Acta Polytechnica


doi:10.14311/AP.2018.58.0104
Acta Polytechnica 58(2):104–108, 2018 © Czech Technical University in Prague, 2018

available online at http://ojs.cvut.cz/ojs/index.php/ap

STABILITY OF SYNTHESIZED SILVER NANOPARTICLES
IN CITRATE AND MIXED GELATIN/CITRATE SOLUTION

Jana Kavuličováa, ∗, Anna Mražíkováb, Oksana Velgosováb,
Dana Ivánováa, Martina Kubovčíkovác

a Institute of Metallurgy, Faculty of Materials, Metallurgy and Recycling, Technical University of Kosice, Slovakia
b Institute of Materials, Faculty of Materials, Metallurgy and Recycling, Technical University of Kosice, Slovakia
c Institute of Experimental Physics, Slovak Academy of Sciences, Kosice, Slovakia
∗ corresponding author: jana.kavulicova@tuke.sk

Abstract. The study focuses on an investigation of the influence of both citrate and mixed gelatin/citrate
as a reductant and stabilizer on the colloidal stability of silver nanoparticles (AgNPs) synthesized by a
chemical reduction of Ag+ ions after a short- (7th day) and long- (118th day) term storage. Formed AgNPs
were characterized by a UV–vis Spectroscopy, Transmission Electron Microscope (TEM), Dynamic light
scattering (DLS) and Zeta-potential (ZP). The obtained results revealed that a short-term stability of the
synthesized AgNPs was greatly influenced by a citrate stabilizer with the absence of gelatin. Smaller-sized
AgNPs (average particle diameter of 3 nm), roughly spherical in a shape, were obtained with a narrow size
distribution. The very negative value of the Zeta-potential confirmed a strong stability of the citrate capped
AgNPs. However, a surface coating of the AgNPs by a gelatin/citrate stabilizer was found to be a dominant
contributor in improving a long-term stability of the AgNPs (average particle diameter of 26 nm). The use of
gelatin in mixed stabilizer solution provided the AgNPs with higher monodispersity and a controllable size
after both the short and long-term storage.
Keywords: silver nanoparticles; sodium citrate; gelatin; size; stability.

1. Introduction
The fabrication of highly characterized nanomaterials
requires a developing of their specific physical and chem-
ical properties. Among these nanomaterials, especially
silver containing nanocomposites has a wide range of po-
tential applications in diverse areas, such as, electronics,
catalysis [1], cosmetics, wastewater treatment, textile
industry and biomedical devices [2].
Many practical applications require stable silver

nanoparticles (AgNPs) dispersions. The improving
of significant parameters, which influence the stability
of silver nanoparticles (AgNPs) against an aggregation
state, is an essential task in the optimization of the
AgNPs synthesis [3, 4]. There are factors that influence
the stability of AgNPs, such as the concentration of the
precursor, pH, temperature, order of mixing of reactants
and stabilizers [6, 7]. For example, different kinds of
stabilizers/capping agents, such as surfactants, polymers
and reducing agents, are used for the surface modification
of AgNPs during their synthesis to control the size,
morphology, stability, and physicochemical properties of
AgNPs [8–10].

Silver nanoparticles are typically prepared through the
reduction of a silver precursor using chemical or physical
means [4, 9]. Chemical reduction of a silver nitrate salt in
an aqueous media by a reducing agent in the presence of
a surfactant is a facile synthetic method. Many authors
have also reported that sodium citrate serves both as
a reducing and stabilizing agent in the preparation of
silver nanoparticles [11–13]. Among stabilizers, gelatin
has also been used as a natural biopolymer for the
stabilization of inorganic nanoparticles and is known to

be biodegradable in physiological environments [4, 14].
The novelty of this study lies in its modified approach

to the synthesis of AgNPs by the surface-protecting
chemical reduction of the silver nitrate using a mixed
gelatin/sodium citrate solution both as a stabilizer and
reducing agent for AgNPs. The effects of reducing agent
and stabilizer on the particle size, size distribution, shape
and stability of AgNPs were evaluated after a short- (7th
day) and long- (118th day) term storage and compared
to those of AgNPs synthesized by using the standard
reduction of silver salt in sodium citrate.

2. Materials and methods
2.1. Chemicals
Silver nitrate (p.a. Mikrochem), sodium citrate (p.a.
Lachema), gelatin (p.a. Lachema) were used for the
synthesis of the silver nanoparticles without any fur-
ther purification. Demineralized water (conductivity
0.3 µS cm−1) from Demiwa 3 rosa (Watek) was used to
prepare the solutions.

2.2. Synthesis of silver nanoparticles
by using sodium citrate method

Chemically synthesized AgNPs were prepared using
a chemical reduction method as demonstrated [15].
Aqueous AgNO3 (0.29 mM) was used as the Ag+ salt
precursor. To this solution, sodium citrate (1 %) was
added drop by drop as a reducing agent. During the
synthesis, the solution was heated to an elevated tem-
perature (∼ 90 °C) and mixed vigorously until a colour
change was evident. The pH of the prepared AgNPs
colloids was close to 7.

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vol. 58 no. 2/2018 Stability of Synthesized Silver Nanoparticles

2.3. Synthesis of silver nanoparticles
by using mixed gelatin/sodium
citrate method

The experiment was modified using the same conditions
of the previous method with a reaction of aqueous
AgNO3 (0.29 mM) and sodium citrate (1 %), but with
gelatin. A 0.01 % gelatin concentration was added to the
solution of AgNO3 prior to the addition of a reducing
agent in order to prevent the particle agglomeration.

2.4. Methods for determination
The formation of AgNPs was monitored by measuring the
UV–vis spectra with the UNICAM UV/vis Spectrometer
UV4. The absorbance was recorded after 5 h, 24 h and
on the 3rd, 7th, 10th, 15th, 23th, 30th, 90th, 118th and
150th day. Transmission Electron Microscope (TEM)
(JEOL model JEM-2000FX microscope operated at an
accelerating voltage of 200 kV) was used to determine the
size and the morphology of AgNPs on the 7th and 118th
day. The samples were prepared by placing a drop of the
colloidal solution of AgNPs on a carbon-coated nickel grid
and completely dried at a room temperature. Dynamic
Light Scattering (DLS) was carried out to estimate the
hydrodynamic size of AgNPs in a suspension on the
7th and 118th day. The samples were measured using
Zetasizer Nano ZS (Malvern Instruments). The zeta
(ζ) potential was determined using the Laser Doppler
Electrophoretic measurement technique with a scattering
angle of 173° at 25 ± 0.1 °C

3. Results and discussion
The inset of Figure 1 shows the photographs of samples
obtained under different conditions. In the reaction
of AgNO3 with citrate without gelatin, silver colloids
turned pale yellow-brown. It implied the formation
of AgNPs, while the addition of gelatin to Ag+ so-
lution led to the change of the solution colour to a
dark brown (after 20 min), indicating the significant
influence of gelatin in terms of its role as a reduc-
ing agent on the final colour of the AgNPs dispersion.
Similarly, the ability of gelatin as a good reducing
agent in the synthesis of AgNPs was reported in the
study [14].

The synthesis of AgNPs was confirmed by a UV-vis
spectroscopy. The surface plasmon resonance (SPR)
bands for AgNPs have been reported to be in the
range of 380–450 nm. The SPR bands are influenced
by size, shape, morphology and composition of the
prepared AgNPs [16]. The symmetry and broadness of
the absorption bands may be related to various shaped
and/or sized AgNPs [17, 18]. The absorption spectra
(Figure 1a) of silver colloids without gelatin displayed the
symmetric and broader SPR peak of a very low intensity
with a no significant spectral shift (from 420 to 422 nm)
after a short storage (range 3–23 days) indicating a
synthesis of uniform and stable AgNPs. Based on the
symmetry of the peak, it is assumed that the majority
of synthesized AgNPs did not aggregate. In the case
of adding gelatin to the Ag+ solution, the asymmetric

Figure 1 

 

             

   

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

300 500 700

A
b

so
rb

a
n

ce
 (

a
.u

)

Wavelength (nm)

a
5 h
3 day
7 day
10 day
15 day
 23 day
30 day
90 day
118 day
150 day

0

0,2

0,4

0,6

0,8

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1,2

1,4

1,6

1,8

300 500 700

A
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so
rb

a
n

ce
 (

a
.u

)

Wavelength (nm)

b

5 h
 24 h
 3 day
 7 day
 10 day
15 day
23 day
30 day
90 day
118 day

Figure 1. UV-vis spectra of AgNPs a) capped with
citrate, b) capped with gelatin/citrate as a function of
reaction time, c) coated with different capping agents
after 7 days. The inset shows the color change upon
synthesis of AgNPs.

and narrower SPR band with a significant spectral shift
(from 414 to 432 nm) after a short storage revealed a
less uniform particle size distribution (Figure 1b) as
compared with those seen in the absence of gelatin.
However, insignificant changes in the red-shift of the SPR
band (from 432 to 434 nm) were observed after a long
storage (range 30–150 days) suggesting a good stability
of the prepared AgNPs for a long time. The intensity of
the SPR bands gradually increased over time due to the
continuous synthesis of AgNPs [19, 20]. As shown in Fig.
1b, the SPR bands were proportionally more heightened
than in Fig.1a, which corresponds to the combined effects
of citrate/gelatin as reducing agents resulting to a more
intensive colour of the AgNPs colloid. According to the
authors [21, 22], the SPR peak located between 410
and 450 nm during the synthesis of AgNPs might be
attributed to spherical or roughly spherical shaped silver
nanoparticles (Figure 1). Figure 1c compares the SPR
bands of the AgNPs coated with citrate, gelatin and
gelatin/citrate after 7 days of storage. It is interesting
to note the significant reduction ability of gelatin in the
synthesis of AgNPs.

The particle size distribution and the shapes of pre-
pared AgNPs were confirmed by a TEM analysis on the
7th and 118th day. The TEM images revealed roughly

105



J. Kavuličová, A. Mražíková, O. Velgosová et al. Acta PolytechnicaFigure 2 

     

 

 

 

 

 

 

 

 

 

 

 

 

a 

d 

c 

b 

Figure 2. TEM images of AgNPs prepared using a)
citrate and b) gelatin/citrate on 7th day, c) citrate and
d) gelatin/citrate on 118th day.

spherical shaped AgNPs in both the absence and the
presence of gelatin (Figure 2). Smaller-sized AgNPs
were obtained after their short storage (Figure 2ab) and
gelatin/citrate capped AgNPs showed a higher degree
of monodispersity (Figure 2bd) than AgNPs without
gelatin, significantly after 118 days (Figure 2c).

The TEM images (on the 7th day) displayed AgNPs
with an average particle diameter of 3 and 15 nm without
and with gelatin, respectively (Figure 3ab). According
to the results, smaller AgNPs were prepared with a
narrow size distribution, the size ranged from 1–22 nm
(Figure 3a) when only citrate was used as a reducing
and stabilizing agent. As indicated above, TEM results
confirmed that the synthesized AgNPs in the absence
of gelatin are more stable after a short storage. The
TEM images (on the 118th day) demonstrated that
a higher percentage of the AgNPs (42 %) synthesized
with gelatin are smaller than those prepared without
gelatin (10 %), with the former in the range of 4 to 55 nm
with an average diameter of 26 nm (Figures 2d and 3d).
The usage of the mixed gelatin/sodium citrate method
allows a synthesis of size-controlled AgNPs. According
to [23], the better control on nucleation and growth
of nanoparticles is caused by a combined effect of two
different reducing agents. The size of AgNPs without
gelatin showed a wider range of particle size distribution
(from 7 to 85 nm in diameter) with an average diameter
of 36 nm (Figures 2c and 3c). This can be particularly
explained by the fact that gelatin forms the coating on
the surface of AgNPs during the particle growth process
and prevents an aggregation through a steric hindrance
due to its high molecular weight and significant surface
activity [4, 24].

It seems that the stability of sterically stabilized silver
colloids in the presence of gelatin predominates over the
electrostatic stabilization caused by a capping of citrate
onto the surface of AgNPs. Moreover, the addition of
excess citrate as a reducing agent in the absence of

Figure 3 

 

 

 

 

 

 

 

 

 

 

 

 

0

5

10

15

20

25

30

1 4 7 10 13 16 19 22

F
re

q
u

e
n

cy
 (

%
)

Particle diameter (nm)

a

0

10

20

30

3 6 9 12 15 18 21 24

F
re

q
u

e
n

cy
 (

%
)

Particle diameter (nm)

b

0
5

10
15
20
25
30

7 17 26 36 46 57 71 85

F
re

q
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e
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cy
 (

%
)

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c

0

10

20

30

4 17 25 36 46 55 71 85

F
re

q
u

e
n

cy
 (

%
)

Particle diameter (nm)

d

Figure 3. The size histogram of AgNPs prepared using
a) citrate and b) gelatin/citrate on the 7th day, c) citrate
and d) gelatin/citrate on the 118th day.

gelatin forms a larger AgNPs in comparison with AgNPs
made with the presence of gelatin (Figure 2). Likewise,
the effect of higher citrate concentrations during the
growth of existing AgNPs nucleis through the surface
reduction of silver ions has also been described by [25].
This supports the observation in UV-vis spectra that
gelatin/citrate capped AgNPs exhibited a long-term
stability.

In addition, the average hydrodynamic diameter,
polydispersity index (PDI) and Zeta potential of AgNPs
in dispersion gathered by the DLS both in the absence
and presence of gelatin are shown in Table 1. The sizes
observed by the DLS (Figure 4ab) are significantly higher
than the sizes obtained by the TEM. This could be
attributed to the effective coating of gelatin and/or
citrate on the surface of AgNPs. The AgNPs in colloids
are electrostatically stabilized because of their negative
surface charge arising from the presence of oxo- or
hydroxo-functional groups in the citrate on the AgNPs
surface [26].

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vol. 58 no. 2/2018 Stability of Synthesized Silver Nanoparticles

Sample
name

pH of
silver
colloid

t = 7 days t = 118 days
Z-average

diameter (nm)
Z-potencial

(mV)
PDI Z-average

diameter (nm)
Z-potencial

(mV)
PDI

TEM DLS TEM DLS
AgNPs (a) 6.77 3 103 −37.3 0.28 36 164 −10.5 0.44
AgNPs (b) 6.93 15 146 −7.8 0.2 26 174 −19.7 0.21

Table 1. Average values of AgNPs diameter determined by DLS method and from TEM images, PDI and Z-potencial of
AgNPs colloids on 7th and 118th day.

Figure 4 

 

 

 

0

2

4

6

8

10

0 200 400 600 800

In
te

n
si

ty
 (

%
)

Size (d.nm)

a

7 d

118 d

0

4

8

12

16

0 200 400 600 800

In
te

n
si

ty
 (

%
)

Size (d.nm)

b

7 d

118 d

Figure 4. DLS size distribution of AgNPs prepared
using a) citrate b) gelatin/citrate on 7th and 118th day.

The Zeta potential analysis also confirmed the strong
stability of AgNPs without gelatin (−37.3 mV) after a
short storage due to the citrate layer on the surface of
AgNPs through an electrostatic interaction. However,
the addition of gelatin resulted in a significantly lower
negative value of the zeta potential (−7.8 mV).

It probably relates to a possible interaction between
amino groups of gelatin and carboxyl groups of citrate
coated on the surface of AgNPs. The stability of AgNPs
coated by organic coatings with varying molecular weight
and functional groups is achieved through the adsorption
or covalent attachment. The attachment of the stabilizer
to AgNPs surface is probably related to their interaction
strength [26]. Effectiveness of the bounded citrate with
gelatin was enhanced on the surface of AgNPs after a
long storage and proved by the fact that the Z-potential
was found to be −19.7 mV.

Electrostatic stabilization of AgNPs can be reduced
by the presence of counterions in the colloid [26]. The
presence of cations (e.g. Na+) in the reducing agent
could partially destabilize the citrate coated AgNPs
after long storage, this is confirmed by the increased
negative value of the zeta potential (−10.5 mV).

A possible reaction scheme expresses the formation of
the citrate capped AgNPs (1) by a faster reduction in
comparison with the formation of the gelatin capped
AgNPs (2) achieved by a slower reduction [14]. If the
course of these reactions is considered, the synthesis of
the gelatin/citrate capped AgNPs can take place through
the formation of a gelatin-silver cation complex and the
gelatin capped AgNPs [27]. The concentration of this
complex is considerably higher than the content of the
gelatin capped AgNPs. This is likely due to the partial
reduction of Ag+ to Ag0 using gelatin during the short
reaction time for the formation of gelatin/citrate-AgNPs.
These reactions can be represented by (3)–(4) (Figure 5).

The AgNPs prepared with gelatin showed considerably
lower PDI of 0.2 and 0.21 as compared with the AgNPs
without gelatin, PDI of 0.28 and 0.44 on the 7th and
118th day, respectively (Table 1). The comparison of
PDI values for AgNPs prepared without and with gelatin
demonstrated that the synthesis of AgNPs in mixed
gelatin/citrate solution produced AgNPs with higher
monodispersity after both short and long-term storage.

The results of this study indicate the importance of
the factors, such as stabilizer type, concentration and
addition order of the reducing and stabilizer agents,
reaction time and ionic strngth, that significantly affect
the stability of the synthesized AgNPs in the colloid.

4. Conclusions
In this study, the influence of citrate and gelatin on the
stability of formed AgNPs in dispersion was investigated.
The modified synthesis of AgNPs through a chemical
reduction of Ag+ in a gelatin/citrate mixed solution
was compared to the standard reduction of Ag+ in
citrate. The surface coating of AgNPs by a citrate
stabilizer proved to be significant for achieving a short-
term stability of AgNPs in colloid. In comparison,
the use of gelatin as a reducing and surfactant agent
greatly increased the long-term stability of AgNPs.
This shows that synthesized AgNPs exhibited improved
stability through synergetic steric/electrostatic effects
of the gelatin/citrate system, respectively. The mixed
gelatin/sodium citrate method offers a size-controlled
synthesis of monodisperse AgNPs with their potential
use in a production of nanocomposite materials.

Acknowledgements
This work was financially supported by the Slovak Grant
Agency (VEGA 1/0197/15).

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J. Kavuličová, A. Mražíková, O. Velgosová et al. Acta Polytechnica

Ag+(aq) + citrate(aq) → citrate-AgNPs(s) + citric acid(aq) (1)
Ag+(aq) + gelatin(gel)(aq) → gelatin-AgNPs(s) (2)

Ag+(aq) + gelatin(gel)(aq) → [Ag(gel)]+(aq) + gelatin-AgNPs(s) (3)
[Ag(gel)]+(aq) + citrate(aq) → gelatin/citrate-AgNPs(s) + citric acid(aq) (4)

Figure 5. A possible reaction scheme for the synthesis of AgNPs coated with the capping agents: citrate (1), gelatin (2)
and gelatin/citrate (3)–(4).

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108

http://dx.doi.org/10.1186/s40643-015-0076-2
http://dx.doi.org/10.1371/journal.pone.0103675
http://dx.doi.org/10.1016/j.chroma.2011.03.034
http://dx.doi.org/10.1021/jp026731y
http://dx.doi.org/10.1039/C3RA44507K
http://dx.doi.org/10.1021/es2037405

	Acta Polytechnica 58(2):104–108, 2018
	1 Introduction
	2 Materials and methods
	2.1 Chemicals
	2.2 Synthesis of silver nanoparticles by using sodium citrate method
	2.3 Synthesis of silver nanoparticles by using mixed gelatin/sodium citrate method
	2.4 Methods for determination

	3 Results and discussion
	4 Conclusions
	Acknowledgements
	References