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Synthesis of Highly Concentrated Suspensions of Silver Nanoparticles by Two

Versions of the Chemical Reduction Method

Article  in  Methods and Protocols · December 2018

DOI: 10.3390/mps2010003

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Protocol
Synthesis of Highly Concentrated Suspensions of
Silver Nanoparticles by Two Versions of the Chemical
Reduction Method
Miguel Gakiya-Teruya, Luis Palomino-Marcelo and Juan Carlos F. Rodriguez-Reyes *
Department of Bioengineering and Chemical Engineering, Universidad de Ingenieria y Tecnologia—UTEC, Jr.
Medrano Silva 165, Barranco, Lima 15063, Peru; mrgteruya@gmail.com (M.G.-T.);
luispalomino026@gmail.com (L.P.-M.)
* Correspondence: jcrodriguez@utec.edu.pe; Tel.: +511-2305000




Received: 24 November 2018; Accepted: 20 December 2018; Published: 24 December 2018


Abstract: In spite of the widespread use of the chemical reduction method to obtain silver
nanoparticles, the nanoparticle yield is often low due to a required addition of small volumes
of diluted metal ions to a solution containing a reducer. Higher yields can be obtained following an
alternative method, in which the reducer is added to a greater volume of silver ions in the solution.
In this study, protocols for both methods are detailed and compared, using characterization tools
such as UV-vis spectrometry, dynamic light scattering (DLS), and zeta potential measurements.
By using this alternative method, the amount of silver in the solution is three times greater, and
nanoparticles with a narrower size distribution are formed (between 6 and 70 nm in size). In contrast,
the regular method produces particles of 3 and 100 nm. Zeta potential measurements indicate that
the nanoparticles synthesized with the alternative method will be more stable than those from the
regular method.

Keywords: Silver nanoparticles; UV-VIS spectrometry; dynamic light scattering; Frens method

1. Introduction
Metallic nanoparticles can be prepared using two approaches: top-down (when the starting
point is a large portion of the material, which is down-sized) and bottom-up (when the nanoparticle
precursors are ions or molecules which will nucleate and grow) [1]. The chemical reduction method,
described in previous reports by other groups [2–4], is a bottom-up method that is relatively simple,
which allows for the control of size and shape of nanoparticles. Nanoparticles produced with this
method are stabilized by (i) maximizing the electrostatic repulsion between nanoparticles using capping
agents (such as anions borohydride or citrate), and (ii) using small concentrations of metal precursors
so that the likeliness of collisions of the growing nanoparticles are small. Thus, the stability of
nanoparticles in suspension can be estimated by measuring the zeta potential (the electrostatic potential
near the surface of a nanoparticle, which is derived from measuring the velocity of the particles in
an applied electric field); greater values of zeta potential indicate greater repulsion and; therefore,
an increased stability of nanoparticles [5]. The size and relative concentration of nanoparticles can be
calculated using the dynamic light scattering (DLS) technique, where the scattered light produced by
moving particles can be measured. The scattering pattern can then be related to the velocity of these
particles and, in turn, to the size of the nanoparticles using the Stokes–Einstein equation [5].
The excessive growth of nanoparticle size is avoided by using low concentrations of metal
precursors (in the mM range) resulting in low concentrations of nanoparticles. For demonstrative and
educational purposes, it is accepted [2]; however, additional work with nanoparticles (for example,

Methods and Protoc. 2019, 2, 3; doi:10.3390/mps2010003 www.mdpi.com/journal/mps

 Methods Protoc. 2018, 1, x FOR PEER REVIEW 2 of 5

for functionalization or for studying interactions with biomolecules) [6,7] in higher concentrations
Methods
may be anddesired.
Protoc. 2019, 2, 3
Surprisingly, there is a lack in the literature with respect to the comparison of 2both of 5
Methods Protoc. 2018, 1, x FOR PEER REVIEW 2 of 5
methods and the characterization and quantification of the resulting nanoparticles from the same
Methods Protoc. 2018, 1, x FOR PEER REVIEW 2 of 5
laboratory.
for Below weorreport
functionalization for a protocol
studying for obtaining
interactions with a biomolecules)
higher concentration [6,7] in ofhigher
Ag nanoparticles
concentrations in
for functionalization or for studying interactions with biomolecules) [6,7] in higher concentrations
suspension,
may be based on a modification of the Frens method [8]. We use UV-Vis and DLS to characterize
bedesired.
desired. Surprisingly,
for functionalization
may or for studying
Surprisingly, there
thereisisainteractions
alack
lackin inthe literature
thewithliterature with
withrespect
biomolecules) respect[6,7] to the
tointhe comparison
higher
comparison of
ofboth
concentrationsboth
the
methods resulting
and solutions. A Nanodrop® spectrometer is also used, as this type of spectrometer allows
may be desired.
methods and the characterization
theSurprisingly,
characterization thereand is aquantification
and lack in the literature
quantification of
of the
the resulting
with
resulting respectnanoparticles
to the comparison
nanoparticles from
from thetheofsame
both
same
for the measurement
laboratory. of higha protocol
concentrations of nanoparticles in suspension,ofas itnanoparticles
can read higher
laboratory. Below we report a protocol for obtaining a higher concentration of Ag nanoparticles in
methods andBelow the we report
characterization and for obtaining
quantification a higher
of the concentration
resulting nanoparticles Ag from the samein
absorbancesbased
suspension, than conventional spectrometers [9].method
For comparison, we include also a protocol similar
laboratory. Below
suspension, basedon onaareport
we modificationa protocol
modification of
oftheforFrens
the obtaining
Frens method [8].
a higher[8]. WeWe use
use UV-Vis
UV-Visand
concentration of Ag
and DLS
DLS totocharacterize
nanoparticles
characterize in
to the
the reported
resulting earlier by
solutions. our group ®[10].
suspension,
the resultingbased solutions. on a A A Nanodrop
modification
Nanodrop® ofspectrometer
the Frens method
spectrometer isisalso
also used,
[8]. We as
used, asthis
use UV-Vis
this type
typeandof
ofspectrometer
DLS to characterize
spectrometer allows
allows
for
thethe
for the measurement
resulting
measurementsolutions. ofofAhigh concentrations
Nanodrop®
high concentrations spectrometer of
of nanoparticles
is also used,
nanoparticles in suspension,
in as this type ofas
suspension, itit can
can read
asspectrometer read higher
allows
higher
2. Experimental Design
absorbances
for the measurement
absorbances than
thanconventional spectrometers
spectrometers[9].
of high concentrations
conventional For
Forcomparison,
of nanoparticles
[9]. comparison, in weweinclude
includealso
suspension, as itaaprotocol
also can readsimilar
protocol higher
similar
to
to the reported
absorbances than earlier by
conventional our group [10].
spectrometers [9]. For comparison, we include also a protocol similar
2.1.the reported earlier by our group [10].
Materials
to the reported earlier by our group [10].
• Experimental
2.2. Silver nitrate
Experimental Design
(J.A. Elmer, 99.9%, Lima, Peru)
Design
• Experimental
2. Sodium citrate Design (Movilab, 99.9%, Lima, Peru)
2.1. Materials
2.1. Materials
2.2. Equipment
•2.1. Materials
Silver nitrate (J.A. Elmer, 99.9%, Lima, Peru)
• Silver nitrate (J.A. Elmer, 99.9%, Lima, Peru)
••MethodsSodium
Protoc.citrate
UV-VIS 2018, 1, (Movilab,
x FOR PEER 99.9%,
spectrophotometer, Elmer,REVIEW ISRLima,
2600 plus Peru)(Shimadzu, Kyoto, Japan) 2 of 5
• Silver
Sodium nitrate
citrate(J.A. (Movilab, 99.9%, Lima, Peru)
• Nanodrop
Sodium 1000(Movilab,
citrate Spectrophotometer 99.9%, Lima, (Thermo
Peru) Fisher Scientific INC., Waltham, MA, USA)
forEquipment
2.2. functionalization or for studying interactions with biomolecules) [6,7] in higher concentrations
• Equipment
2.2. Dynamic light scattering (DLS) Möbiuζ® (Wyatt Technology, Santa Barbara, CA, USA)
may be desired. Surprisingly, there is a lack in the literature with respect to the comparison of both
• Equipment
•2.2. Vacuumspectrophotometer,
UV-VIS Pump RV 3 (Edwards, ISR 2600 Westplus Sussex, UK)
(Shimadzu, Kyoto, Japan)
•methods UV-VIS andspectrophotometer,
the characterization ISRand 2600 plus (Shimadzu,
quantification of the Kyoto, Japan)
resulting nanoparticles from the same
• Nanodrop 1000 Spectrophotometer (Thermo Fisher Scientific INC., Waltham, MA, USA)
•laboratory.
UV-VISBelow
Nanodrop spectrophotometer,
1000 we Spectrophotometer
report a ISR 2600
protocol for plus
(Thermo (Shimadzu,
obtaining Fisher a higher Kyoto,
Scientific Japan)
INC.,
concentration Waltham,of AgMA, USA)
nanoparticles in
••3. Procedure
Dynamic
Nanodrop
Dynamic light1000
light scattering
Spectrophotometer
scattering (DLS)
(DLS) Möbiuζ
Möbiuζ®
® (Wyatt Technology, Santa Barbara, CA, USA)
(Thermo (WyattFisher Scientific
Technology, INC.,
Santa Waltham,
Barbara, MA,
CA, USA)
USA)
suspension, based on a modification of the Frens method [8]. We use UV-Vis and DLS to characterize
••the Vacuum
DynamicPump light RV 33A(Edwards,
scattering (DLS)Using West
Möbiuζ® Sussex, UK)
(Wyatt isTechnology, asSanta Barbara,
Vacuum
resulting
3.1. Synthesis Pump
ofsolutions.
Silver RV (Edwards,
Nanodrop®
Nanoparticles West theSussex,
spectrometer
Regular UK) Methodalso (Time
used, for this type
Completion: 1 CA,
h) USA) allows
of spectrometer
•for theVacuum Pump RV 3 (Edwards, West Sussex, UK)
measurement of high concentrations of nanoparticles in suspension, as it can read higher
3.3.
1.Procedure
Place the solution of AgNO3 2 mM on a dropper.
Procedure
absorbances than conventional spectrometers [9]. For comparison, we include also a protocol similar
2. Place 50 mL of sodium citrate 7mM in an Erlenmeyer flask with a magnetic pill (see Figure 1).
3.toProcedure
the reported
3.1. Synthesis of Silver earlier by our group
Nanoparticles [10].the Regular Method (Time for Completion: 1 h)
Using
3. Synthesis
3.1. Cover the of flask
Silverwith aluminum
Nanoparticles foil and
Using heat it in
the Regular a water
Method (Timebathforuntil boiling point.
Completion: 1 h)
3.1.
1.4. Synthesis
When
Place the
the of Silver
solution
solution Nanoparticles
of
of sodium
AgNO 2 Using
citrate
mM the
has
on a Regular
reached
dropper. Method
the boiling (Time for
point Completion:
add dropwise 1 h) 8.8 mL of AgNO3.
1.2. Experimental
Place the solution Design 3
of AgNO3 2 mM on a dropper.
CRITICAL
2.1. Place 50 themL STEP: The
of sodium
solution solution
of AgNO citrate of
7mM
3 2 7mM
AgNO
mM on in an 3 has to be added dropwise
Erlenmeyer flask with a magnetic pill (see Figureto obtain a good distribution
1).
2. Place 50 mL of sodium citrate in aandropper.
Erlenmeyer flask with a magnetic pill (see Figure 1).
2.1.
3.3.
2. Coverof
Coversilver
Materials
Place 50 the nanoparticles.
flask with aluminum foil and heat it in a water bath until boiling point.
themL of sodium
flask with aluminum citrate 7mM foil andin anheat Erlenmeyer
it in a water flaskbath with a magnetic
until pill (see Figure 1).
boiling point.
5. When
4.4. Stir the the solution
solution forof 40 min. citrate
sodium has reached
3.
• When Cover
Silverthe the
nitrate flask
solution with
(J.A.of aluminum
sodium
Elmer, 99.9%, foil
citrate and Peru) the boiling point add dropwise8.8
heat
has reached
Lima, it the
in a boiling
water point
bath add
until dropwise
boiling 8.8mL
point. mLof ofAgNO
AgNO33..
PAUSE
4.• CRITICAL
When STEP:
thecitrate After
solution stirring,
of sodium allow
citrate the suspension
has 3reached to
theadded cool
boiling down to
point add ambient temperature.
Sodium STEP:(Movilab,
The solution 99.9%, of AgNO
AgNO
Lima, has to
Peru) to be be dropwise todropwise
obtain aa good 8.8 mL
good of AgNO3.
distribution
6. CRITICAL
CentrifugeSTEP:
CRITICAL 1 mL
STEP:
The
The ofsolution
the solution
solution
of
of AgNO at33 has
2040to RCF
has be
added
added
dropwise
(relative
dropwise
to
centrifugal
to
obtain
obtain force,
a good
distribution
following
distributionthe
of silver
of silver nanoparticles.
nanoparticles.
recommendation
2.2. Equipment
of silver nanoparticles. of the company Citodiagnostics [11]) for 30 min, discard the supernatant and
5.5. Stir Stir the solution
the
resuspend
solution
in 1
for 40
for
mL
40 min.
min.
distilled water.
5. PAUSEStir theSTEP: solution for 40
After stirring, min. allow the suspension to coolKyoto, down Japan)to ambient temperature.
• PAUSE UV-VIS spectrophotometer,
STEP: After The solution can ISRbe 2600
stored plus
at 4(Shimadzu,
°C into the dark for toup to 6 months.
6.• PAUSE
PAUSE
Centrifuge STEP:
STEP: 1 After
mL stirring,
stirring,
of the allow
allow
solution the
Nanodrop 1000 Spectrophotometer (Thermo Fisher Scientific INC.,the suspension
suspension
at 2040 RCF to cool
cool down
(relativedown toambient
ambient
centrifugal Waltham,
temperature.
temperature.
force, MA, following
USA) the
6.6. Centrifuge
Centrifuge
recommendation 11 mLmL of of
ofthe the
the solution
companysolution at
at 2040
2040
Citodiagnostics RCF
RCF (relative
[11])(relative
for 30 centrifugal
centrifugal
min, discard force,
force,
the following
following
supernatant the
the
and
• Synthesis
3.2. Dynamic of light
Silverscattering
Nanoparticles (DLS) Möbiuζ®
Using the Frens (Wyatt
Method Technology,
(Timefor for30 Santa Barbara,
Completion: 40 min) CA, USA)
recommendation
recommendation of
of the
the company
company Citodiagnostics
Citodiagnostics [11])
[11]) for 30 min,min, discard
discard the the supernatant
supernatant and
• resuspend
Vacuum Pump in 1 mL RVdistilled
3 (Edwards, water. West Sussex, UK)
and
1. PAUSEPlace resuspend
resuspend50 mLinof
STEP: 1 in
ThemL1solution
AgNO mL3 1mMdistilled
distilled can inbe
water. water.
an Erlenmeyer
stored at 4 °Cflask in thewith darka for magnetic
up to 6pill (see Figure 1).
months.
2.3. Procedure
Cover
PAUSE the
STEP: flask The with aluminum
solution can befoil and
stored
PAUSE STEP: The solution can be stored at 4 C in the dark for up to 6 months. heat
at 4 ◦ it
°C until
in the boiling
dark point.
for up to 6 months.
3. Synthesis
3.2. When the solution
of Silver reaches theUsing
Nanoparticles boiling point Method
the Frens add 500 µL of
(Time for sodium
Completion: citrate
40 min)0.189 M (obtained
3.2.
3.2.
3.1. Synthesis
Synthesis
from [12]).
Synthesis ofofofSilver
Silver
Silver Nanoparticles
Nanoparticles
Nanoparticles Using
Using
Using the
the
the Frens
Frens
RegularMethod
Method Method (Time
(Time
(Timefor
forCompletion:
Completion:
for Completion: 40
40min)
min)
1 h)
1. Place 50 mL of AgNO3 1mM in an Erlenmeyer flask with a magnetic pill (see Figure 1).
1.2. CRITICAL
1.1. Place STEP: To obtain a good distribution and small size of silver nanoparticles, the
Place50
Place
Cover 50the mL
mL of
of AgNO
flask
solution AgNO
with 33 1mM3 in
ofaluminum
AgNO an
2 mMfoilErlenmeyer
and
on a heat it flask
dropper. until with boiling aa magnetic
point. pill
magnetic pill (see
(see Figure
Figure 1). 1).
2.3. concentration
2.2. Cover
Cover themL flask ofwith
the aluminum
sodium
aluminum citrate has
foil and
and to be itas
heat itadd mentioned
until boiling above.
point.
Place the
When 50 flaskof with
solution sodium reaches citrate the7mMfoil
boiling in an heat
point until
Erlenmeyer 500boiling
µL of
flask point.
sodium
with citrate pill
a magnetic 0.189 (seeMFigure
(obtained
1).
4.
3. Stir
When the solution
the solution for 20
reachesmin. the boiling point add 500 µL of sodium citrate 0.189 M (obtained
3.3. When from
Cover [12]).
the
thesolution
flask with reaches
aluminum the boiling
foil andpoint heat add it in a500 waterµL of bathsodium citrate point.
until boiling 0.189 M (obtained
5. Centrifuge
from 1 mL of the solution at 2040 RCF (relative centrifugal force) for 30 min, discard the
from
4. CRITICAL
When [12]). STEP: To
the solution obtain acitrate
of sodium goodhas distribution
reached theand boiling small pointsizeadd ofdropwise
silver nanoparticles,
8.8 mL of AgNO the3.
supernatant and resuspend in 1 mL distilled water.
CRITICAL
concentration
CRITICAL STEP:
STEP: of the To
The obtain
sodium
solution a good
citrate
of AgNO distribution
has to be to
3 has as be and
mentioned small
added dropwise size
above.size of silver
to obtain nanoparticles,
a good the
distribution
CRITICAL
PAUSE STEP: STEP: To
The solution obtain can abegood storeddistribution
at 4 °C in theand dark. small of silver nanoparticles,
4. the concentration
Stir the solution of for
the20 sodium citrate has to be as mentioned above.
of silver nanoparticles.
concentration of themin. sodium citrate has to be as mentioned above.
4.5. Centrifuge
5. Stir
Stir the
the solution1
solution mL for
of
for 20
the
40 min.
solution at 2040 RCF (relative centrifugal force) for 30 min, discard the
min.
4. Stir the solution for 20 min.
5. PAUSECentrifuge
supernatant STEP: 1andmL
After of stirring,
the solution
resuspend in atthe
1 mL
allow 2040 RCFwater.
distilled
suspension (relative to cool centrifugal
down to force) ambient fortemperature.
30 min, discard the
supernatant
PAUSE STEP: and The resuspend
solution in
can 1 mL
be distilled
stored at
6. Centrifuge 1 mL of the solution at 2040 RCF (relative centrifugal force, following the4 water.
°C in the dark.
PAUSE STEP: Theof
recommendation solution
the company can be stored at 4 °C in [11])
Citodiagnostics the dark. for 30 min, discard the supernatant and
resuspend in 1 mL distilled water.
PAUSE STEP: The solution can be stored at 4 °C in the dark for up to 6 months.

3.2. Synthesis of Silver Nanoparticles Using the Frens Method (Time for Completion: 40 min)
 3. Cover the flask with aluminum foil and heat it in a water bath until boiling point.
4. When the solution of sodium citrate has reached the boiling point add dropwise 8.8 mL of AgNO3.
CRITICAL STEP: The solution of AgNO3 has to be added dropwise to obtain a good distribution
of silver nanoparticles.
5. Stir the solution for 40 min.
Methods and Protoc. 2019, 2, 3 3 of 5
PAUSE STEP: After stirring, allow the suspension to cool down to ambient temperature.
6. Centrifuge 1 mL of the solution at 2040 RCF (relative centrifugal force, following the
5. Centrifuge
recommendation 1 mL of ofthe
thesolution
company atCitodiagnostics
2040 RCF (relative [11])centrifugal
for 30 min,force) for the
discard 30 min, discard the
supernatant and
supernatant and resuspend
resuspend in 1 mL distilled water. in 1 mL distilled water.
PAUSE
PAUSE STEP:
MethodsSTEP:
The1,solution
The
Protoc. 2018,
solution can
x FOR PEERcan
be stored
be
REVIEW
storedatat44◦°C in the
C in the dark.
dark for up to 6 months. 3 of 5

3.2. Synthesis of Silver Nanoparticles Using the Frens Method (Time for Completion: 40 min)
1. Place 50 mL of AgNO3 1mM in an Erlenmeyer flask with a magnetic pill (see Figure 1).
2. Cover the flask with aluminum foil and heat it until boiling point.
3. When the solution reaches the boiling point add 500 µL of sodium citrate 0.189 M (obtained
from [12]).
CRITICAL STEP: To obtain a good distribution and small size of silver nanoparticles, the
concentration of the sodium citrate has to be as mentioned above.
4. Stir the solution for 20 min.
5. Centrifuge 1 mL of the solution at 2040 RCF (relative centrifugal force) for 30 min, discard the
supernatant and resuspend in 1 mL distilled water.
PAUSE STEP: The solution can be stored at 4 °C in the dark.

Figure
Figure 1. Setup1. Setup for nanoparticle
for nanoparticle synthesisusing
synthesis using the
themethods
methods described in this
described in protocol. The picture
this protocol. The in
picture in
the center compares the appearance of nanoparticle suspensions with these methods. Notice that the
the center compares the appearance of nanoparticle suspensions with these methods. Notice that the
characteristic color of colloidal silver is more intense for the suspension obtained with the Frens method.
characteristic color of colloidal silver is more intense for the suspension obtained with the Frens method.
4. Results
4. Results
4.1. Calculation of Nanoparticle Concentration.
4.1. Calculation of Nanoparticle Concentration
In this work we are reporting the use of two different amounts of silver nitrate (e. g., 50 ml
In AgNO
this work we are reporting the use of two different amounts of silver nitrate (e.g., 50 mL AgNO3
3 1 mM), which is mixed with 500 µL of sodium citrate 0.189 M. The concentration of silver in

1 mM), the
which
final is mixed(inwith
solution 500 µL
grams/L) canof
besodium
estimatedcitrate 0.189
using the M. Theformula:
following concentration of silver in the final
solution (in grams/L) can be estimated using the following formula:
M(Ag) = C(Ag) × V(Ag) × 108 / V(total)
Where M(Ag) is the weight M(Ag) = C(Ag)of×silver
concentration V(Ag) × 108/V(total)
in the final solution, C(Ag) and V(Ag) are the
molar concentration (1 × 10−3 M) and the volume of the precursor solution (50 × 10−3 L), respectively,
where M(Ag)
V(total)isis the weight
the total concentration
volume of silver
of the suspension in the
(the sum final solution,
of volumes of silverC(Ag) andsodium
nitrate and V(Ag)citrate,
are the molar
concentration
50.5 × 10(1−3 L) 10−108
×and 3 M) and the volume of the precursor solution (50 × 10−3 L), respectively,
represents the atomic weight of silver (108 g/mol). Using the values from the
V(total)Frens
is themethod, it resultsofinthe
total volume a solution with a(the
suspension concentration of 0.107 gof
sum of volumes Ag/L (or nitrate
silver 107 mg and
Ag/L). In the citrate,
sodium
case
− 3of the regular method the concentration is 0.032 g Ag/L (or 30 mg Ag/L).
50.5 × 10 L) and 108 represents the atomic weight of silver (108 g/mol). Using the values from the
Frens method, it results in a solution with a concentration of 0.107 g Ag/L (or 107 mg Ag/L). In the
4.2. Characterization through Absorption Spectroscopy and Dynamic Light Scattering.
case of the regular method the concentration is 0.032 g Ag/L (or 30 mg Ag/L).
Silver nanoparticle suspensions using regular and Frens methods were characterized using UV-
Vis spectrometry with a Shimadzu spectrometer and with a Nanodrop. The resulting spectra are
4.2. Characterization through Absorption Spectroscopy and Dynamic Light Scattering
shown in Figure 2 and the information is summarized in Table 1.
Silver nanoparticle suspensions using regular and Frens methods were characterized using UV-Vis
Table 1. Comparison of intensity (arbitrary units, a.u.), position (nm), and full width at half maximum
spectrometry with a Shimadzu spectrometer and with a Nanodrop. The resulting spectra are shown in
(FWHM, nm) for nanoparticles synthesized via the regular and Frens methods. Results from using
Figure 2 and the information is summarized in Table 1.
UV-Vis spectrometry and Nanodrop are included.

Peak
Table 1. Comparison Position (nm)
of intensity (arbitrary units,Intensity (a.u.) (nm), and fullFWHM
a.u.), position width (nm)
at half maximum
(Nanodrop) (UV-Vis) (Nanodrop) (UV-Vis) (Nanodrop)
(FWHM, nm) for nanoparticles synthesized via the regular and Frens methods. Results (UV-Vis)
from using
Frens
UV-Vis spectrometry and Nanodrop are included.
418 ± 4 419 ± 3 0.9 ± 0.1 0.6 ± 0.5* 116 ± 10 106 ± 5
method
Regular Peak Position (nm) Intensity (a.u.) FWHM (nm)
427 ± 7 429 ± 5 0.2 ± 0.1 1.5 ± 0.6 123 ± 7 118 ± 6.1
method (Nanodrop) (UV-Vis) (Nanodrop) (UV-Vis) (Nanodrop) (UV-Vis)
* Sample was diluted (ten-fold).
Frens method 418 ± 4 419 ± 3 0.9 ± 0.1 0.6 ± 0.5 * 116 ± 10 106 ± 5
Regular metho 427 ± 7 429 ± 5 0.2 ± 0.1 1.5 ± 0.6 123 ± 7 118 ± 6.1
* Sample was diluted (ten-fold).
 Methods and Protoc. 2019, 2, 3 4 of 5
Methods Protoc. 2018, 1, x FOR PEER REVIEW 4 of 5
Methods Protoc. 2018, 1, x FOR PEER REVIEW 4 of 5

Figure Absorbance spectra

Figure 2. Absorbance spectraofofAg
Agnanoparticles
nanoparticles obtained
obtained with
with the the regular
regular method
method and the
and with withFrens
the
Figure 2. Absorbance spectra of Ag nanoparticles obtained with the regular method and with the
Frens
method,method,
using ausing a Nanodrop
Nanodrop instrument
instrument (a) and (a) and a spectrometer
a UV-Vis UV-Vis spectrometer (b). The suspension
(b). The suspension obtained
Frens method, using a Nanodrop instrument (a) and a UV-Vis spectrometer (b). The suspension
obtained via the
via the Frens Frens exceeded
method method exceeded the maximum
the maximum measurement
measurement of the UV-VIS
of the UV-VIS spectrometer
spectrometer (b); for (b);
this
obtained via the Frens method exceeded the maximum measurement of the UV-VIS spectrometer (b);
for this reason,
reason, the suspension
the suspension was diluted
was diluted by aoffactor
by a factor 10. of 10.
for this reason, the suspension was diluted by a factor of 10.
From the
From the intensities
intensitiesofofsignals,
signals,it is
it clear thatthat
is clear the Frens method
the Frens produces
method suspensions
produces with higher
suspensions with
From the intensities of signals, it is clear that the Frens method produces suspensions with
concentrations
higher of nanoparticles.
concentrations In addition,
of nanoparticles. this method
In addition, produces
this method nanoparticles
produces with a smaller
nanoparticles with a
higher concentrations of nanoparticles. In addition, this method produces nanoparticles with a
diameterdiameter
smaller than those
thanobtained with the with
those obtained regularthemethod
regular(lower
method absorption wavelengths
(lower absorption are associated
wavelengths are
smaller diameter than those obtained with the regular method (lower absorption wavelengths are
with smaller sizes of nanoparticles). Finally, from the analysis of the full width
associated with smaller sizes of nanoparticles). Finally, from the analysis of the full width at half at half maximum,
associated with smaller sizes of nanoparticles). Finally, from the analysis of the full width at half
which is related
maximum, whichtois the dispersion
related of nanoparticle
to the dispersion size, it is size,
of nanoparticle possible
it is to see that
possible to the
see Frens method
that the Frens
maximum, which is related to the dispersion of nanoparticle size, it is possible to see that the Frens
producesproduces
method nanoparticles with a narrower
nanoparticles size distribution.
with a narrower size distribution.
method produces nanoparticles with a narrower size distribution.
The broad
The broad spectra
spectra shown
shown inin Figure
Figure 2 suggest
suggest that nanoparticle
nanoparticle suspensions
suspensions are are polydispersed,
polydispersed,
The broad spectra shown in Figure 2 suggest that nanoparticle suspensions are polydispersed,
with contributions centered below 400 nm and above 450 nm. These These positions
positions areare usually
usually associated
associated
with contributions centered below 400 nm and above 450 nm. These positions are usually associated
with nanoparticles that are smaller than than 10 10 nm
nm andand above
above 5050 nm.
nm. InInorder
orderto toconfirm
confirmthisthissuggestion,
suggestion,
with nanoparticles that are smaller than 10 nm and above 50 nm. In order to confirm this suggestion,
experiments using
experiments using DLS
DLS were
were conducted
conductedand andtwo twogroups
groupsof ofsizes
sizeswere
werefound,
found,as asshown
shownin inFigure
Figure3.3.
experiments using DLS were conducted and two groups of sizes were found, as shown in Figure 3.
While with thethe regular
regularmethod
methodtwotwosize
sizedistributions
distributionsare arealso obtained,
also obtained, around
around 3 and 102102
3 and nm,nm,
with the
with
While with the regular method two size distributions are also obtained, around 3 and 102 nm, with
Frens method the two size distributions are centered around 5
the Frens method the two size distributions are centered around 5 and 68 nm. This and 68 nm. This observation agrees
agrees
the Frens method the two size distributions are centered around 5 and 68 nm. This observation agrees
with the fact that the FWHM of spectra using
with using the Frens method
method is narrower
narrower than than the one obtained
obtained
with the fact that the FWHM of spectra using the Frens method is narrower than the one obtained
with the regular method. Figure
with Figure 3b3b shows
shows aa TEM micrograph
micrograph for Ag nanoparticles
nanoparticles obtained
obtained using
using
with the regular method. Figure 3b shows a TEM micrograph for Ag nanoparticles obtained using
the Frens method, showing that “large” nanoparticles had spherical and cylinder
the Frens method, showing that “large” nanoparticles had spherical and cylinder shapes. Finally, the shapes. Finally,
the Frens method, showing that “large” nanoparticles had spherical and cylinder shapes. Finally, the
the Zeta
Zeta potential
potential waswas alsoalso measured,
measured, finding
finding a value
a value −1.3
ofof−1.3 ±± 0.30.3
mVmVfor forthe
theregular
regular method
method and
and
Zeta potential was also measured, finding a value of −1.3 ± 0.3 mV for the regular method and
− 27.7 ± 0.8 mV for the Frens method. Thus, the analysis of zeta potential suggests
−27.7 ± 0.8 mV for the Frens method. Thus, the analysis of zeta potential suggests that suspensions that suspensions
−27.7 ± 0.8 mV for the Frens method. Thus, the analysis of zeta potential suggests that suspensions
prepared via the Frens method are more stable.
prepared via the Frens method are more stable.

Figure 3.
Figure Determination of
3. Determination of the
the nanoparticle
nanoparticle size
size obtained
obtained byby using
using the
the methods
methods described
described in
in this
this
Figure 3. Determination of the nanoparticle size obtained by using the methods described in this
protocol: (a)
protocol: (a) Using
Using dynamic
dynamic light
light scattering
scattering (DLS);
(DLS); (b)
(b) by
by transmission
transmission electron
electron microscopy.
microscopy.
protocol: (a) Using dynamic light scattering (DLS); (b) by transmission electron microscopy.
 Methods and Protoc. 2019, 2, 3 5 of 5

5. Summary and Conclusions

Two methods for the synthesis of silver nanoparticles were tested and compared in this study.
Nanoparticle suspensions with higher concentration, improved stability, and smaller size distribution
were obtained by a modified Frens method.

Author Contributions: Methodology, writing the manuscript, conceptualization, data curation—all authors;
performed the experiments—L.P.-M. and M.G.-T.; supervision and funding acquisition—J.C. and R.-R.
Funding: This work has been funded by CONCYTEC (155-2015-FONDECYT) and by the partnership Cleveland
Clinic—Universidad de Ingenieria y Tecnologia—UTEC.
Acknowledgments: Vijay Krishna, Saad Ahsan, and Jeanna Li (Cleveland Clinic) are acknowledged for useful
discussions and for the use of the DLS instrument. Angela Pinedo and Bryan Alcazar are acknowledged for
the training in the synthesis of nanoparticles. Karinna Visurraga and Luz Perez (UTEC) are acknowledged for
administrative and technical support.
Conflicts of Interest: The authors declare no conflicts of interest.

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