar- different mixing ratios of Neem extract to 0.01 M
ticles (Shankar et al. 2003). AgNO3 (1:4, 2:3, 1:1, 3:2 and 4:1) at 20 and 40%
Among the various bioreductants, Neem leaves extract concentrations without varying the other
extract was chosen for the present study, since (i) conditions.
Neem is a quite commonly available plant, (ii) it In another variation of the synthesis process,
excludes addition of external stabilizing agent during aqueous Neem extract (20% w/v) was prepared with
synthesis and (iii) antimicrobial properties of the leaves shade dried for a duration of 7 days, 30 days
synthesized silver nanoparticles (one of the major end and 60 days, keeping the other synthesis parameters
uses) might be enhanced due to synergistic effects of constant.
Neem leaves. To follow the kinetics of the biosynthesis of
The principal objective of this work was to study nanoparticles, the Neem leaf extract (20% concen-
in detail the influence of various process variables tration) was interacted with 0.01 M AgNO3 in 1:4

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mixing ratio for different time intervals ranging from The purified powders obtained after 4 h interaction
30 min to 66 days to observe the effect of short-term were also subjected to X-ray diffraction analysis
and long-term kinetics (ageing). (JEOL—JDX 8030 diffractometer) and the target was
´
To observe the effect of pH of medium, the pH of Cu Ka (k = 1.54 Å). The generator was operated at
AgNO3 aqueous solution was varied as 2, 6, 7, 8, 9 45 kV and with a 30 mA current. The scanning range
and 10 using dilute sulphuric acid and ammonium (2h) was selected from 10° to 100°.
hydroxide solutions. Neem leaf aqueous extract was The transmission electron micrographs of the
interacted with 0.01 M AgNO3 solution of different purified, dried silver nanoparticles were taken using
pH levels for 6 and 24 h, respectively. the Tecnai G-20, a 200 kV TEM with a W-source and
The dry powders of the silver nanoparticles were an ultra high resolution pole piece with a point–point
obtained in the following manner: after desired resolution of 1.9 Å.
reaction period, the broth containing silver nanopar-
ticles was centrifuged at 10,000 rpm for 15 min,
following which the pellet was re-dispersed in sterile Results and discussion
distilled water to get rid of any uncoordinated
biological molecules. The process of centrifugation On adding the aqueous extract of Neem leaves to
and re-dispersion in sterile distilled water was AgNO3 solution, the colour of the reaction medium
repeated thrice to ensure better separation of free changed rapidly from colourless to brown. The
entities from the metal nanoparticles. The purified appearance of brown colour was due to the excitation
pellets were then freeze dried using a Lyophilizer of surface plasmon vibrations, typical of silver
(Thermo Electron Corporation—Micro Modulyo 230 nanoparticles (Ahmad et al. 2003). The localized
freeze dryer). SPRs are collective oscillations of the conduction
All the chemicals used were of reagent grade. electrons confined to metallic nanoparticles. Excita-
tion of the localized surface plasmons causes strong
Characterization light scattering by an electric field at a wavelength
where resonance occurs; this phenomenon results in
Preliminary characterization of the silver nanoparti- the appearance of strong SPR bands. The optical
cles was carried out using UV–visible spectroscopy. absorption spectrum of metal nanoparticles is domi-
Noble metals, especially gold (Au) and silver (Ag) nated by the SPR, which exhibits a shift towards the
nanoparticles exhibit unique and tunable optical red end or blue end depending upon the particle size,
properties on account of their surface plasmon shape, state of aggregation and the surrounding
resonance (SPR), dependent on shape, size and size dielectric medium (Mock et al. 2002; Mulvaney
distribution of the nanoparticles (Mulvaney 1996). 1996). In this study, the formation of silver nanopar-
The reduction of Ag? ions was monitored by ticles was initially confirmed using SPR phenomenon.
measuring the UV–visible spectra of the solutions For silver nanoparticles, kmax values were reported in
after diluting a small aliquot (0.2 mL) of the sample the visible range of 400–500 nm (Sastry et al. 1997).
20 times. UV–visible spectra were recorded on Figure 1a shows the UV–visible spectra of the
Hitachi double beam spectrophotometer (model U- nanoparticles obtained on varying the Neem broth
2800) from 200 to 600 nm. The distilled deionized concentrations. The particles synthesized with 5%
water was used as blank. broth concentration gave a very weak plasmon
The purified powders of silver nanoparticles were resonance band (absorbance value 0.509) at
subjected to FTIR spectroscopy measurement. These 417 nm. On increasing the concentration to 20%,
measurements were carried out on a Perkin–Elmer kmax increased to 444 nm with an absorbance 0.815.
Spectrum-One instrument in the diffuse reflectance On further increasing the concentration to 40% a
mode at a resolution of 4 cm-1 in KBr pellets. For small change in kmax to 441 nm was noted though
comparison, a drop of 20% Neem broth was mixed absorbance declined to 0.476. The observed changes
with KBr powder and pelletized after drying prop- in the kmax values were not very significant taking
erly. The pellets were later subjected to FTIR into consideration ±5% experimental error range.
spectroscopy measurement. The size of silver nanoparticles depends on the ratio

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Fig. 1 a UV–vis spectra recorded as a function of concentra- function of mixing ratio between two concentrations of Neem
tion of Neem leaves extract in a reaction with an aqueous leaves extract with an aqueous solution of 10-2 M AgNO3
solution of 10-2 M AgNO3. b UV–vis spectra recorded as a

of silver ions to capping agent/stabilizing agent. The time tests. With increasing interaction time from
slight variations in kmax values signify changes in 30 min to 4 h 15 min, a red shift in kmax from 420 to
particle size owing to changing concentration ratios 444 nm was noted. On increasing interaction time
between Neem extract and silver ions. For further further up to 24 h, the kmax value did not vary
experiments, 20% Neem leaf broth was used as significantly, though absorbance values increased.
reductant. Figure 1b shows the spectral data for 20 The maximum absorbance was noted at 24 h, at 0.77.
and 40% Neem extract concentrations at two differ- Biomineralization is a process of spatially controlled
ent mixing ratios (1:4 and 2:3) with 0.01 M AgNO3 crystallization, in terms of both the development of
solution. The maximum absorbance was noted for crystal faces, their orientation and also synthesizing
20% concentration at 1:4 Neem extract to AgNO3 particles of defined shape. The organics present in the
mixing ratio. Further experiments were carried out Neem leaf broth controlled both nucleation and
with this combination. growth. Here, the organics stopped growth when a
Figure 2a shows the UV–visible spectra from the desired dimension of nanoparticles was reached.
silver nanoparticles obtained from short interaction These initial processes resulted in preliminary change

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in kmax values in the first 24 h. The previous study on peak at 380 nm appeared as a shoulder in the UV–
synthesis using Neem broth reported a 24 h interac- visible spectra after 7 days of the reaction. This
tion time, though they did not carry out a detailed shoulder might correspond to the transverse plasmon
kinetic study on biosynthesis (Shankar et al. 2004). vibration in the silver nanoparticles, whereas the peak
The long-term interaction study was carried out at 440–460 nm was primarily due to excitation of
varying time intervals from 1 to 66 days (Fig. 2b) to longitudinal plasmon vibrations (Shankar et al. 2003).
investigate the ageing effect on biosynthesized The onset of transverse plasmon vibration can be
nanoparticles and to study their stability. The red explained in terms of departure from spherical shape
shift (from 440 to 462 nm) and broadening of the for a few particles (Mulvaney 1996). The red shift,
SPR was observed in Fig. 2 with increasing interac- broadening and splitting of the SPR was probably due
tion time. In addition to the peak at 440 nm another to the dampening of the SPR caused by the combined

Fig. 2 a UV–vis spectra recorded as a function of interaction ratio. b UV–vis spectra recorded as a function of interaction
time (4–24 h) of reaction between 20% Neem leaves extract time (24–66 h) of reaction between 20% Neem leaves extract
with an aqueous solution of 10-2 M AgNO3 in 1:4 mixing with an aqueous solution of 10-2 M AgNO3 in 1:4 mixing ratio

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effect of increasing particle size of the silver nano- nanoparticles, respectively. Both of them showed the
particles in colloidal solution and anisotropy of their presence of bonds due to O–H stretching (around
shapes. An increase in absorbance value correspond- 3,430 cm-1), aldehydic C–H stretching (around
ing to kmax was observed till 29th day of interaction, 2,910 cm-1), C=C group (around 1,600 cm-1) and
thereafter, the absorbance dropped slightly. geminal methyl group (around 1,380 cm-1). These
It is well known that the experimental data bands are indicative of terpenoid group of compounds
obtained from X-ray diffraction (XRD) of very small present in the aqueous Neem extract (Siddiqui et al.
particles is not easy to analyze. Nanoparticle XRD 2004). Some of the major chemical constituents
patterns exhibit several different size-dependent fea- present in neem leaves have been identified through
tures leading to anomalous peak positions, heights detailed studies using 1D, 2D NMR and FT-IR as
and widths (Hall 2000; Chen et al. 2002; Buffat quercetin rhamnoside (0.45%), a flavonoid, quercetin
2003). The occurrence of these so-called multiply- (0.257%) and nimbin (0.19%). A few other constit-
twinned particles (MTPs) mixed with the usual fcc uents also present are nimbocinone (250 ppm), a
structure makes the XRD data interpretation more protomeliacin (precursor for limnoids), nimbandiol
complex for metal nanoparticles (Hall 2000; Buffat (130 ppm) and nimbal, 6-deacetyl (120 ppm) (Ross
2003). To study the crystalline nature of the silver 2003; Siddiqui et al. 1986). From FT-IR results it can
nanoparticles, the XRD analysis was undertaken, be inferred that some of the bio-organics from Neem
revealing four peaks [38.0635° (111), 44.1392° (200), broth formed a strong coating/capping on the
64.4001° (220) and 77.3686° (311)] corresponding to nanoparticles.
four diffraction facets of silver. A few intense When the metal nanoparticles form in solution,
additional and yet unassigned peaks were also noticed they must be stabilized against the Van der Waals
in vicinity of the characteristic peaks of silver force that may cause coagulation. The stabilization
(32.1895°, 46.1712° and 76.7038°). These sharp may occur in a number of ways: physisorbed surfac-
Bragg peaks might have resulted from some bio- tant and polymers may create steric or electrosteric
organic compounds/protein(s) present in the neem barriers or purely electrostatic barriers around the
leaf broth. The intensity of the Bragg reflections particle surface (Mulvaney 1996). In many cases, the
suggests strong X-ray scattering centres in the distinction between chemical adsorption (involving
crystalline phase and could possibly arise from direct covalent bonding with the surface metal atoms)
metalloproteins in the broth (Shankar et al. 2003). and more subtle electrostatic mechanisms (e.g.,
As detailed in the experimental section, the silver charge-induced dipole mechanisms and dispersion
nanoparticles after their formation were repeatedly force mechanisms) is largely a matter of degree. The
centrifuged and re-dispersed in sterile distilled deion- problem distinguishing chemisorption and physisorp-
ized water as a part of purification, thus ruling out the tion is further exacerbated in the case of metal clusters
presence of any free compound/protein that might containing only a few tens of metal atoms, where it is
independently crystallize and give rise to the not clear whether the stabilizing molecules should be
observed Bragg reflections. The XRD results there- denoted as ‘‘adsorbates’’ or ‘‘ligands’’. In this case, the
fore suggest that the crystallization of the bioorganic bioorganic capping agents around the nanoparticles
phase occurs on the surface of the silver nanoparticles were responsible for their stability.
(or vice versa) (Shankar et al. 2003). Figure 4a–d represents TEM images of the nano-
The aqueous extract of shade-dried Neem leaves particles formed after 4 h, 24 h, 7 days and 30 days
for different drying periods did not yield any interaction, respectively. Mostly spherical and near
absorbance band in the visible range 440–460 nm spherical shapes can be observed for interaction time
region, confirming failure of silver nanoparticle 4 h (Fig. 4a) in 10–20 nm size range. When the
formation. Thus, it can be inferred that the shade interaction time was increased till 24 h, nanoparticles
drying of Neem leaves had destroyed/decreased mostly of spherical shape having different sizes were
significantly the activity of reducing components in obtained. A few non-spherical polyhedral particles
the Neem leaf extract. were also found. With increase in interaction time the
Figure 3a, b shows the FTIR spectra of aqueous aggregation and anisotropy in shape increased. From
extract of fresh Neem leaves and purified silver Fig. 4c, d nanoparticles of increased size and

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Fig. 3 a FT-IR spectrum recorded by making KBr disc with the fresh neem leaves extract. b FT-IR spectrum recorded by making
KBr disc with as synthesized silver nanoparticles

different shapes were noted. Number of polyhedral For the reductants with redox potentials deviating
shaped particles increased, with a few showing from the potential of zero charge of polycrystalline
capsular shape for 7 day interaction time. Large silver (-0.7 V NHE), the synthesized sols (nanopar-
aggregated particles having near or more than 100- ticles) will always have residual electrical charge on
nm-size, having polyhedral or capsular shape were surface and thus will give plasmon absorption bands
obtained when the interaction time was increased till shifted from the position expected for an electrically
30 days. The TEM images were commensurate with neutral particle. An uncharged silver colloid should
red shift, broadening and splitting of the SPR, as have a maximum wavelength at 382 ± 1 nm; longer
observed from UV–visible spectroscopy. wavelengths can be attributed to incomplete

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Fig. 4 a–d TEM images of

as synthesized nanoparticles
formed after 4 h, 24 h,
7 days, and 30 days
interaction, respectively

reduction of silver ions (Mock et al. 2002). Figure 5 surface charge on the nanoparticles. The stability of
demonstrates the effect of change in initial pH of the cluster distribution was enhanced at alkaline pH
AgNO3 solution on the UV–visible spectra of the range owing to complete charging of the clusters thus
silver nanoparticles synthesized by 4 h interaction. maximizing the repulsive electrostatic/electrosteric
For 4 h interaction, a blue shift (a sudden drop in the interactions. This enhanced stability and decreased
kmax value) was observed from pH 6 to pH 7; the aggregation diminished broadening in spectra and
changes in kmax value were within experimental blue shifted the UV–visible peak.
limits beyond pH 7 unto pH 10. When the particles In this context, it also needs to be mentioned that
were synthesized by 24 h interaction, the sudden Ag (I) ions are unable to hydrolyze in these acid
decline in the kmax value was noted from pH 6 to pH surroundings. At pH values above 8, Ag (I) ions in
8, beyond which no significant changes were solution partly hydrolyze to form Bio-organic–Ag
observed (not in figure). A large fall in the floccu- (OH)x or Bio-organic–Ag (NH3)2? complex on the
lation parameter in alkaline pH ranges has been surface of the particles and AgOH/Ag2O colloid in
reported (Sastry et al. 1997), which might have the medium. The degree of hydrolysis and colloid
caused decrease in aggregation. In our experiments formation increases with increase in pH of the
with Ag sol having initial pH 2, we could not get solution. This effect may prevent further decrease
nanoparticles and flocculation was clearly observed in the kmax value, therefore, negligible changes were
in the medium. The stability of the nanoparticles in observed in highly alkaline range. The second
the presence of additives has been found to be a important observation was a drop in pH during the
function of sol pH since OH- ions can change the synthesis, when the initial pH was in the alkaline

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J Nanopart Res (2010) 12:237–246 245

characterizing the reducing and stabilizing compo-

nents from the Neem leaf broth is underway.

Acknowledgments The authors would like to thank Pro-

Chancellors of VIT-University, Dean of School of
Biotechnology and Division Leader of Environmental
Biotech group for wholehearted cooperation. They also wish
to acknowledge Ms Charumathi for carrying out a few
preliminary experiments on pH effect.

Fig. 5 UV–vis spectra recorded as a function of initial sol pH

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