Journal of Science: Advanced Materials and Devices 4 (2019) 66e71

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Journal of Science: Advanced Materials and Devices

journal homepage: www.elsevier.com/locate/jsamd

Original Article

Effects of reaction temperatures and reactant concentrations on the

antimicrobial characteristics of copper precipitates synthesized using
L-ascorbic acid as reducing agent
~ ol a, Nacita B. Lantican b,
Ana Rose Ramos a, Alvin Karlo G. Tapia a, Chrysline Margus N. Pin
Ma. Lourdes F. del Mundo , Ronniel D. Manalo , Marvin U. Herrera a, *
b c

a
Institute of Mathematical Sciences and Physics, University of the Philippines Los Ban ~ os College, Laguna, 4031, Philippines
b ~ os College, Laguna, 4031, Philippines
Institute of Biological Sciences, University of the Philippines Los Ban
c
Department of Forest Products and Paper Science, University of the Philippines Los Ban ~ os College, Laguna, 4031, Philippines

a r t i c l e i n f o a b s t r a c t

Article history: Copper-based precipitates were synthesized using a straightforward wet chemical reduction method
Received 18 October 2018 with copper sulfate salts as precursor and L-ascorbic acid as reducing agent. Scanning electron micro-
Received in revised form graphs of the precipitates reveal particle-like structures with edges. The percent age yield was observed
23 December 2018
to increase with the reaction temperature and the reductant-to-precursor concentration ratio. Com-
Accepted 29 December 2018
Available online 5 January 2019
parison of samples prepared at different times showed that the bulk of the precipitates was formed
during the first 24 hours. New yield added to the bulk decreased over time. Here, the ascorbic acid served
as a capping agent, which impeded the oxidation. This capping was not visible in the electron micro-
Keywords:
Wet-chemical method
graphs but may be inferred from the infrared spectra. Faster capping reduced the amount of oxides in the
Capping precipitates. This was seen in samples prepared at a higher temperature and with a lower reductant-to-
Antimicrobial precursor concentration ratio. Antimicrobial testing under dynamic contact conditions showed that the
Staphylococcus aureus copper-based precipitates were more effective against Staphylococcus aureus (a gram-positive bacteria)
Escherichia coli than Escherichia coli (a gram-negative bacteria). Copper-based precipitates collected after the first 24
hours were more potent than those collected after eight days. Furthermore, samples prepared at room
temperature and lower reductant-to-precursor concentration ratios were found to be more effective in
reducing the number of S. aureus after one hour of contact. The synthesized copper-based precipitates
was observed to reduce the population of S. aureus (CFU/mL) by up to 98% after one hour of contact.
© 2019 The Authors. Publishing services by Elsevier B.V. on behalf of Vietnam National University, Hanoi.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

1. Introduction
Copper is a popular cheaper alternative to silver. Often, it is used
as a conductor of heat and electricity. Like silver, antimicrobial
properties of copper [1e6] are well known. Knowledge of its
sanitizing ability dates back to Ancient Egypt, when it was used to
clean drinking water but it was not until the 19th and early 20th
centuries when its applications in the field of medicine became
prevalent [5].
Antimicrobial properties may be attributed to the ability of
charges (e.g., ions, oxidation states, free electrons, radicals) to
directly or indirectly disrupt the vital charge transports within the
* Corresponding author.
E-mail address: muherrera@up.edu.ph (M.U. Herrera).
Peer review under responsibility of Vietnam National University, Hanoi.
cells. Thus, when placed on different materials and surfaces, anti-
microbial agents can minimize, inhibit, or stop the spread of infec-
tious diseases. Antimicrobial agents may be in the form of liquids,
gels, sheets, and powders. Unlike liquids and gels, powders do not
evaporate and, thus, have longer effective lifetime. Compared to
sheets, powders have the advantage of being easily integrated,
mixed, and incorporated in different materials.
Copper particles or powder may be produced using physical,
electrochemical, and chemical means [7e21]. Physical methods
(e.g., pulse laser ablation or deposition, ball milling method, and
pulsed wire discharge method) and electrochemical methods (e.g.,
chemical reduction) may be faster. However, these methods usually
require more expensive equipment and/or higher energy cost.
Chemical methods (e.g., chemical reduction) may offer cheaper
alternatives. But these, at times, use reagents and/or produce by-
products which are harmful to the environment.

https://doi.org/10.1016/j.jsamd.2018.12.009
2468-2179/© 2019 The Authors. Publishing services by Elsevier B.V. on behalf of Vietnam National University, Hanoi. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
 A.R. Ramos et al. / Journal of Science: Advanced Materials and Devices 4 (2019) 66e71
Fig. 1. X-ray diffraction patterns of precipitates produced at room temperature
(~25
C) after 24 hours using 0.2M copper sulfate solution and 1.2M ascorbic acid
solution.
This work is an investigation of the effects of the processing
parameters (e.g., reaction temperatures and reactant concentra-
tions) on the characteristics of copper-based particles synthesized
using chemical reduction technique, a simple chemical method
that involves the use of reducing agents to trigger the formation
of copper metal. One non-toxic, abundant, and cheap reducing
agent used in the synthesis of copper-based particles is the
ascorbic acid [22e26]. Ascorbic acid, like copper, is known for its
antimicrobial properties. But, aside from being a reductant,
ascorbic acid also acts as a capping agent, which affects the
Fig. 2. Scanning electron micrographs of the precipitates produced at room temperature (~25
C) after 24 hours using 0.2 M copper sulfate solution and 1.2 M ascorbic acid solution.
The magnifications were (a) 1000, (b) 2000, (c) 5000 and (d) 8000.
67
growth, the aggregation, and the interaction of the synthesized
particles with the external environment [26].
2. Methodology
Copper particles were synthesized via straightforward wet-
chemical reduction with copper sulfate pentahydrate (Technical
grade) and L-ascorbic acid powder (US Pharmacopoeia grade) as
the precursor and reducing agent, respectively. Different concen-
trations of the copper sulfate solution were prepared by dissolving
copper sulfate pentahydrate salt in water. The copper sulfate so-
lution was added dropwise to a 1.2M ascorbic acid solution, which
was made by dissolving an appropriate amount of L-ascorbic acid
powder in water. Equal volumes of the two solutions were mixed at
room temperature (~25
C). Another sample was prepared at a
higher temperature (85
C). The precipitates were gathered by
suction filtration 24 hours after the first drop of the copper sulfate
solution was added to the ascorbic acid solution and air-dried
immediately. After eight days, precipitates were collected again
using suction filtration and likewise air-dried.
The percentage yield (%yield) was computed using the equation:
output
%yield ¼  100;
input
where the ‘output’ is the mass of the precipitates while the ‘input’ is
the total mass of copper element in the precursor. The phases
present in the samples were identified using an X-ray Diffractom-
eter. The infrared spectra of the precipitates were also determined
using Fourier-Transform Infrared (FTIR) spectroscopy.
68 A.R. Ramos et al. / Journal of Science: Advanced Materials and Devices 4 (2019) 66e71

Table 2
Amount of precipitate collected after 24 hours at different temperatures using 0.2M
copper sulfate solution and 1.2M ascorbic acid solution.

Temperature,
C Yield, g Percent Yield, %

25 0.5072 15.96
85 2.9092 91.56

The sample's infrared spectrum presented in Fig. 3 shows the

Fig. 3. Infrared spectrum of precipitates produced at 25
C after 24 hours using 0.2M
copper sulfate solution and 1.2M ascorbic acid solution.
Finally, antimicrobial properties of the precipitates against one
gram-positive (Staphylococcus aureus) and one gram-negative
(Escherichia coli) bacteria were tested under dynamic contact con-
ditions (ASTM E2149-01).
3. Results and discussion
3.1. Copper-based precipitates
X-ray diffraction patterns confirm the presence of copper in the
samples. Fig. 1 shows the pattern obtained from precipitates pro-
cessed at room temperature (25
C) using the 0.2 M copper sulfate
solution and the 1.2 M ascorbic acid solution. The diffraction peaks
at 2q ¼ 43.28
and 50.41
reveal that the precipitates are mainly
composed of copper metal. The peaks at 2q ¼ 36.4
and 42.3

indicate small amounts of cupric oxide (CuO) and cuprous oxide
(Cu2O) in the samples. The majority of the structures seen in the
scanning electron micrographs of the samples (Fig. 2) are particle-
like. This means that there is no excessive elongation in any of the
dimensions. The particles are not perfect spheres. Instead, they
have edges. This implies that the particle growth may have some
degree of preference in certain planes. The average size of the
precipitates was 10 mm.
Table 1
Peaks of the infrared spectra of precipitates produced at 25
C after 24 hours using
0.2M copper sulfate solution and 1.2M ascorbic acid solution and their corre-
sponding interpretation.
vibrational modes associated with the presence of the poly-
hydrated ascorbic acid molecules. The peaks of the infrared spec-
trum and their corresponding interpretations are detailed in
Table 1. An L-ascorbic acid molecule contains two hydroxyl (OH)
groups attached to double bonded carbon atoms. nfrared spectrum
of the synthesized particle, however, does not show vibration
modes associated with the C¼C bond. The ascorbic acid may be
oxidized into dehydroascorbic acid. Dehydroascorbic acids in the
aqueous solution may have undergone hydration, thus forming
polyhydrated ascorbic acid molecules. The broad peak at 3198 cm1
associated with O-H stretching modes found in the intermolecular
bonded alcohol groups suggests the formation of networks of the
ascorbic acid molecules.
The infrared spectrum in Fig. 3 also shows unidentified peaks in
the range from 2569 to 1980 cm1. These peaks are attributed to a
possible bond or a complex formed between the Cu atoms and the
polyhydrated ascorbic acid molecules. The ascorbic acid did not
merely coat the copper metal. Otherwise, it would have dissolved in
water. Instead, it formed a complex, which capped the molecules.
This capping may be only a few molecules thick and is too thin to be
visible in the electron micrographs.
3.2. Variation of the reaction temperature
Table 2 presents a comparison of the yield obtained at different
reaction temperatures. This reflects the amount of synthesized
copper particles per unit of available copper ions. The precipitates
were collected 24 hours after mixing 0.2M copper sulfate and 1.2M
ascorbic acid solutions. Samples prepared at 85
C generated a
percentage yield of 91.56% while those produced at room temper-
ature (~25
C) gave only a yield of 15.96%. The energy needed to
trigger the reduction of copper ions into copper metal decreases
with the temperature. Moreover, at higher temperature, the prob-
ability of one molecule colliding and reacting with another in-
creases. With the increased probability of collision and reaction
among molecules in the mixture, precipitates are also more likely
to be capped before they interact with oxygen molecules in the

Wavenumber, cm1 Assignment

3568e3211 O-H stretching of

free alcohol group/water
3198 O-H stretching of intermolecular
bonded alcohol group
2893 O-H stretching of intramolecular
bonded alcohol group
2830 C-H asymmetrical stretching in CH2 group
2569e1980 not identified but attributed to the presence
of complex between the Cu atoms and
polyhydrated ascorbic acid molecules
1623 C-O stretching
1562 C-C stretching
1398 O-H bending
1303 C-OH stretching
1220 O-H in-plane bending
1080 C-O stretch of 1
alcohol
1018 C-H in-plane bending
804 C-C in-plane bending
Fig. 4. X-ray diffraction patterns of precipitates produced at (a) 85
C and (b) ~25
C
631 O-H out-of-plane bending
using 0.2M copper sulfate solution and 1.2M ascorbic acid solution.
 A.R. Ramos et al. / Journal of Science: Advanced Materials and Devices 4 (2019) 66e71
Table 3
Antimicrobial properties of the precipitates against S. aureus and E.coli under dy-
namic contact conditions. The samples were synthesized at different reaction
temperatures using 0.2M copper sulfate solution and 1.2M ascorbic acid solution.
Temperature,
C Reduction, % (after one hour)
S. aureus
25 98
85 83
atmosphere. The effect of faster capping at higher temperatures is
seen in the XRD patterns of the samples (Fig. 4). Both show strong
diffraction peaks associated with the presence of copper metal.
However, the peaks associated with CuO differ in intensity. The
sample synthesized at higher temperature has a weaker diffraction
peak at 36.4
. Samples prepared at room temperature contain more
oxides. It also has a peak at 42.3
, which is associated with presence
of Cu2O.
Table 3 shows the antimicrobial properties of the different
precipitates against S. aureus and E. coli under dynamic contact
conditions. The copper-based particles synthesized at 85
C, has
reduced the population of S. aureus (CFU/mL) by 83% after one hour
of contact. This value is lower compared to the effect of copper-
based particles that was prepared at 25
C (98%). At higher tem-
peratures, the increase in probabilistic collisions between ascorbic
acid molecules and copper-based particles may lead to a more
extensive capping of precipitates. Capping minimizes interaction
between the precipitates and S. aureus, thus, reducing its antimi-
crobial activity against the gram-positive bacteria. The same was
observed when the samples prepared at different temperatures
were tested against E.coli. However, in general, the precipitates
were more effective in reducing the number of S. aureus (gram-
positive) than that of E.coli (gram-negative) after one hour of
contact.
3.3. Variation of the reaction concentrations
Table 4 summarizes the effect of different reductant-to-
precursor concentration ratios on the yield. At higher copper sul-
fate concentrations, more copper ions are available for reduction.
This enhances the yield. However, in terms of percentage, the yield
becomes less. This downward trend in the percentage yield may be
attributed to the decrease in the number of available ascorbic acid
molecules for every unit of copper ions.
In the experiment, the L-ascorbic acid solution was maintained
at 1.2M while the copper sulfate concentration was varied to ach-
ieve 12:1, 6:1, and 3:1 reductant-to-precursor concentration ratios.
As the concentration of copper sulfate was increased, the ratio of
the reductants to the precursors decreased. A greater percentage
yield was obtained by limiting the precursor., thus, reducing the
amount of copper sulfate in the solution.
Fig. 5 presents a comparison of the XRD patterns. All show
strong diffraction peaks confirming the presence of copper metal in
the precipitates. Traces of oxides are also seen in the samples.
Table 4
Amount of precipitate produced at ~25
C using different concentrations of copper
sulfate solution and an ascorbic acid solution of 1.2 M.
Copper sulfate Reductant-to- Yield, g
concentration precursor
(M) concentration ratio
0.1 12:1 0.2777
0.2 6:1 0.5072
0.4 3:1 0.7271
69
E. coli
33
32
Fig. 5. X-ray diffraction patterns of precipitates produced at ~25
C reaction temper-
ature using 1.2 M ascorbic acid solution and varying copper sulfate concentration (a)
0.4M, (b) 0.2 M and (c) 0.1 M.
However, the associated peaks are very low for the precipitates
produced using 0.4M copper sulfate concentration or 3:1
reductant-to-precursor ratio. Capping impedes oxidation. Based on
the results, capping may have proceeded faster when the dispro-
portion between the concentrations of ascorbic acid (the capping
agent) and copper sulfate became less.
The antimicrobial properties of these samples were likewise
tested against the S. aureus and E. coli under dynamic contact
conditions. The precipitates were found to be more effective against
S. aureus. The data in Table 5 also show the effect of varying reactant
concentrations on the antimicrobial properties of the samples. The
precipitates prepared using high reductant-to-precursor concen-
tration ratio (12:1) demonstrated the least activity against S. aureus,
with only 68% reduction after one hour of contact. In the previous
section, the antimicrobial activity was related to the degree of
capping. In this case, while it may have proceeded faster at 3:1
reductant-to-precursor ratio, capping may not be as extensive.
So far, the data presented correspond to precipitates gathered
after the first 24 hours (or one day). The yield is proportional to the
reaction time. The amount of precipitates yielded more than
doubled after eight days. However, the reaction rates usually slow
down with time due to the consumption of reactants. From Table 6,
it can be inferred that around 32% of the total product was formed
on the first day and, on the average, only about 0.1939 g or 12.5%
was added each day after.
The precipitates were again found to be more effective in
reducing the number of S. aureus compared to that of E.coli after one
hour of contact (Table 7). The samples collected after 8 days,
however, showed poor antimicrobial activity against both bacteria.
This decrease in the antimicrobial activity may still be attributed to
extensive capping. Samples collected after 8 days were found to
have significantly lower oxide content compared to those collected
after the first 24 hours. The diffraction peaks associated with oxides
Table 5
Antimicrobial properties of the precipitates synthesized at ~25
C using different
reductant-to-precursor concentration ratios against S. aureus and E.coli under dy-
namic contact conditions.
Copper sulfate Reductant-to-precursor Reduction, % (after
Percent
concentration (M) concentration ratio one hour)
Yield, %
S. aureus E.coli
17.48 0.1 12:1 68 37
15.96 0.2 6:1 98 33
11.44 0.4 3:1 93 31
70 A.R. Ramos et al. / Journal of Science: Advanced Materials and Devices 4 (2019) 66e71
Table 6
Amount of precipitate produced at ~25
C using 3:1 reductant-to-precursor con-
centration ratio at different collection times.
Collection Amount of precipitate (g)
1st (after 1 day) 0.7271
2nd (after 8 days) 1.5512
have become very small and almost indistinguishable (Fig. 6). After
the first 24 hours, conditions may have favored capping over
further aggregation and improving the yield. Capping may have
continued and become more extensive lessening oxidation and
lowering the antimicrobial property of the precipitates.
In general, the synthesized copper-based particles have better
antimicrobial properties against S. aureus than against E. coli. The
higher percentage reduction observed in S. aureus as compared to
the percentage reduction of E. coli may be attributed to the struc-
tural difference in their cell wall. Gram-positive bacteria such as
S. aureus have abundant pores, which may facilitate foreign parti-
cles to pass through. On the other hand, gram-negative bacteria
such as E. coli have lipopolysaccharide, lipoproteins and phospho-
lipids, which make up a better barrier against foreign particles. The
higher antimicrobial activity of different materials against S. Aureus
than against E.coli is also observed in other works [27e30].
4. Conclusion
A straightforward wet chemical reduction technique was
employed to produce copper-based particles. Copper sulfate salts
and L-ascorbic acid were used as precursor and reducing agent,
respectively. Synthesis of copper-based precipitates was confirmed
through an X-ray Diffractometer. Scanning electron micrographs of
the precipitates showed particle-like structures with edges. The
amount of synthesized copper particles per unit of available copper
ions varied with the reaction temperature and the reactant con-
centration. Greater percent age yield was obtained when the
sample preparation was performed above room temperature and a
higher reductant-to-precursor concentration ratio was used.
Infrared spectra of the samples suggest formation of networks of
ascorbic acid molecules and complex between the Cu atoms and
the polyhydrated ascorbic acid molecules.
Ascorbic acid served as a capping agent. Faster capping reduced
the amount of oxides in the precipitates, particularly in samples
prepared at a higher temperature and with a lower reductant-to-
precursor concentration ratio. The rate of capping, however, may
not be directly related to its extent. Antimicrobial testing under
dynamic contact conditions showed that the copper-based pre-
cipitates were more effective against S. aureus (a gram-positive
bacteria) than against E. coli (a gram-negative bacteria). Samples
prepared at room temperature and lower reductant-to-precursor
concentration ratio were more successful in reducing the number
of S. aureus after one hour of contact. Capping may limit the
interaction between the precipitates and the bacteria. Therefore,
capping may have been less extensive in the aforementioned cases
Table 7
Antimicrobial properties of the precipitates synthesized at ~25
C using 3:1
reductant-to-precursor ratio, against S. aureus and E.coli under dynamic contact
conditions.
Time Collection Reduction, % (after one hour)
S. aureus E. coli
After first 24 hours 93 31
After eight days 45 27
Fig. 6. X-ray diffraction patterns of precipitates that were taken from the second
collection of the samples. The samples were produced at 25
C reaction temperature
using 1.2 M of ascorbic acid solution and (a) 0.4M, (b) 0.2M and (c) 0.1M copper sulfate
solutions. The corresponding reductant-to-precursor ratios are (a) 3:1, (b) 6:1 and (c)
12:1, respectively.
than in the samples prepared at a higher temperature and using a
greater reductant-to-precursor concentration ratio.
Comparison of precipitates collected at different times suggests
that about 47% of the total product resulted from reactions occur-
ring within the first 24 hours. However, in the succeeding days,
these reactions slowed down. The conditions may have favored
capping thus lowering the amount of oxide in the samples and
decreasing its antimicrobial ability.
Acknowledgements
This research is funded by Philippine Council for Industry, En-
ergy, and Emerging Technology Research and Development
(PCIEERD), Department of Science and Technology (DOST) with
project number 04013.
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