Sensitive and selective detection of trace copper in standard alloys, food and biological samples using a bulk optode based on N,N′-(4,4′- ethylene biphenyl) bis(3-methoxy salicylidine imine) as neutral carrier

Article abstract of DOI:10.1016/j.saa.2013.01.051

In this paper, an optical sensing film has been proposed for sensitive determination of copper (II) ion in aqueous solutions. The copper sensing membrane was prepared by incorporating N,N′-(4,4′-ethylene biphenyl) bis(3-methoxy salicylidine imine) as ionophore in the plasticized PVC membrane containing bis(2-ethylhexyl) sebacate (DOS) as plasticizer. This proposed membrane works on the basis of a cation-exchange mechanism and shows a significant absorbance signal change on exposure to acetate buffer solution of pH 4.0 containing copper ion. The proposed sensing film displays a linear range of 0.01-32.0 μg mL^-1 with a limit of detection 0.008 μg mL ^-1. Moreover, upon the introduction of a negatively charged lipophilic additive (oleic acid) into the membrane, the optode displayed enhanced sensitivity. In addition, satisfactory analytical sensing characteristics for determining copper (II) ion were obtained in terms of the selectivity, stability and reproducibility. The response time of the optode was less than 3 min, depending on the concentration of Cu(II) ions. The optode membrane has been applied to determine Cu(II) in various real samples.

Full text of DOI:10.1016/j.saa.2013.01.051

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 280–288
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Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
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Sensitive and selective detection of trace copper in standard alloys, food and
biological samples using a bulk optode based on N,N0-(4,40-ethylene biphenyl)
bis(3-methoxy salicylidine imine) as neutral carrier
⇑
Majid Arvand , Zahra Lashkari
Department of Chemistry, Faculty of Science, University of Guilan, Namjoo Street, P.O. Box 1914, Rasht, Iran
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
Absorption spectra of ionophore in the solution at the presence of different cations.
"
"
"
"
An optical sensing film has been
proposed for sensitive determination
of Cu(II) ion.
The copper sensing membrane
works on the basis of a cation-
exchange mechanism.
Satisfactory analytical sensing
characteristics for determining Cu(II)
ion were obtained.
The optode membrane has been
applied to determine Cu(II) in
various real samples.
a r t i c l e i n f o
a b s t r a c t
Article history:
In this paper, an optical sensing film has been proposed for sensitive determination of copper (II) ion in
aqueous solutions. The copper sensing membrane was prepared by incorporating N,N0-(4,40-ethylene
biphenyl) bis(3-methoxy salicylidine imine) as ionophore in the plasticized PVC membrane containing
bis(2-ethylhexyl) sebacate (DOS) as plasticizer. This proposed membrane works on the basis of a cat-
ion-exchange mechanism and shows a significant absorbance signal change on exposure to acetate buffer
solution of pH 4.0 containing copper ion. The proposed sensing film displays a linear range of 0.01–
Received 2 December 2012
Received in revised form 10 January 2013
Accepted 19 January 2013
Available online 4 February 2013
Keywords:
Optical sensing film
Copper (II) ion
Ionophore
UV–Vis spectrophotometry
32.0 l l
g mLꢀ1 with a limit of detection 0.008 g mLꢀ1. Moreover, upon the introduction of a negatively
charged lipophilic additive (oleic acid) into the membrane, the optode displayed enhanced sensitivity.
In addition, satisfactory analytical sensing characteristics for determining copper (II) ion were obtained
in terms of the selectivity, stability and reproducibility. The response time of the optode was less than
3 min, depending on the concentration of Cu(II) ions. The optode membrane has been applied to deter-
mine Cu(II) in various real samples.
Ó 2013 Elsevier B.V. All rights reserved.
Introduction
optical signal measurement are those of the advanced techniques
in analytical chemistry and they have been accepted as advanta-
In recent years, there has been a growing need for constructing
chemical or electrochemical sensors for fast and economical mon-
itoring of environmental samples especially for heavy metal ions in
real time [1–18]. In this connection, chemical sensors based on
geous because they can be miniaturized and manufactured at
low cost [19].
For the preparation of an ion-selective optode membrane, an ion-
ophore can be incorporated into a hydrophobic membrane such as a
plasticized poly(vinylchloride) (PVC) membrane, and utilized in
contact with a sample solution containing a primary ion. Ionophores
are lipophilic complexing agents having the capability of binding
ions reversibly and to transport them across organic membranes
⇑
Corresponding author. Tel./fax: +98 131 3233262.
E-mail address: arvand@guilan.ac.ir (M. Arvand).
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.01.051

M. Arvand, Z. Lashkari / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 280–288
281
by carrier translocation. Ideally, selective ion carriers render a mem-
brane permeable for one given sort of ion I only. For an analytically
relevant application of ionophores in solvent polymeric membranes
for bulk optodes or ion selective electrodes some requirements such
as sufficient lipophilicity, sufficiently reversible exchange kinetics
and high selectivity have to be met simultaneously [20–27].
membrane phase and form a complex, which results a decrease in
the ionophore absorbance and produce a new absorbance peak at a
higher wavelength. The sensor has longer linear dynamic range
than the previously reported copper optical sensors [30–37], plus
lower limit of detection. In addition, selectivity of the film optode
based on EBSI was investigated. As a result, EBSI shows a striking
selectivity for Cu2+ complexation and is more selective than the
previously reported sensors [31–34]. To the best of our knowledge,
this compound has not previously been used in the development of
a Cu(II) optical sensing film or in any other optical sensors.
Copper is one of the heavy metals, low concentration of which is
essential for plants and animals whereas, it is highly toxic to aqua-
tic plants and bacteria in a high concentration. Copper is an essen-
tial trace element for biological processes, even at an ultra trace
level [28]. Due to its mobilization and redox activity, copper is be-
lieved to play a central role in the formation of reactive oxygen
species, such as OÅ2 and OHÅ radicals. These radicals bind very rap-
idly to DNA, and can cause damage by breaking the DNA strands or
modifying the bases and/or deoxyribose, therefore leading to carci-
nogenesis [29]. Moreover, it is an essential constituent of about
thirty enzymes and glycoproteins and is required for the synthesis
of hemoglobin and for some biological processes. It also promotes
iron absorption from the gastrointestinal system, is involved in the
transport of iron from tissues into plasma, helps to maintain mye-
lin in the nervous system, is important in the formation of bone
and brain tissues and is necessary for other many important func-
tions [30]. Thus, due to the urgent need for selective copper deter-
mination in many biological, geological, environmental and
industrial samples, various analytical techniques have been pro-
posed for determination of copper including spectrophotometry
[31–38], atomic absorption spectrometry (AAS) [39], inductively
coupled plasma-emission spectrometry (ICP-ES) [40], potentiome-
try [41–45], and anodic stripping voltammetry [46]. These meth-
ods usually have a sufficiently low detection limit and specificity,
but also have drawbacks such as high costs for equipment and as-
says, time-consuming, and complicated operation. So these meth-
ods are unsuitable for simple, low cost, and remote determination
of copper especially in field analysis strategy.
Experimental
Reagents
All chemicals were of the best available analytical reagent
grade and all solutions were prepared with double distilled
water. Relatively high-molecular-weight poly(vinylchloride)
(PVC), nitrobenzene (NB), acetophenone (AP), dibuthyl phthalate
(DBP), bis(2-ethylhexyl) sebacate (DOS), dibuthyl sebacate (DBS),
tetrahydrofuran (THF), triethylammonium phosphate (TEAP),
oleic acid (OA), sodium tetraphenyl borate (NaTPB) and all other
reagents were obtained from Merck (Darmstadt, Germany) or
Fluka (Buchs, Switzerland). A stock solution of 100 l
g mLꢀ1 cop-
per (II) solution was prepared by dissolving appropriate amount
of Cu(NO3)2ꢁ3H2O (Merck) in 100 mL volumetric flask and di-
luted with doubly distilled water. Working standard solutions
of lower concentrations were prepared by suitable dilutions of
the stock solution with water. The pH adjustments were
made with acetate buffer solution (1 ꢂ 10ꢀ4
M CH3COOH –
M CH3COONa) to achieve the desired pH, but in
5.5 ꢂ 10ꢀ4
studying of pH effect, the pH of solutions was adjusted with
either HCl or NaOH solutions.
Following to our recent studies concern to the application of a
new class of ionophores containing nitrogen atoms such as 6-(4-
nitrophenyl)-2-phenyl-4-(thiophen-2-yl)-3,5-diaza-bicyclo[3.1.0]-
hex-2-ene [47], 6-(4-nitrophenyl)-2,4-diphenyl-3,5-diaza-bicyclo
[3.1.0] hex-2-ene [48], 6-(4-nitrophenyl)-2-phenyl-4,4-dipropyl-
3,5-diaza-bicyclo [3,1,0] hex-2-ene [49], 2-(4-methoxy phenyl) 6-
(4-nitrophenyl)-4-phenyl-1,3-diazabicyclo [3.1.0] hex-3-ene [50],
(2E, 4E)-5-(2,4-dinitrophenyl amino)penta-2,4-dienal [51] and
N5-(2,4-dinitro-phenyl)-N1,N1-diethyl-penta-1,3-diene-1,5-diamine
[52] as carrier in developing of ion-selective electrodes [47–50]
and optodes [51,52], we have been motivated to investigate the
application of N,N0-(4,40-ethylene biphenyl) bis(3-methoxy sali-
cylidine imine) (EBSI, Scheme 1) as complexing agent for sensitive
determination of copper (II) ion in aqueous solutions. This iono-
phore can extract Cu(II) from aqueous sample solution into organic
Synthesis of the N,N0-(4,40-ethylene biphenyl) bis(3-methoxy
salicylidine imine) (EBSI) as ionophore
The ionophore EBSI (Scheme 1), used in this effort, was synthe-
sized according to the reported method with some modification in
the described procedure [53]. 0.7 g (3.3 mmol) of the 4,40-diamino
bibenzyl was dissolved in 20 mL ethanol (96% v/v). To this was
added dropwise solution of (1.0 g, 6.6 mmol) o-vanilin in 2 mL eth-
anol over period of 10 min. This solution was stirred for 15 min at
room temperature. The color of solution changed to orange. The
crude precipitate was filtered and recrystallized from toluene.
The precipitate was washed with cold ethanol. The melting point
of the orange crystalline form of the EBSI ionophore was 224–
226 °C; IR (KBr): 3450, 1620, 1470, 1260 cmꢀ1
.
1H NMR
(500 MHz, CDCl3/DMSO-d6): d = 13.4 (s, 1H), d = 8.3 (s, 1H),
d = 6.91 (s, 4H), d = 6.7 (dd, J = 1.1 Hz, J = 7.8 Hz, 1H), d = 6.6 (d,
J = 7.8 Hz, 1H), d = 6.5 (t, J = 7.8 Hz, 1H), d = 3.58 (s, 3H), d = 2.64
(s, 2H) ppm; 13C NMR (125 MHz, DMSO-d6): 38.1, 57.9, 118.6,
120.1, 121.8, 123.4, 129.7, 139.5, 148.6, 148.8, 150.9, 161.1 ppm;
C30H28N2O4 (480.56): calcd. C 74.98, H 5.87, N 5.83, O 13.32; found
C 75.21, H 5.71, N 6.11, O 12.97.
N
N
Apparatus
OH
HO
A Cary 100 UV–Vis spectrophotometer (Agilent Technologies,
Santa Clara, United States) with a 1 cm cell was used for recording
all spectra and absorbance measurements. A Metrohm pH meter
(model 827, Swiss made) with a combined double junction glass
electrode was used for monitoring pH values. All measurements
were made in the absorbance mode.
OMe
MeO
Scheme 1. Structure of EBSI used as ionophore.

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Membrane preparation
Recommended procedure
The optimized composition for the preparation of the optode
membrane consisted of 2 mg of EBSI as ionophore, 61 mg of DOS
as plasticizer, 4 mg of OA as additive and 30 mg PVC. The mem-
brane components were dissolved in 1.5 mL freshly distilled THF
in a glass vial. The solution was immediately shaken vigorously
For simple and easy use of the ion-sensing film optode, the PVC
membrane was attached to the surface of an 8 mm ꢂ 25 mm thin
glass plate whose size fits into a conventional glass vessel used
as a standard optical cell for a spectrophotometer as shown in
Fig. 1. Consequently, the determination of an analyte ion using
the film optode can be achieved by simply immersing it into the
glass vessel for monitoring the absorbance change as the response
of the optode.
to achieve complete homogeneity. An aliquot of 60 lL of this solu-
tion was poured and uniformly spread on a dust free glass plate
(8 mm ꢂ 25 mm) which mounted on a spin device (rotating fre-
quency ꢃ2600 rpm). After a spinning time of about 15 s, the glass
support plate with sensing membrane was removed and allowed
to stand in ambient air for 24 h before use. The thickness of dry
The prepared membrane was put vertically inside the sample
cell containing 3 mL acetate buffer solution with pH = 4.0, and a
membrane (without ionophore) at the same conditions was used
as a blank membrane. The standardized metal ion solutions were
added to the sample cell and the absorbance value of the system
was measured after 15 min (required to reach the equilibrium) at
kmax = 400 nm. The limiting absorbances A0 and Ac were deter-
mined with the optode membrane in contact without and with
membrane was estimated to be ꢃ4
lm.
Sample preparation and determination
Preparation of standard alloys
A 0.1 g sample of the standard alloy was completely dissolved in
6 mL of (1 + 1) hydrochloric acid by heating in a water bath and
adding 1 mL of 30% (v/v) hydrogen peroxide. The excess peroxide
was decomposed by heating the sample in the water bath. The
solution was cooled, filtered if needed and diluted to 200 mL with
distilled water in a standard flask after adjusting the pH to 4 [31].
An aliquot of this sample was taken and the copper content was
then determined according to the recommended procedure.
64.0
l
g mLꢀ1 Cu2+. By plotting the calibration curve of the absor-
(Eq. (3)) versus the logarithmic concentration
bance signal value
a
of Cu ion (pCu), the unknown Cu concentration can be read. All
measurements were carried out at room temperature.
Results and discussion
Spectral characteristics of ionophore in the solution
Preparation of water sample
One liter of mineral water sample was adjusted to pH 4.0 with
acetate buffer, filtered through a Millipore 0.45 lm pore size mem-
brane into previously cleaned polyethylene bottle and was ana-
lyzed within 6 h of collection [31]. A defined volume treated by
the recommended procedure for the determination of copper.
The membrane sensor proposed is based on the incorporation of
a Schiff base, EBSI with two hydroxyl functional groups in its struc-
ture as an ion exchanger. This ionophore (EBSI) is insoluble in
water in its neutral form. In preliminary experiments, the com-
plexation of EBSI with different cations was investigated spectro-
photometrically in ethanol solvent at 25 0.1 °C. A 1.0 ꢂ 10ꢀ5
M
Determination of copper in hair
ionophore solution was prepared by dissolving 0.24 mg of the ion-
ophore in 50 mL of ethanol. The solutions of different cations with
equal concentrations were selected. Then, 3 mL of them and 3 mL
of ionophore solution were dropped into the glass vial. The solu-
tions were immediately shaken vigorously to achieve complete
homogeneity. Finally, obtained solutions were injected to the cells,
and absorbance values were measured after 15 min (because of the
time required to reach the equilibrium). The reference cell contains
3 mL cation solution and 3 mL ethanol. The absorbance was mea-
sured over the wavelength range of 300–700 nm (Fig. 2).
The spectral change (decrease in absorption band at 330 nm
and increase in the absorption band at 400 nm) shows that the ion-
ophore is a selective complexing agent for copper ions. As can be
seen, the complexation was accomplished by a relatively strong
shift of the absorption band of the ligand H2L (EBSI), with kmax at
330 nm toward higher wavelength, 400 nm, for the complex, at
which the ligand has low absorbance. Such a pronounced effect
on the electronic spectra of the ligand could be related to strong
complexation with Cu(II) ions.
A 1.0 g of sample was decomposed by heating with 30 mL of
concentrated nitric acid and 3 mL of 60% perchloric acid in a Kjel-
dahl flask. The solution was cooled, filtered and diluted to 200 mL
with water in a calibrated flask after adjusting the pH to 4 [54]. An
aliquot of this solution was taken and copper was determined by
the general procedure.
Determination of copper in multivitamin capsule
Five capsules of multivitamin was accurately weighed and
placed into ceramic crucible. Five milliliter of concentrated nitric
acid was added to it and heated to near dryness. After cooling,
the residue was dissolved in another 5 mL of concentrated HNO3
and the solution was gently evaporated using a water bath. The
residue was again heated with 50 mL distilled water, filtered off
and diluted to 100 mL in a calibrated flask after adjusting the pH
to 4 [55]. A defined volume treated by the recommended proce-
dure for the determination of copper.
Determination of copper in foods
20.0 g of each sample was accurately weighed and placed into
quartz crucible. Ten milliliter of concentrated sulfuric acid was
added to it and evaporated to near dryness; then 10 mL of nitric
acid (1 + 1, volume ratio) was added and evaporated to dryness.
Under the heating conditions, concentrated hydrogen peroxide
was added by drop till the solution clearness and evaporated.
Water was added and continued to heat to remove the hydrogen
peroxide. The residue cooled and was transferred into a 50 mL cal-
ibrated flask and diluted to the mark with water after adjusting the
pH to 4 [56]. A defined volume was taken for the determination of
copper via the recommended procedure under the established
optimum conditions.
Fig. 1. Absorbance measuring system for the film optode.

M. Arvand, Z. Lashkari / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 280–288
283
Fig. 2. Absorption spectra of ionophore in the solution at the presence of different cations. Ionophore concentration was 1.0 ꢂ 10ꢀ5 M and absorbance values were measured
after 15 min to reach the equilibrium.
In order to determine the stoichiometry of the resulting com-
plex, the absorption spectra containing 1.0 ꢂ 10ꢀ4 M of the ligand
solution at a fixed ionic strength of 0.05 M (maintained by TEAP)
and varying amounts of the Cu ion were obtained at 400 nm in eth-
anol solution. The absorbance versus [Cu(II)]/[EBSI] mole ratio plot
revealed an inflection point at [Cu(II)]/[EBSI] mole ratio of about 1,
emphasizing the formation of a 1:1 (metal-to-ligand) complex in
the solution.
Measuring principle
Absorption spectrum of EBSI (H2L) in Cu2+ solution showed a
maximum at 400 nm. By addition of a metal ion, it forms a complex
with H2L and increases its absorption at kmax = 400 nm. Under the
experimental condition used and supposing the formation of a 1:1
complex between EBSI and Cu(II) ion in the organic phase, the re-
sponse mechanism of this optical sensor can be described by the
following ion-exchange mechanism:
H2Lorg þ Cu2þ ꢀ CuLorg þ 2Hþ
ð1Þ
aq
aq
Thus, based on the ion-exchange mechanism for the optical sen-
sor, its measuring principle can be explained as follows. Upon the
establishment of the complexation equilibrium between Cu2+ ion
in aqueous sample solution and the ionophore (H2L) in the organic
membrane phase (Eq. (1)), CuL complex can be formed
Spectral characteristics of ionophore in the membrane
After EBSI incorporated in the PVC membrane, the spectral
properties of the ligand in the membrane remained the same as
those measured in ethanol solvent (Fig. 3). This is the basis of the
optical sensing device in this investigation. Therefore, the wave-
length 400 nm has been used in all subsequent measurements of
absorbance. The absorption spectra of the optical sensor in buffer
solution of pH 4.0 and in the presence of increasing concentration
of Cu2+ ion is shown in Fig. 4. As it is obvious, the absorption inten-
sity of ionophore is decreased considerably upon increasing con-
2
2þ
Kexch ¼ ½CuLꢄorg½Hþꢄaq=½H2Lꢄ ½Cu
ꢄ
ð2Þ
org
aq
The relative absorption intensity,
a, is defined as the ratio of
complexed ionophore in the membrane phase, [CuL]org, to its total
uncomplexed form [H2L]org, so
a
¼ ½CuLꢄorg=½H2Lꢄorg ¼ ðA ꢀ A0Þ=ðA1 ꢀ A0Þ
ð3Þ
centration of Cu(II) ion in the buffer solution and
a new
absorption band was appeared at higher wavelength due to com-
plex formation between ionophore and copper ion.
A0 and A1 are the limiting absorbance intensities of the optical sen-
sor at = 0 (i.e. uncomplexed H2L) and = 1 (i.e. totally complexed
a
a
Fig. 3. Absorption spectra for Cu–EBSI complex in the membrane containing 2 mg EBSI, 61 mg DOS, 4 mg OA and 30 mg PVC. Inset: Absorption spectra for Cu–EBSI complex in
the solution containing 1.0 ꢂ 10ꢀ5 M EBSI and 6.4
l
g mLꢀ1 Cu2+ at pH = 4.0.

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M. Arvand, Z. Lashkari / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 280–288
Fig. 4. Absorption spectra for the ionophore EBSI incorporated into membrane and varying amounts of Cu(II) ions (a–g: 0.0, 1.3, 3.4, 5.4, 6.3, 8.7, 13.4 l
g mLꢀ1) in aqueous
solution with pH = 4.0; Membrane composition: 2 mg of EBSI, 61 mg of DOS, 4 mg of OA, and 30 mg PVC.
H2L), respectively. The relationship between
a values and the con-
centration of Cu2+ in aqueous sample solution [Cu2+
tained from Eqs. (2) and (3):
]
can be ob-
aq
2
2þ
a=ð1 ꢀ
a
Þ ¼ ðKexch=½Hþꢄ Þ ½Cu
ꢄ
ð4Þ
aq
Eq. (4) could be used as a basis for the quantitative determination of
copper ions using the proposed optical membrane.
Effect of pH on the membrane response
Sensitivity to Cu(II) depends the pH in the aqueous solution.
This implies that the binding curves for Cu(II) should be measured
with pH-buffered solutions. Fig. 5 shows the effect of pH values on
the dynamic range of the optical membrane in the presence of
Cu(II) ions. The response curve data were obtained by measuring
the absorbance values for Cu(II) at different pH values. At the pH
value in the range of 1.0–7.0, the working dynamic range reached
the widest value, 0.01–32.0 l
g mLꢀ1. As it is seen, the best result
Fig. 5. Effect of pH on the response of membrane in the presence of 3.3–
6.8
g mLꢀ1 Cu(II) at 400 nm; Membrane composition: 2 mg of EBSI, 61 mg of DOS,
4 mg of OA, and 30 mg PVC.
was obtained at pH 4.0. This is in agreement with proposed pro-
ton/copper exchange mechanism. However, in pH lower than 4.0
the response of sensor in test solution, decreases. It is probably,
due to chemical decomposition of ionophore under acidic condi-
tion that is a result of the acidic hydrolysis of EBSI or electrostatic
repulsion between Cu2+ and protonated ligand in highly acidic
media. Hence the dynamic range would of course also decrease.
At pH values higher than 4.0 also the response of membrane de-
creased that may be due to the hydrolysis of metal ion.
l
as DBS, DOS, DBP, NB and AP were tested as potential plasticizers
for preparing the membrane. The membranes were prepared from
a mixture of PVC (30 mg), plasticizer (61 mg), additive (4 mg) and
ionophore (2 mg) in THF (1.5 mL). The membranes containing DOS
revealed the best physical properties with maximum sensitivities
and wide concentration range (Fig. 6c). Membranes obtained from
AP and DBS as plasticizers did not show any absorption change
against Cu(II) ion.
For determination of the effect of plasticizer amount on the op-
tode response, membranes that contain different amounts solvent
mediator DOS (30, 61, 72 mg) were prepared. Absorption measure-
ments showed that membrane containing 61 mg DOS revealed the
best physical properties and wide concentration range (Fig. 7A).
Effect of membrane composition
Choice of solvent mediator (plasticizer)
It is obvious that the selectivity and sensitivity obtained for a gi-
ven ionophore depends significantly on the membrane composi-
tion and the nature of solvent mediator and additives used [57].
Thus, the influence of membrane composition on the response
behavior and leaching of sensor was investigated.
The membrane composition and the nature of the plasticizer
largely influence the response characteristics and the working con-
centration range of the optical sensors [57]. Optode films with a
high amount of plasticizer have optimum physical properties and
ensure relatively high mobility of their constituents. In order to
give a homogeneous organic phase, the membrane solvent must
be physically compatible with the polymer. Five plasticizers such
Effect of the PVC amount
It is noteworthy that the best membrane characteristic is re-
ported to usually obtain at a plasticizer/PVC ratio of 1.6–2.2. In
general, the thickness and hardness of the membrane depend on
the amount of PVC used. At lower PVC contents, the membrane be-
comes mechanically weak and swells up easily in aqueous solution,

M. Arvand, Z. Lashkari / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 280–288
285
c DOS
b DBP
a NB
c
a
Fig. 6. Effect of the type of plasticizer on the response of the optical sensing film in aqueous solution with pH = 4.0; Membrane composition: 2 mg of EBSI, 61 mg of
plasticizer, 4 mg of OA, and 30 mg PVC.
Fig. 7. Effect of the amount of plasticizer (A), ionophore (B) and additive (C) on the response of the optical sensing film in aqueous solution with pH = 4.0.
and leaching of the membrane is significant. By increasing the
amount of PVC, the membrane becomes dense, preventing the
leaching of ionophore from membrane into aqueous solution. In
this work, we examined the various amounts of PVC on the re-
sponse characteristic of membrane. The results illustrated that
increasing the amount of PVC (more than 35 mg) did not improve
the sensitivity and were not suitable because of the decreasing of
ion diffusion into the bulk of membrane.
was taken as the reference. As it is seen from Fig. 7B, the presence
of 2 mg of ionophore in the PVC membrane resulted in the best
optical response of the Cu ion-sensing film. Higher amounts of ion-
ophore (more than 2 mg) did not improve the sensitivity and were
not suitable due to the leaching of ionophore.
Effect of the additive
In order to facilitate the establishment of a thermodynamic
equilibrium between the bulk liquid membrane optodes and the
sample solution of interest, a rather fast mass transfer of analyte
from the sample into the membrane is required [58]. Thus, in this
proposed Cu optical sensing film containing EBSI as an ionophore,
the incorporation of a suitable additive was necessary to ensure
the fast establishment of the corresponding ion-exchange equilib-
rium. OA and NaTPB were tested as additive for preparing the
membrane. The membranes were prepared from a mixture of
PVC (30 mg), plasticizer (61 mg), additive (4 mg) and ionophore
(2 mg) in THF (1.5 mL). The results showed that the membrane
containing OA revealed good response and transparency. The en-
hanced sensitivity may be attributed to the OA existing in the
PVC membrane, changing the structure of the membrane and
improving the passing efficiency of Cu into the organic membrane.
It favorably makes much more Cu(II) extract into the membrane,
and leads to a higher sensitivity. For study of the effect of additive
Effect of amount of the ionophore
Ionophores play a key role in the selectivity of ion-selective op-
todes. The rational design of synthetic carriers takes advantages of
the different elements of molecular recognition. The creation of
suitable binding sites and proper topology in the ionophore that
are complementary to the size and charge of a particular ion can
lead to very selective interaction.
The optical sensing film proposed is based on the incorporation
of EBSI with two hydroxyl functional groups in its structure as an
ion exchanger. This ionophore possesses a double binding property
towards Cu(II) ion in sensing membrane. This ionophore is insolu-
ble in water in its neutral form, while it is quite soluble in organic
solvents. The effect of amount of the ionophore on the response of
the membrane is illustrated in Fig. 7B. The blank membrane (mem-
brane without ionophore in presence of Cu(II) ion with pH = 4.0)

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M. Arvand, Z. Lashkari / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 280–288
amount on the optode response, membranes that contain different
amounts OA (2, 4, 6 mg) were prepared. Absorption measurements
showed that membrane containing 4 mg OA revealed best re-
sponse (Fig. 7C).
preparing five membranes from the same mixture and the repro-
ducibility was obtained by determining the signal of the mem-
brane to 6.4 l
g mLꢀ1 solution of Cu(II). The mean absorbance
was found to be 0.357 with RSD = 1.94%. The results show that
the reproducibility is satisfactory.
Calibration range and detection limit
Response time
The optical response of the proposed Cu(II) sensitive membrane
under optimum experimental conditions was obtained. The plot of
normalized absorbance value against pCu can be used as a calibra-
tion plot for determination of Cu ions in the concentration range
From the literature and preliminary experiments, the amounts
of PVC and plasticizer can influence the response time of the mem-
brane. The response time of the present optical sensing film is con-
trolled by the time of required for the analyte to diffuse from the
bulk of the solution toward the membrane interface to associate
with the ligand. The results revealed that the membranes contain-
ing 30 mg PVC and 61 mg plasticizer gave the shortest response
time. The data show that the response time of membrane can reach
to 98% of its final (steady state) value in a time of less than 3 min.
(0.01–32.0
l
g mLꢀ1
)
with equation of Y = –0.2686X + 1.8683
and X is negative logarithm of Cu ion
(R2 = 0.9888), where Y is
a
concentration in molar. Detection limit of the sensing film, which
is defined as the concentration of the sample yielding a signal
equal to the blank signal three times of its SD, was found to be
0.008 l
g mLꢀ1. The relative standard deviations for the copper
determinations were 0.41% (n = 10) at 2.0 l
g mLꢀ1 Cu(II).
Analytical application
Selectivity
The proposed sensor was also applied to the direct determina-
tion of copper in various real samples. The samples were prepared
as described in section 2.5 and determined with both the proposed
sensor and AAS. From the results of three replicate measurements
given in Tables 2 and 3, it is immediately obvious that there is sat-
isfactory agreement between the results obtained by the Cu(II)–
selective optode and AAS. The results were also statistically com-
pared by the paired t-test. This test revealed the absence of signif-
icant differences between the results obtained by both methods (at
95% confidence level). There was thus no evidence of the presence
of systematic error in the results. Table 4 presents a comparison
between the proposed method and other spectrophotometric
methods for the determination of Cu [30–38]. The results show
that proposed method is comparable to the existing spectrophoto-
metric methods.
The selectivity of optical sensor, which reflects the relative re-
sponse of the sensor for primary ion over divers ions present in
solution, is perhaps the most important characteristics of an opti-
cal sensor. In order to access the possible analytical application of
this sensing method, the effects of some alkali, alkaline earth and
heavy metal ions were investigated. The experiment was carried
out with a fixed concentration of Cu(II) at 0.64 l
g mLꢀ1 and then
measuring the changes in absorbance intensity before and after
adding different foreign interferents in the Cu2+ solution buffered
at pH = 4.0. The tolerance limit was set as the amount of foreign
ion causing 5% error in determination of Cu. The results are sum-
marized in Table 1. As can be seen from Table 1, several metal ions
do not interfere even at high concentrations. In addition to Cu, the
sensing film also produces a response to some other metal ions
(Hg2+, Ni2+) that form complex with EBSI at pH = 4.0. The results
obtained may be important in terms of application of the sensing
film to determination of other ions.
Conclusions
On the basis of the results presented in this work, the proposed
Cu(II) ion-selective optode has many advantages including: easy
preparation, low cost, fast response time, wide dynamic range,
Reversibility, reproducibility, short-term stability and lifetime
Some reagents including HCl, HNO3, H3PO4, H2SO4, EDTA and
(NH2)2CS were studied as regenerating and masking reagents. It
was found that none of the above reagents or their mixtures could
regenerate optode membrane completely and thus the membrane
could be used as a probe for Cu ion determination.
Table 2
Determination of copper in standard alloys.
Sample
Composition (%)
Concentration (%)
The short-term stability of the optical sensing film was deter-
mined by its absorbance intensity measurements in contact with
a buffer solution (pH = 4) in a cuvette. The signal was recorded
every 30 min at wavelength of 330 nm (kmax of ionophore) over a
period of about 5 h. It was found that no significant loss of the ion-
ophore occurs during this time. The membrane was stable over a
period of 3 months when not in use (membrane was kept in air)
and the signal value of the membrane did not change. The differ-
ence in the response of individual membranes was evaluated by
Certified
value
Founda
JSS 653-7
stainless
steel
C, 0.068; Si, 0.63; Cr, 22.53; Co,
0.35; Mn, 1.72; Ni, 13.91; N,
0.0276
0.030
0.027 0.003
NKK No. 920
aluminum
alloy
Si, 0.78; Fe, 0.72; Mg, 0.46; Cr,
0.27; Zn, 0.80; Ti, 0.15; Sn, 0.20;
Pb, 0.10; Sb, 0.10; Bi, 0.06; Ga,
0.05; Ca, 0.03; Co, 0.10; Mn,
0.20; Ni, 0.29; V, 0.15
0.71
0.74 0.04
0.28 0.02
2.68 0.08
NKK No. 916
aluminum
alloy
Si, 0.41; Fe, 0.54; Mg, 0.10; Cr,
0.05; Zn, 0.30; Ti, 0.10; Sn, 0.05;
Pb, 0.04; Sb, 0.01; B, 0.0006; Zr,
0.05; Bi, 0.03; Co, 0.03; Mn,
0.11; Ni, 0.06; V, 0.02
0.27
2.72
Table 1
Effect of some diverse ions on the optode response for the determination of
0.64 l
g mLꢀ1 Cu2+. The reasonably less than 5% relative error was tolerated.
Foreign ions
Tolerance limit
NKK No. 1021 Si, 5.56; Fe, 0.99; Mg, 0.29; Zn,
(Wion/WCu(II)
)
Al, Si, Cu,
Zn, Cr,
alloy
1.76; Ti, 0.04; Sn, 0.10; Pb, 0.18;
Sb, 0.01; Zr, 0.01; Bi, 0.01; V,
0.007; Ca, 0.004; Mn, 0.11; Ni,
0.14
Mg2+, Ca2+, Cd2+, Co2+, Fe2+, Pb2+, Na+, Mn2+, Sr2+, Bi3+
,
1000
Zn2+, Li+, Clꢀ, Iꢀ, SO24ꢀ, NO3ꢀ
Cr3+, Al3+, Sn2+, Ag+
Hg2+, Ni2+
100
10
a
Average of five determination standard deviation.

M. Arvand, Z. Lashkari / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 280–288
287
Table 3
Results of three replicate determinations of Cu in mineral water, human hair, foods and pharmaceuticals.
a
Sample
Concentration (
l
g gꢀ1
)
t–testb
F–test
Proposed method
AAS method
Hair
Rice
Tea
Tomato sauce
Celery
11.32 0.15
3.02 0.13
4.3 0.4
0.50 0.03
0.76 0.08
0.20 0.07
0.43 0.03
11.21 0.12
2.81 0.08
4.8 0.2
0.99
2.38
1.94
0.96
1.87
0.60
2.08
0.38
1.56
2.64
4.0
2.25
1.26
1.96
2.78
1.89
0.48 0.02
0.89 0.09
0.23 0.05
0.50 0.05
Cabbage
Multivitamin capsule
Mineral waterc
0.97 0.11 (
l
g mLꢀ1
)
0.94 0.08 (l )
g mLꢀ1
a
b
c
Average of three determination standard deviation.
The theoretical values of t and F at P = 0.05 are 2.57 and 19.00, respectively.
The water obtained from Vata spring (Ardebil, Iran).
Table 4
Comparisons of the proposed method with the existing spectrophotometric methods mentioned in the literature.
Method
LDR (l
g mLꢀ1
)
LLD (l
g mLꢀ1
)
RSD (%)
Type of samples
Ref.
Spectrophotometry
Spectrophotometry
Spectrophotometry
Spectrophotometry
Spectrophotometry
Spectrophotometry
Spectrophotometry
Spectrophotometry
Spectrophotometry
Spectrophotometry
0.25–6.35
0.04–5.0
0.1–10.0
0.5–4.0
0.021
0.03
0.01
–
0.19 (n = 9)
1.4 (n = 10)
2.0 (n = 10)
0.057 (n = 10)
0.67 (n = 5)
0.45 (n = 5)
5.0 (n = 6)
4.68 (n = 5)
0.5 (n = 7)
0.41 (n = 10)
Natural waters, certified vitamin and simulated samples
Standard alloys
Natural waters and pharmaceutical samples
–
Synthetic mixture, sea and well water samples
Certified standard material and water samples
Water and alloy samples
Vegetables and tea
Tap water, drinking water and wastewater
Standard alloys, mineral water, human hair, foods and pharmaceuticals
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
1.015–8.122
3.19 ꢂ 10ꢀ2
0.1–5.0 (
l
g Lꢀ1
)
0.025 (
0.05
l
g Lꢀ1
)
0.48–12.8
0–1.024
7.03 ꢂ 10ꢀ4
2.5–75 (
l
g Lꢀ1
)
0.85 (
0.008
l
g Lꢀ1
)
0.01–32.0
This work
LDR: linear dynamic range; LDL: lower limit of detection.
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determine the concentration of copper (II) ions in real samples.
This sensing membrane has potential application for determina-
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