# Synthesis and Antimicrobial Activity of Silver Citrate Complexes

Authors: Stojan Djokić
Journal: Bioinorganic Chemistry and Applications
 (2008)

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## Abstract

Formation of silver citrate/citric acid complexed solutions was investigated.  Although, silver citrate is minimally soluble in water, it can successfully be dissolved in citric acid solutions.  The maximum concentration of Ag(I) in solution is estimated at 23 to 25 g/L if the concentration of citric acid is at least 4 mol/L or higher. The dissolution of silver citrate in citric acid solutions was attributed to the formation of silver citrate complexes of a general formula[Ag3(C6H5O7)n+1]3n−.  The silver citrate/citric acid solutions, containing more than about 13 g/L Ag+ ion, have exhibited a decrease in Ag(I) concentration in solution over time, due to crystallization.  The crystallization product was attributed to the formation of [Ag3C6H5O7]x⋅nH2O. Importantly, the diluted silver citrate/citric acid complexed solutions have exhibited very strong bacteriostatic and bactericidal activities.


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## Body

## 1. Introduction

It is well established that only silver in its ionic or complexed forms is
antimicrobially active, while the elemental silver, even in the so-called
“nanocrystalline” state is not [1].  Silver-containing
compounds are attractive because of the fact that in the range of the
applicable concentrations, silver ions do not exhibit toxicity and carcinogenic
activities [2].Various
forms of silver and its compounds have been investigated in the past few
decades due to the antimicrobial activity of silver ions, and there is an
increased interest in the potential use of silver(I) as a therapeutic agent for
different antimicrobial applications. Researchers have found out that in the
treatment of infections the availability of silver ions is completely
independent on the total amount of silver chelates [3]. The silver complexes, for
example, silver chelates have been described as more effective therapeutic
agents than free silver ions [4].A few amino acids with N- and O-donor ligands, which show a very wide spectrum of
effective antimicrobial activities against bacteria and yeast, were used to
obtain water-soluble silver complexes [5]. O-donor ligands, such as α-hydroxycarboxylic
acids (mandelic, glycolic, malic, tartaric, etc.) also form complexes with Ag(I).It was disclosed in the literature that simple carboxylic acids show an unexpected
ability to enhance the antimicrobial power of a wide range disinfectants and/or
antibiotic agents [6]. The presence of sodium citrate was demonstrated to be
necessary to yield potent inhibition of growth of certain pathogenic organisms
[7]. Similarly, the antimicrobial antioxidant effects of citrate ions have been
confirmed to be very efficient against the proliferation of various spoilage
micro-organisms [8]. The taurolidine-citrate solution was suggested as
promising combination agent for the prevention of intravascular catheter-related
infections [9].The synthesis, structure, and antimicrobial activity of silver complexes with
mandelic acid were described in the recent publication [10]. The silver
mandelate compound was found to be a very successful agent against Mycobacterium tuberculosis.It is believed that a combination ofAg+ ions with citrate ions (C6H5O73−)
can be very attractive for the future biomedical or pharmaceutical/therapeutic
applications. Based on the literature
review, it is reasonable to assume that silver citrate complexes would act
synergistically as antimicrobial agents (due to the presence of Ag(I) ions) and
also as enhancers of antimicrobial activity, antioxidants, and anticancer
agents (due to the presence of C6H5O73− ions) [1–10].Silver citrate is a white substance with a very limited solubility in water. Under the normal physicochemical conditions, 1 part of silver citrate is soluble in 3500
parts of water, which corresponds to 285 ppm of Ag(I) ion in the solution [11].Dissolution of silver nitrate into citric acid solution or other hydroxycarboxylic acids does not lead to the precipitation silver citrate or other hydroxycarboxylates
(e.g., malate, tartrate, lactate, etc.) [12]. In the case of silver nitrate and
citric acid, this behavior can be explained by the following reaction:
(1)3AgNO3+H3C6H5O7⟷Ag3C6H5O7↓+3HNO3.Due
to the dissolution of silver citrate inHNO3, the precipitation of
Ag3C6H5O7 does not occur. Therefore, it can be concluded that the
equilibrium of the reaction (1) is shifted to the left (in the reverse
direction).Silver citrate can successfully be produced using the routes as described in the
following text.(a) Sodium citrate (Na3C6H5O7) route:(2)AgNO3+Na3C6H5O7⟶Ag3C6H5O7↓+NaNO3.(b) Sodium hydroxide (NaOH) route:(3)2AgNO3+2NaOH⟶Ag2O↓+2NaNO3+H2O,3Ag2O↓+3H3C6H5O7⟶2Ag3C6H5O7↓+3H2O.When an adequate
filtration and washing with water is applied during the processing, this route
offers a production of silver citrate with a high purity.(c) Ammonium hydroxide (NH4OH) route:(4)AgNO3+3NH4OH⟶[Ag(NH3)2]OH+NH4NO3+2H2O,[Ag(NH3)2]OH+2H3C6H5O7⟶Ag3C6H5O7↓+(NH4)3C6H5H5O7+3NH4OH.Since ammonium citrate and ammonium hydroxide are soluble in water, further steps
involve filtration and washing, in order to produce pureAg3C6H5O7.Other approaches for the production of silver citrate, based on the routes described
here, are also possible. They may include silver oxide(s) or any silver salt
which can be further manipulated in order to produce silver citrate.In the present work, synthesis, structure, and some properties of silver citrate (Ag3C6H5O7)
are discussed. Formation of water-soluble silver citrate complexes, containing
as high as 25 g/L silver is described. These silver citrate complexed solutions
have not been reported in literature. The stability of silver citrate complexed
solutions and their antimicrobial activity have also been investigated.


## 2. Experimental

### 2.1. Synthesis

Silver
citrate (Ag3C6H5O7) precipitate was
produced using routes (a), (b), and (c), described in Section 1. These routes are in the further text
designated as Na3C6H5O7 (a), NaOH
(b), and NH4OH (c), unless otherwise specified.  In each route, the stoichiometric amounts of
the required chemicals were dissolved in 100 mL of water. These solutions were
then mixed in a 400 mL beaker and magnetically stirred for 30 minutes. The
white precipitate of silver citrate was separated from the solution by
filtration, rinsed with water, dried and used for the further analysis.


### 2.2. Materials and Methods

In all experiments, reverse osmosis (RO) water and stoichiometric amounts of
analytical grade chemicals were used.  In
order to study the dissolution, specific amounts of silver citrate (as
documented in Section3)
were mixed with water or citric acid solutions of various concentrations. The concentration
of Ag+ ions in the solution was determined after 24 hours, a time
for which it is believed that the equilibrium was achieved. Analytical methods
employed in the present work have included volumetric determination of total
silver in the solution or in the silver citrate precipitate, X-ray diffraction
(XRD), differential scanning calorimetry (DSC), and scanning electron
microscopy (SEM).


### 2.3. Biological Screening

Based
on the experimental observations for the dissolution ofAg3C6H5O7 in citric acid solutions, aqueous silver citrate complexes were prepared and
used further for testing
the stability and for investigating the antimicrobial activity.Simple
bacteriostatic tests [13] against Pseudomonas
aeruginosa were used in order to demonstrate antimicrobial activity of
silver citrate complexed solutions synthesized in the present work. For this purpose, silver citrate/citric acid
solution containing 18 g/L silver ions was further diluted with water in order
to obtain a solution containing about 100 ppm silver ions. A 100 μL of this diluted solution was placed
on a sterile 13 mm Whatman antibiotic assay disk in order to achieve a complete
wetting of the disk. The wetted disks were then placed in a Petri dish containing
Mueller Hinton agar and seeded with Pseudomonas
aeruginosa. After the incubation at 37°C for 24 hours, zones of
inhibition (areas surrounding the tests samples where bacterial growth is
inhibited or does not occur) were examined. The corrected zone of inhibition
(CZOI) was calculated by subtracting the diameter of the test sample from the
diameter of the zone of inhibition.The bactericidal activity of silver
citrate/citric acid solution containing 100 ppm Ag(I) was investigated
following the standard procedure [14] using Pseudomonas
aeruginosa.  It is important to note,
that for this purpose the originally prepared solution of silver citrate/citric
acid containing 18 g/L silver ions was further diluted with a 0.1% citric acid
solution. The target was to obtain a solution containing about 100 ppm of Ag(I) ions in the final dilution.


## 2.1. Synthesis

Silver
citrate (Ag3C6H5O7) precipitate was
produced using routes (a), (b), and (c), described in Section 1. These routes are in the further text
designated as Na3C6H5O7 (a), NaOH
(b), and NH4OH (c), unless otherwise specified.  In each route, the stoichiometric amounts of
the required chemicals were dissolved in 100 mL of water. These solutions were
then mixed in a 400 mL beaker and magnetically stirred for 30 minutes. The
white precipitate of silver citrate was separated from the solution by
filtration, rinsed with water, dried and used for the further analysis.


## 2.2. Materials and Methods

In all experiments, reverse osmosis (RO) water and stoichiometric amounts of
analytical grade chemicals were used.  In
order to study the dissolution, specific amounts of silver citrate (as
documented in Section3)
were mixed with water or citric acid solutions of various concentrations. The concentration
of Ag+ ions in the solution was determined after 24 hours, a time
for which it is believed that the equilibrium was achieved. Analytical methods
employed in the present work have included volumetric determination of total
silver in the solution or in the silver citrate precipitate, X-ray diffraction
(XRD), differential scanning calorimetry (DSC), and scanning electron
microscopy (SEM).


## 2.3. Biological Screening

Based
on the experimental observations for the dissolution ofAg3C6H5O7 in citric acid solutions, aqueous silver citrate complexes were prepared and
used further for testing
the stability and for investigating the antimicrobial activity.Simple
bacteriostatic tests [13] against Pseudomonas
aeruginosa were used in order to demonstrate antimicrobial activity of
silver citrate complexed solutions synthesized in the present work. For this purpose, silver citrate/citric acid
solution containing 18 g/L silver ions was further diluted with water in order
to obtain a solution containing about 100 ppm silver ions. A 100 μL of this diluted solution was placed
on a sterile 13 mm Whatman antibiotic assay disk in order to achieve a complete
wetting of the disk. The wetted disks were then placed in a Petri dish containing
Mueller Hinton agar and seeded with Pseudomonas
aeruginosa. After the incubation at 37°C for 24 hours, zones of
inhibition (areas surrounding the tests samples where bacterial growth is
inhibited or does not occur) were examined. The corrected zone of inhibition
(CZOI) was calculated by subtracting the diameter of the test sample from the
diameter of the zone of inhibition.The bactericidal activity of silver
citrate/citric acid solution containing 100 ppm Ag(I) was investigated
following the standard procedure [14] using Pseudomonas
aeruginosa.  It is important to note,
that for this purpose the originally prepared solution of silver citrate/citric
acid containing 18 g/L silver ions was further diluted with a 0.1% citric acid
solution. The target was to obtain a solution containing about 100 ppm of Ag(I) ions in the final dilution.


## 3. Results and Discussion

### 3.1. Characterization of Silver Citrate Produced via Different Routes

In
Table1 are given results for the yield and silver content in silver citrate
obtained by different routes (i.e., via Na3C6H5O7 (a), via NaOH (b), or via NH4OH (c)), as described in Section 1. It
is obvious from Table 1 that the yield for the production of Ag3C6H5O7 via different routes is above 98%. Discrepancies from 100% are related to the
experimental error.Table 1
The yield and the silver content in silver citrate produced via different routes.
RouteExpected  compoundYield %Ag content %Theoretical Ag content %Na3C6H5O7Ag3C6H5O799.2563.0663.13NaOHAg3C6H5O798.3462.9563.13NH4OHAg3C6H5O798.7562.5063.13The
chemical analysis showed that all the samples of silver citrate produced in the
present work contained the amount of silver close to the theoretical value of
63.13%, suggesting a relatively high purity of the product.Overlaid
XRD patterns of theAg3C6H5O7 produced by the routes (a), (b), and (c) are shown in Figure 1. As illustrated in
Figure 1, the peaks in the XRD patterns of the white precipitate exactly
coincide and match the lines for the Ag3C6H5O7.Figure 1
Comparison of the XRD patterns of silver citrate (Ag3C6H5O7)
produced via (a) Na3C6H5O7, (b) NaOH, and (c) NH4OH routes.In
Figure2 are presented DSC curves of Ag3C6H5O7 produced using Na3C6H5O7 (a), NaOH
(b), or NH4OH (c) routes. When silver citrate samples produced in
the present work are heated in an argon atmosphere, the obtained DSC curves
exhibit a clearly distinguished exothermic maximum in the range from 200°C to 210°C, as illustrated in Figure 2. The energy of this exothermic
process is estimated at about 220 J/g for all the tested samples. This exotherm,
in analogy with the behavior of other silver salts during heating in an inert
environment, is attributed to the decomposition of silver citrate to the
elemental silver.Figure 2
Comparison of DSC curves of silver citrate (Ag3C6H5O7)
produced via (a) Na3C6H5O7, (b) NaOH, and (c) NH4OH routes (heating rate 10°C/min, Ar
atmosphere).Figure3 presents SEM micrographs of Ag3C6H5O7 samples produced via Na3C6H5O7 (a),
via NaOH (b), or via NH4OH (c) routes. As can be seen from these
micrographs, there are no significant differences in the shape of the
individual particles of silver citrate produced in different ways. The size of
these individual particles is estimated to be in the range from 0.2 μm to 0.5 μm.Comparison
of SEM micrographs of silver
citrate (Ag3C6H5O7) produced via (a) Na3C6H5O7, (b) NaOH, (c) and NH4OH routes (magnifications are given on
images).
(a)(b)(c)


### 3.2. Dissolution of Silver Citrate in Citric Acid Solutions

As
mentioned above, silver citrate is very little soluble in water. However, the
experiments in the present work have found that silver citrate can quite
successfully be dissolved in the citric acid aqueous solutions. This observation
is summarized in Figure4, where a dependence of the concentration of silver
ions in solution on the concentration of citric acid, for a fixed amount of
silver citrate, is presented. As shown in Figure 4, an increase in the
concentration of citric acid, for the fixed amount of Ag3C6H5O7,
leads to an increase in the concentration of silver ions in solution. These
results strongly suggest that an increase in the concentration of citric acid
in solution leads to an increase in the solubility of silver citrate.Figure 4
Concentration ofAg+ when a constant mass of silver citrate is dissolved in solutions with different
citric acid concentrations.The
maximum concentration of silver ions in a saturated solution of silver citrate in
water can be estimated at about 0.3 g/L (2.8×10−3 mol/L), which is
in agreement with the literature data [11].Silver citrate dissociates in aqueous solutions according to the following reaction:(5)Ag3C6H5O7⟷3Ag++C6H5O73−.
Consequently,
the equation for the solubility product of silver citrate can be written as
(6)Ksp(Ag3C6H5O7)=[Ag+]3+[C6H5O73−].Based
on the results presented in Figure4, the solubility product of Ag3C6H5O7 is estimated to be in the order of magnitude 10−12 to 10−11. Of course, for a precise determination of the
solubility product of silver citrate, more sophisticated experiments are
required.  It is to be mentioned that the
value of solubility product has not been found in the open literature.In Figure5 are presented results of
silver ion concentration on the amount of silver citrate dissolved in 50 cm3 solutions with different concentrations of citric acid. Based on the dependences presented in Figure
5, the similar can be concluded as explained above for Figure 4. Generally, in
a solution with a constant concentration of citric acid, an increase in the
amount of silver citrate leads to an increase in the concentration of silver
ions in solution. However, the maximum amount of silver citrate which can be
dissolved depends on the citric acid concentration. For example, the maximum
amount of silver citrate soluble in a 50 cm3 of 0.5 M citric acid
solution is estimated at about 0.4 g, which corresponds to the Ag+ ion concentration of about 5 g/L. A
further increase in the amount of silver citrate in a 0.5 M citric acid
solution, as illustrated in Figure 5, does not lead to an increase in the
concentration of Ag+ ions, suggesting that the dissolution does not
take place and that the saturation is achieved.Figure 5
Concentration ofAg+ as a function of the amount of silver citrate dissolved in aqueous solutions of
citric acid with different concentrations.However,
a rise in the concentration of citric acid leads to an increase in the amount
of silver citrate which can be dissolved. In concentrated citric acid solutions
(3 mol/L to 4 mol/L), which corresponds to about 576 to 768 g/L, the maximum
concentration of silver ions is estimated to about 22–25 g/L (0.204–0.232
mol/L). These concentrations of silver
ions correspond to the amount of silver citrate in the range from 34.85 g/L to
39.60 g/L. A further addition of silver citrate to the concentrated citric acid
solutions does not lead to dissolution, suggesting that the saturation is
achieved. On the other hand, the
preparation of citric acid solutions with concentrations more than 4 mol/L
faces experimental difficulties, even with heating, since the crystallization
of citric acid occurs due to the saturation.How can the dissolution of silver
citrate in citric acid solutions be described?
Citric acid,H3C6H5O7, contains
three carboxylic groups (–COOH) and, consequently, three hydrogen ions can be
replaced with metal ions, as it is observed in the precipitation of silver
citrate, Ag3C6H5O7.  When one or two hydrogen ions from the
carboxylic groups of citric acid are replaced with Ag+ ions AgH2C6H5O7 or Ag2HC6H5O7 should be produced,
respectively. The formation of these compounds can be described with the
following reactions:
(7)Ag3C6H5O7↓+2H3C6H5O7⟶3AgH2C6H5O7,(8)2Ag3C6H5O7↓+H3C6H5O7⟶3Ag2HC6H5O7.Based
on the stoichiometry of the reactions (7) and (8) and an assumption that AgH2C6H5O7 or Ag2HC6H5O7 is soluble in water,
it can easily be calculated that the amounts of citric acid for the dissolution
of 100 g of Ag3C6H5O7 (0.195 mol)
are, respectively, 74.89 g (0.384 mol) for the reaction (7) and 18.72 g
(0.0975 mol) for the reaction (8).However,
the experimental observations have shown that thisis not the case. As experimentally found, significantly larger
amounts of citric acid are required for the dissolution of Ag3C6H5O7 than those calculated on the basis of the stoichiometry of the reactions (7) and (8). Consequently,
if AgH2C6H5O7 and Ag2HC6H5O7 are soluble in water, it is unlikely that the dissolution of silver citrate proceeds
via reactions (7) and (8). On the other hand, chemical analysis of
“undissolved” precipitate found that it (precipitate) contained about 63% Ag,
which corresponds to Ag3C6H5O7 (see
Table 1) and not to AgH2C6H5O7 or Ag2HC6H5O7.Considering the fact that significantly
larger amounts of citric acid are required for the dissolution of silver
citrate, it is reasonable to assume, based on the silver analysis and
stoichiometry of the chemical equations, that citric acid produces complexes
with silver citrate according to the following reaction:(9)Ag3C6H5O7+nH3C6H5O7⟷[Ag3(C6H5O7)n+1]3n−+3nH+.The
complexes with a general formula stated as[Ag3(C6H5O7)n+1]3n−,
where n is an integer, that is, n = 1, 2, 3…, are expected to be soluble and
stable in water. These complexes, of course, should further be studied using
spectroscopic and other analytical techniques. On the other hand, the reaction
(9) is reversible, and as seen from Figures 4 and 5, depends on both amounts of silver
citrate and citric acid concentration. The structure of the proposed [Ag3(C6H5O7)n+1]3n− complex ion should further be investigated using spectroscopic or other
analytical techniques.


### 3.3. Stability of Silver Citrate/citric Acid Complexes

The
solutions of silver citrate/citric acid have exhibited a relatively good
stability. Figure6 shows a dependence
of silver ion concentration on
time for a silver citrate dissolved in a 4 mol
H3C6H5O7 solution left to stay for
13 weeks. It is obvious from Figure 6 that the concentration of silver ion in
solution decreased from 19 g/L to about 13 g/L during 4 weeks. A further aging,
that is, after 4 weeks, did not lead to any significant changes in the Ag+ ion concentration, and as illustrated in Figure 6, it remained practically the
same (about 13 g/L). The decrease in the silver ion concentration is attributable
to the recrystallization of Ag3C6H5O7 in saturated silver citrate/citric acid solution.Figure 6
Dependence of silver
ion concentration on time for a silver citrate complexed solution where the
initialAg+ ion concentration was about 19 g/L.Indeed, a precipitation (formation
of colorless crystals) in this solution was observed. Chemical analysis has
found that these crystals contain about 63% Ag, which is comparable to the
silver content in silver citrate (see Table1).Do
these crystals, formed during the aging of concentrated silver citrate
complexed solutions, really representAg3C6H5O7 (one that is produced via routes (a), (b), or (c), described in Section 1)? Based on
the XRD patterns obtained for the crystals formed during the aging silver
citrate/citric acid complexed solutions, it seems unlikely that these crystals
are Ag3C6H5O7. As illustrated in Figure 7, there is a
significant difference in the XRD patterns of crystals produced during the
aging of silver citrate/citric acid solutions (a) and silver citrate produced
via NaOH route (b).Figure 7
Comparison of XRD
patterns of crystals produced during the aging of (a) silver citrate/citric acid
solutions and (b) silver citrate (Ag3C6H5O7) produced via NaOH route.When considering the interactions of
silver(I) with hydroxycarboxylic acids, little information is available in the
open literature [15]. In general terms, only weak complexes are formed, with a
tendency to undergo reduction to silver(0) in aqueous solutions, as it is seen
with ascorbate and tartrate. On the other hand, the citrate ions do not show
tendency to be oxidized by the Ag(I) ions. Despite the resistivity of Ag(I)
ions to be reduced by the citrate, and commercial availability of silver
citrate, very little knowledge on this class of compounds is available in the
literature.Crystals  of polymeric ammonium silver citrate
hydrate, namely,{NH4[Ag2(C6H5O7)](H2O)}n were grown successfully [16]. Similarly, the crystals of silver glycolate
hemihydrate ([Ag2(HOCH2CO2)2]n⋅nH2O)
[15], or silver mandelate complex [10] formulated as [Ag(C8H7O3)]n have been reported in the literature.
Based on the experimental observations, literature [10, 15, 16] and the
fact that the investigated system contained only H2O,  C6H5O73− and Ag+ can be postulated that the formation of crystals in the
saturated silver citrate/citric acid solutions can be described by the
following equation:
(10)x[Ag3(C6H5O7)n+1]3n−⟶[Ag3C6H5O7]x↓+nC6H5O73n−.In this way, the
XRD pattern presented in Figure7(a) can be attributed to [Ag3C6H5O7]x,
or most likely its hydrated form, for example, [Ag3C6H5O7]x⋅nH2O.
It is obvious that further XRD studies are required in order to determine the
exact structure of this compound.Based
on the results presented in Figure6, it was assumed that if the solutions of
silver citrate/citric acid are prepared with lower concentration of Ag+,
they should exhibit a better stability than the solutions containing more than
15 g of Ag+/L.
In other words, under these conditions, the
precipitation should not be observed, of course, if the concentration of citric
acid is sufficient “to allow” the existence of silver citrate complexes in
solution. In fact, the originally prepared silver citrate/citric acid solution
containing about 20 g/L was diluted with water to about 13 g/L and left to stay
in clear bottles exposed to daylight at room temperature. As shown in Figure 8,
changes in the concentration of silver ions in solutions have not been observed
for over 10 weeks.  Based on the
experimental results of the present work, it seems that the silver
citrate/citric acid complexed solutions are quite stable for a relatively long
period of time, when the concentration of Ag+ ion is less than 15 g/L and the concentration of citric acid is more than 3 mol/L (576 g/L). These
solutions have not exhibited changes in color and, also, a formation of
precipitate did not occur. Contrarily, these solutions were clear and colorless
for more than 6 months, suggesting as well a good stability when exposed to
light.Figure 8
Dependence of silver
ion concentration on time for a silver citrate complexed solution where the
initialAg+ ion concentration was about 13 g/L.


### 3.4. Antimicrobial Activity of Silver Citrate Complexes

In Figure9 are shown photographs (after
incubation for 24 hours at 37°C) of disks placed onto an agar medium
seeded with Pseudomonas aeruginosa.
It is obvious that a clear zone surrounding the test sample where bacterial
growth does not occur (or is inhibited) was obtained for the antibiotic disk assay
wetted with 100 μL of silver citrate/citric acid solution containing about 100 ppm Ag(I) ion. The CZOI was estimated at about 9 mm. This result suggests a
strong bacteriostatic activity of silver citrate/citric acid complexed
solution.Bacteriostatic activity
of (a) silver citrate/citric acid complexed solution and (b) control.
(a)(b)For comparison, in Figure9(b) is
also shown a photograph of the antibiotic assay disk (not treated with silver
citrate/citric acid solutions). In this case, a clear zone surrounding the test
sample was not obtained. Contrarily, bacterial growth was observed on the disk.The
results of the bactericidal activity are summarized in Table2. As can be seen
from Table 2, a log reduction of 7.39 was achieved with silver citrate/citric
acid solution (practically speaking, no surviving organisms were observed),
suggesting a total kill of the tested organisms (Pseudomonas aeruginosa). Importantly, the results in Table 2
suggest a very strong bactericidal activity of silver citrate/citric acid
solution. On the other hand, silver nitrate solution containing the same
concentration of Ag(I) ion as silver citrate/citric acid solution has shown the
log reduction of only 0.16, suggesting a very low killing rate. This result is
in the agreement with the previously observed behavior that the silver
complexes (as in the present work, silver citrate/citric acid solutions) are
more effective agents than free silver ions (silver nitrate) [4]. It is obvious
that silver citrate/citric acid solution has exhibited a superior bactericidal
activity over silver nitrate solution.Table 2
Bactericidal activity of silver citrate/citric acid complexed solutions againstPseudomonas aeruginosa (30 minutes
exposure).
SampleAg content (ppm)CFU/plateDensity CFU/mLLog densityLog reductionControl0272.5×1077.392622AgNO3
solution100161.7×1077.230.161818Silver citrate/citric acid solution1000007.3900


## 3.1. Characterization of Silver Citrate Produced via Different Routes

In
Table1 are given results for the yield and silver content in silver citrate
obtained by different routes (i.e., via Na3C6H5O7 (a), via NaOH (b), or via NH4OH (c)), as described in Section 1. It
is obvious from Table 1 that the yield for the production of Ag3C6H5O7 via different routes is above 98%. Discrepancies from 100% are related to the
experimental error.Table 1
The yield and the silver content in silver citrate produced via different routes.
RouteExpected  compoundYield %Ag content %Theoretical Ag content %Na3C6H5O7Ag3C6H5O799.2563.0663.13NaOHAg3C6H5O798.3462.9563.13NH4OHAg3C6H5O798.7562.5063.13The
chemical analysis showed that all the samples of silver citrate produced in the
present work contained the amount of silver close to the theoretical value of
63.13%, suggesting a relatively high purity of the product.Overlaid
XRD patterns of theAg3C6H5O7 produced by the routes (a), (b), and (c) are shown in Figure 1. As illustrated in
Figure 1, the peaks in the XRD patterns of the white precipitate exactly
coincide and match the lines for the Ag3C6H5O7.Figure 1
Comparison of the XRD patterns of silver citrate (Ag3C6H5O7)
produced via (a) Na3C6H5O7, (b) NaOH, and (c) NH4OH routes.In
Figure2 are presented DSC curves of Ag3C6H5O7 produced using Na3C6H5O7 (a), NaOH
(b), or NH4OH (c) routes. When silver citrate samples produced in
the present work are heated in an argon atmosphere, the obtained DSC curves
exhibit a clearly distinguished exothermic maximum in the range from 200°C to 210°C, as illustrated in Figure 2. The energy of this exothermic
process is estimated at about 220 J/g for all the tested samples. This exotherm,
in analogy with the behavior of other silver salts during heating in an inert
environment, is attributed to the decomposition of silver citrate to the
elemental silver.Figure 2
Comparison of DSC curves of silver citrate (Ag3C6H5O7)
produced via (a) Na3C6H5O7, (b) NaOH, and (c) NH4OH routes (heating rate 10°C/min, Ar
atmosphere).Figure3 presents SEM micrographs of Ag3C6H5O7 samples produced via Na3C6H5O7 (a),
via NaOH (b), or via NH4OH (c) routes. As can be seen from these
micrographs, there are no significant differences in the shape of the
individual particles of silver citrate produced in different ways. The size of
these individual particles is estimated to be in the range from 0.2 μm to 0.5 μm.Comparison
of SEM micrographs of silver
citrate (Ag3C6H5O7) produced via (a) Na3C6H5O7, (b) NaOH, (c) and NH4OH routes (magnifications are given on
images).
(a)(b)(c)


## 3.2. Dissolution of Silver Citrate in Citric Acid Solutions

As
mentioned above, silver citrate is very little soluble in water. However, the
experiments in the present work have found that silver citrate can quite
successfully be dissolved in the citric acid aqueous solutions. This observation
is summarized in Figure4, where a dependence of the concentration of silver
ions in solution on the concentration of citric acid, for a fixed amount of
silver citrate, is presented. As shown in Figure 4, an increase in the
concentration of citric acid, for the fixed amount of Ag3C6H5O7,
leads to an increase in the concentration of silver ions in solution. These
results strongly suggest that an increase in the concentration of citric acid
in solution leads to an increase in the solubility of silver citrate.Figure 4
Concentration ofAg+ when a constant mass of silver citrate is dissolved in solutions with different
citric acid concentrations.The
maximum concentration of silver ions in a saturated solution of silver citrate in
water can be estimated at about 0.3 g/L (2.8×10−3 mol/L), which is
in agreement with the literature data [11].Silver citrate dissociates in aqueous solutions according to the following reaction:(5)Ag3C6H5O7⟷3Ag++C6H5O73−.
Consequently,
the equation for the solubility product of silver citrate can be written as
(6)Ksp(Ag3C6H5O7)=[Ag+]3+[C6H5O73−].Based
on the results presented in Figure4, the solubility product of Ag3C6H5O7 is estimated to be in the order of magnitude 10−12 to 10−11. Of course, for a precise determination of the
solubility product of silver citrate, more sophisticated experiments are
required.  It is to be mentioned that the
value of solubility product has not been found in the open literature.In Figure5 are presented results of
silver ion concentration on the amount of silver citrate dissolved in 50 cm3 solutions with different concentrations of citric acid. Based on the dependences presented in Figure
5, the similar can be concluded as explained above for Figure 4. Generally, in
a solution with a constant concentration of citric acid, an increase in the
amount of silver citrate leads to an increase in the concentration of silver
ions in solution. However, the maximum amount of silver citrate which can be
dissolved depends on the citric acid concentration. For example, the maximum
amount of silver citrate soluble in a 50 cm3 of 0.5 M citric acid
solution is estimated at about 0.4 g, which corresponds to the Ag+ ion concentration of about 5 g/L. A
further increase in the amount of silver citrate in a 0.5 M citric acid
solution, as illustrated in Figure 5, does not lead to an increase in the
concentration of Ag+ ions, suggesting that the dissolution does not
take place and that the saturation is achieved.Figure 5
Concentration ofAg+ as a function of the amount of silver citrate dissolved in aqueous solutions of
citric acid with different concentrations.However,
a rise in the concentration of citric acid leads to an increase in the amount
of silver citrate which can be dissolved. In concentrated citric acid solutions
(3 mol/L to 4 mol/L), which corresponds to about 576 to 768 g/L, the maximum
concentration of silver ions is estimated to about 22–25 g/L (0.204–0.232
mol/L). These concentrations of silver
ions correspond to the amount of silver citrate in the range from 34.85 g/L to
39.60 g/L. A further addition of silver citrate to the concentrated citric acid
solutions does not lead to dissolution, suggesting that the saturation is
achieved. On the other hand, the
preparation of citric acid solutions with concentrations more than 4 mol/L
faces experimental difficulties, even with heating, since the crystallization
of citric acid occurs due to the saturation.How can the dissolution of silver
citrate in citric acid solutions be described?
Citric acid,H3C6H5O7, contains
three carboxylic groups (–COOH) and, consequently, three hydrogen ions can be
replaced with metal ions, as it is observed in the precipitation of silver
citrate, Ag3C6H5O7.  When one or two hydrogen ions from the
carboxylic groups of citric acid are replaced with Ag+ ions AgH2C6H5O7 or Ag2HC6H5O7 should be produced,
respectively. The formation of these compounds can be described with the
following reactions:
(7)Ag3C6H5O7↓+2H3C6H5O7⟶3AgH2C6H5O7,(8)2Ag3C6H5O7↓+H3C6H5O7⟶3Ag2HC6H5O7.Based
on the stoichiometry of the reactions (7) and (8) and an assumption that AgH2C6H5O7 or Ag2HC6H5O7 is soluble in water,
it can easily be calculated that the amounts of citric acid for the dissolution
of 100 g of Ag3C6H5O7 (0.195 mol)
are, respectively, 74.89 g (0.384 mol) for the reaction (7) and 18.72 g
(0.0975 mol) for the reaction (8).However,
the experimental observations have shown that thisis not the case. As experimentally found, significantly larger
amounts of citric acid are required for the dissolution of Ag3C6H5O7 than those calculated on the basis of the stoichiometry of the reactions (7) and (8). Consequently,
if AgH2C6H5O7 and Ag2HC6H5O7 are soluble in water, it is unlikely that the dissolution of silver citrate proceeds
via reactions (7) and (8). On the other hand, chemical analysis of
“undissolved” precipitate found that it (precipitate) contained about 63% Ag,
which corresponds to Ag3C6H5O7 (see
Table 1) and not to AgH2C6H5O7 or Ag2HC6H5O7.Considering the fact that significantly
larger amounts of citric acid are required for the dissolution of silver
citrate, it is reasonable to assume, based on the silver analysis and
stoichiometry of the chemical equations, that citric acid produces complexes
with silver citrate according to the following reaction:(9)Ag3C6H5O7+nH3C6H5O7⟷[Ag3(C6H5O7)n+1]3n−+3nH+.The
complexes with a general formula stated as[Ag3(C6H5O7)n+1]3n−,
where n is an integer, that is, n = 1, 2, 3…, are expected to be soluble and
stable in water. These complexes, of course, should further be studied using
spectroscopic and other analytical techniques. On the other hand, the reaction
(9) is reversible, and as seen from Figures 4 and 5, depends on both amounts of silver
citrate and citric acid concentration. The structure of the proposed [Ag3(C6H5O7)n+1]3n− complex ion should further be investigated using spectroscopic or other
analytical techniques.


## 3.3. Stability of Silver Citrate/citric Acid Complexes

The
solutions of silver citrate/citric acid have exhibited a relatively good
stability. Figure6 shows a dependence
of silver ion concentration on
time for a silver citrate dissolved in a 4 mol
H3C6H5O7 solution left to stay for
13 weeks. It is obvious from Figure 6 that the concentration of silver ion in
solution decreased from 19 g/L to about 13 g/L during 4 weeks. A further aging,
that is, after 4 weeks, did not lead to any significant changes in the Ag+ ion concentration, and as illustrated in Figure 6, it remained practically the
same (about 13 g/L). The decrease in the silver ion concentration is attributable
to the recrystallization of Ag3C6H5O7 in saturated silver citrate/citric acid solution.Figure 6
Dependence of silver
ion concentration on time for a silver citrate complexed solution where the
initialAg+ ion concentration was about 19 g/L.Indeed, a precipitation (formation
of colorless crystals) in this solution was observed. Chemical analysis has
found that these crystals contain about 63% Ag, which is comparable to the
silver content in silver citrate (see Table1).Do
these crystals, formed during the aging of concentrated silver citrate
complexed solutions, really representAg3C6H5O7 (one that is produced via routes (a), (b), or (c), described in Section 1)? Based on
the XRD patterns obtained for the crystals formed during the aging silver
citrate/citric acid complexed solutions, it seems unlikely that these crystals
are Ag3C6H5O7. As illustrated in Figure 7, there is a
significant difference in the XRD patterns of crystals produced during the
aging of silver citrate/citric acid solutions (a) and silver citrate produced
via NaOH route (b).Figure 7
Comparison of XRD
patterns of crystals produced during the aging of (a) silver citrate/citric acid
solutions and (b) silver citrate (Ag3C6H5O7) produced via NaOH route.When considering the interactions of
silver(I) with hydroxycarboxylic acids, little information is available in the
open literature [15]. In general terms, only weak complexes are formed, with a
tendency to undergo reduction to silver(0) in aqueous solutions, as it is seen
with ascorbate and tartrate. On the other hand, the citrate ions do not show
tendency to be oxidized by the Ag(I) ions. Despite the resistivity of Ag(I)
ions to be reduced by the citrate, and commercial availability of silver
citrate, very little knowledge on this class of compounds is available in the
literature.Crystals  of polymeric ammonium silver citrate
hydrate, namely,{NH4[Ag2(C6H5O7)](H2O)}n were grown successfully [16]. Similarly, the crystals of silver glycolate
hemihydrate ([Ag2(HOCH2CO2)2]n⋅nH2O)
[15], or silver mandelate complex [10] formulated as [Ag(C8H7O3)]n have been reported in the literature.
Based on the experimental observations, literature [10, 15, 16] and the
fact that the investigated system contained only H2O,  C6H5O73− and Ag+ can be postulated that the formation of crystals in the
saturated silver citrate/citric acid solutions can be described by the
following equation:
(10)x[Ag3(C6H5O7)n+1]3n−⟶[Ag3C6H5O7]x↓+nC6H5O73n−.In this way, the
XRD pattern presented in Figure7(a) can be attributed to [Ag3C6H5O7]x,
or most likely its hydrated form, for example, [Ag3C6H5O7]x⋅nH2O.
It is obvious that further XRD studies are required in order to determine the
exact structure of this compound.Based
on the results presented in Figure6, it was assumed that if the solutions of
silver citrate/citric acid are prepared with lower concentration of Ag+,
they should exhibit a better stability than the solutions containing more than
15 g of Ag+/L.
In other words, under these conditions, the
precipitation should not be observed, of course, if the concentration of citric
acid is sufficient “to allow” the existence of silver citrate complexes in
solution. In fact, the originally prepared silver citrate/citric acid solution
containing about 20 g/L was diluted with water to about 13 g/L and left to stay
in clear bottles exposed to daylight at room temperature. As shown in Figure 8,
changes in the concentration of silver ions in solutions have not been observed
for over 10 weeks.  Based on the
experimental results of the present work, it seems that the silver
citrate/citric acid complexed solutions are quite stable for a relatively long
period of time, when the concentration of Ag+ ion is less than 15 g/L and the concentration of citric acid is more than 3 mol/L (576 g/L). These
solutions have not exhibited changes in color and, also, a formation of
precipitate did not occur. Contrarily, these solutions were clear and colorless
for more than 6 months, suggesting as well a good stability when exposed to
light.Figure 8
Dependence of silver
ion concentration on time for a silver citrate complexed solution where the
initialAg+ ion concentration was about 13 g/L.


## 3.4. Antimicrobial Activity of Silver Citrate Complexes

In Figure9 are shown photographs (after
incubation for 24 hours at 37°C) of disks placed onto an agar medium
seeded with Pseudomonas aeruginosa.
It is obvious that a clear zone surrounding the test sample where bacterial
growth does not occur (or is inhibited) was obtained for the antibiotic disk assay
wetted with 100 μL of silver citrate/citric acid solution containing about 100 ppm Ag(I) ion. The CZOI was estimated at about 9 mm. This result suggests a
strong bacteriostatic activity of silver citrate/citric acid complexed
solution.Bacteriostatic activity
of (a) silver citrate/citric acid complexed solution and (b) control.
(a)(b)For comparison, in Figure9(b) is
also shown a photograph of the antibiotic assay disk (not treated with silver
citrate/citric acid solutions). In this case, a clear zone surrounding the test
sample was not obtained. Contrarily, bacterial growth was observed on the disk.The
results of the bactericidal activity are summarized in Table2. As can be seen
from Table 2, a log reduction of 7.39 was achieved with silver citrate/citric
acid solution (practically speaking, no surviving organisms were observed),
suggesting a total kill of the tested organisms (Pseudomonas aeruginosa). Importantly, the results in Table 2
suggest a very strong bactericidal activity of silver citrate/citric acid
solution. On the other hand, silver nitrate solution containing the same
concentration of Ag(I) ion as silver citrate/citric acid solution has shown the
log reduction of only 0.16, suggesting a very low killing rate. This result is
in the agreement with the previously observed behavior that the silver
complexes (as in the present work, silver citrate/citric acid solutions) are
more effective agents than free silver ions (silver nitrate) [4]. It is obvious
that silver citrate/citric acid solution has exhibited a superior bactericidal
activity over silver nitrate solution.Table 2
Bactericidal activity of silver citrate/citric acid complexed solutions againstPseudomonas aeruginosa (30 minutes
exposure).
SampleAg content (ppm)CFU/plateDensity CFU/mLLog densityLog reductionControl0272.5×1077.392622AgNO3
solution100161.7×1077.230.161818Silver citrate/citric acid solution1000007.3900


## 4. Conclusions

Silver citrate can successfully be
deposited using silver nitrate as a source of silver ions and salts of citric
acid as sources of citrate ions. Although, silver citrate is very little
soluble in water, it can be successfully dissolved in citric acid solutions. An
increase in the concentration of citric acid leads to an increase in the amount
of silver citrate that can be dissolved. The maximum concentration of Ag(I) in the
solution that can be achieved is estimated at about 23 g/L to 25 g/L if the
concentration of citric acid is at least 4 mol/L or higher.The dissolution of silver citrate in
citric acid solutions was attributed to the formation of silver citrate
complexes of a general formula[Ag3(C6H5O7)n+1]3n−.
These solutions have shown a reasonable stability over time. In concentrated
solutions, containing more than 13 g/L of Ag(I) ion, the crystallization was
observed. Based on the XRD and chemical analyses, the crystallization product
was attributed to the formation of a compound [Ag3C6H5O7]x⋅nH2O.Importantly, the diluted silver
citrate/citric acid complexed solutions have exhibited very strong
bacteriostatic and bactericidal activities.



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*Source: 436458-2008-11-27.xml*