An efficient, cost effective, sensing behaviour liquid-liquid extraction and spectrophotometric determination of copper(II) incorporated with 4-(4′-chlorobenzylideneimino)-3-methyl-5-mercapto-1, 2, 4-triazole: Analysis of food samples, leafy vegetables, fertilizers and environmental samples

Article abstract of DOI:10.1016/j.saa.2017.08.054

The aim of the present work is to develop an efficient, simple and selective moreover cost-effective method for the extractive spectrophotometric determination of copper(II) by using the Schiff base 4-(4′-chlorobenzylideneimino)-3-methyl-5-mercapto-1, 2, 4-triazole [CBIMMT]. This chromogenic reagent forms a yellow coloured complex with copper(II) in acetate buffer at pH 4.2. The copper(II) complex with ligand is instantly extracted into chloroform and shows a maximum absorbance at 414 nm which remains stable for > 48 h. The composition of extracted complex is found to be 1:2 [copper(II): reagent] which was ascertained using Job's method of continuous variation, mole ratio method and slope ratio method. Under optimal conditions, the copper(II) complex in chloroform adheres to Beer's law up to 17.5 μg mL^{? 1} of copper(II). The optimum concentration range obtained from Ringbom's plot is from 5 μg mL^{? 1} to 17.5 μg mL^{? 1}. The molar absorptivity, Sandell's sensitivity and enrichment factor of the extracted copper(II) chelate are 0.33813 × 10⁴ L mol^{? 1} cm^{? 1}, 0.01996 μg cm^{? 2} and 2.49 respectively. In the extraction of copper(II), several affecting factors including the solution pH, ligand concentration, equilibrium time, effect of foreign ions are optimized. The interfering effects of various cations and anions were also studied and use of masking agents enhances the selectivity of the method. The chromogenic sulphur containing reagent, 4-(4′-chlorobenzylideneimino)-3-methyl-5-mercapto-1, 2, 4-triazole has been synthesized in a single step with high purity and yield. The synthesized reagent has been successfully applied first time for determination of copper(II). The reagent forms stable chelate with copper(II) in buffer medium instantly and quantitatively extracted in chloroform within a minute. The method is successfully applied for the determination of copper(II) in various synthetic mixtures, complexes, fertilizers, environmental samples such as food samples, leafy vegetables, and water samples. The results are compared with those obtained with a reference procedure. Good agreement was attained. All the obtained results are indicative of a convenient, fast method for the extraction and quantification of micro levels of copper(II) from various environmental matrices without use of sophisticated instrumentation and procedure. The method showed a relative standard deviation of 0.42%.

Full text of DOI:10.1016/j.saa.2017.08.054

Accepted Manuscript
An efficient, cost effective, sensing behaviour liquid-liquid
extraction and spectrophotometric determination of copper(II)
incorporated
with
4-(4′-
chlorobenzylideneimino)-3-methyl-5-mercapto-1, 2, 4-triazole:
Analysis of food samples, leafy vegetables, fertilizers and
environmental samples
Umesh B. Barache, Abdul B. Shaikh, Tukaram N. Lokhande,
Ganesh S. Kamble, Mansing A. Anuse, Shashikant H. Gaikwad
PII:
S1386-1425(17)30683-2
doi: 10.1016/j.saa.2017.08.054
SAA 15411
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date:
Revised date:
Accepted date:
30 March 2017
15 August 2017
19 August 2017
Please cite this article as: Umesh B. Barache, Abdul B. Shaikh, Tukaram N. Lokhande,
Ganesh S. Kamble, Mansing A. Anuse, Shashikant H. Gaikwad , An efficient, cost
effective, sensing behaviour liquid-liquid extraction and spectrophotometric determination
of copper(II) incorporated with 4-(4′-chlorobenzylideneimino)-3-methyl-5-mercapto-1, 2,
4
-triazole: Analysis of food samples, leafy vegetables, fertilizers and environmental
samples, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2017),
doi: 10.1016/j.saa.2017.08.054
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ACCEPTED MANUSCRIPT
An efficient, cost effective, sensing behaviour liquid-liquid extraction and
spectrophotometric determination of copper(II) incorporated with 4-(4'-
chlorobenzylideneimino)-3-methyl-5-mercapto-1, 2, 4-triazole: Analysis of
food samples, leafy vegetables, fertilizers and environmental samples
a
a
a
b,c
Umesh B. Barache , Abdul B. Shaikh , Tukaram N. Lokhande , Ganesh S. Kamble ,
b
a*
Mansing A. Anuse , Shashikant H. Gaikwad
aChemistry Research Laboratory, Department of Chemistry, Shri Shivaji Mahavidyalaya,
Barshi, 413411, Maharashtra, India.
bAnalytical Chemistry Research Laboratory, Department of Chemistry, Shivaji University,
Kolhapur, 416004, Maharashtra, India.
cKolhapur Institute of Technology’s, College of Engineering, Kolhapur, 416234,
Maharashtra, India.
Abstract
The aim of the present work is to develop an efficient, simple and selective moreover
cost-effective method for the extractive spectrophotometric determination of copper(II) by
using the Schiff base 4-(4’-chlorobenzylideneimino)-3-methyl-5-mercapto-1, 2, 4-triazole
[
CBIMMT]. This chromogenic reagent forms a yellow coloured complex with copper(II) in
acetate buffer at pH 4.2. The copper(II) complex with ligand is instantly extracted into
chloroform and shows a maximum absorbance at 414 nm which remains stable for more than
4
8 h. The composition of extracted complex is found to be 1:2 [copper(II) : reagent] which
was ascertained using Job’s method of continuous variation, mole ratio method and slope
ratio method. Under optimal conditions, the copper(II) complex in chloroform adheres to
-1
Beer’s law up to 17.5 µg mL of copper(II). The optimum concentration range obtained from
-1
-1
Ringbom’s plot is from 5 µg mL to 17.5 µg mL . The molar absorptivity, Sandell’s
1

ACCEPTED MANUSCRIPT
4
-1
sensitivity and enrichment factor of the extracted copper(II) chelate are 0.33813 x 10 L mol
-1
-2
cm , 0.01996 µg cm and 2.49 respectively. In the extraction of copper(II), several affecting
factors including the solution pH, ligand concentration, equilibrium time, effect of foreign
ions are optimized. The interfering effects of various cations and anions were also studied
and use of masking agents enhances the selectivity of the method. The chromogenic sulphur
containing reagent, 4-(4’-chlorobenzylideneimino)-3-methyl-5-mercapto-1, 2, 4-triazole has
been synthesized in a single step with high purity and yield. The synthesized reagent has been
successfully applied first time for determination of copper(II). The reagent forms stable
chelate with copper(II) in buffer medium instantly and quantitatively extracted in chloroform
within a minute. The method is successfully applied for the determination of copper(II) in
various synthetic mixtures, complexes, fertilizers, environmental samples such as food
samples, leafy vegetables, and water samples. The results are compared with those obtained
with a reference procedure. Good agreement was attained. All the obtained results are
indicative of a convenient, fast method for the extraction and quantification of micro levels of
copper(II) from various environmental matrices without use of sophisticated instrumentation
and procedure. The method showed a relative standard deviation of 0.42%.
Keywords:
CBIMMT;
Copper(II);
Environmental
samples;
Food
samples;
Spectrophotometric determination.
Abbreviations:
CBIMMT:
RSD:
4-(4’-Chlorobenzylideneimino)-3-methyl-5-mercapto-1, 2, 4-triazole
Relative standard deviation
UV-Vis.:
AAS:
Ultraviolet visible
Atomic absorption spectrophotometer
2

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LOD:
LOQ:
Limit of detection
Limit of quantification.
*
Corresponding author: rasayanshg@gmail.com, rasayanshg1@gmail.com (S. H. Gaikwad)
. Introduction
Copper is one of the very essential element that plays a vital role in the biological
1
systems. Copper occurs naturally in many vegetables, meat and grains. In biological systems,
copper counter acts the toxicity of zinc suggesting Cu–Zn antagonism [1]. Copper and its
salts are more toxic to some lower organism(s) than to human beings. It turns toxic at higher
level due to its accumulation in cell membranes and prevents transport across the wall of the
cell [2]. Hereditary disease such as Wilson’s disease occurs due to excess amounts of copper
in the normal human body. It causes fatal consequences like liver damage. Due to elevated
levels of copper, violent tendencies are observed in males that may leads to depression and
schizophrenia [3].
As a pollutant, copper is of mainly concern because of its high degree of toxicity.
+
2+
There are several toxic copper species which include Cu(OH) , Cu
2
(OH)
2
and CuCO .
3
2
+
However, there is no doubt, that Cu ion, which are present in various aqueous solutions, are
considered to be poisonous due to dissolved copper species. Excess of copper in water is not
only harmful to human beings but also to many aquatic organisms [4]. It acts as a main
cofactor in many enzymes and plays a key role in protein and cell division [5]. It may cause
generalized cancer in the laboratory animals and has been linked epidemiologically with
certain human cancers [6]. Most serious diseases are cirrhosis and skin cancer which causes
gradually weaken bone structure and cause the diminution of stature that ultimately leads to
total collapse of the entire skeletal systems of humans. Higher toxicity of copper towards
marine and fresh water species is also observed [7].
3

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An increase in copper concentrations in the environment results from industrial and
domestic waste discharge, disposal of mining washings, refineries into the water streams
which consequently enter in several food materials. World Health Organization (WHO)
-1
guidelines recommend the permissible limit of copper in drinking water is 2.0 mg L [8].
Recently a variety of analytical advanced methods requiring high-priced
instrumentation are reported to be dealing with the determination of copper(II) from various
matrices at trace level. These methods include: neutron activation analysis [9], X-ray
fluorescence [10], pulse polarography [11], inductively coupled plasma emission
spectrophotometry (ICP-ES) [12], graphite furnace atomic absorption spectrophotometry
(GF-AAS) [13], flame atomic absorption spectrometry (FAAS) [14-16]. Although these
analytical techniques are highly sensitive and selective, they are quite expensive for the
developing countries and require well qualified laboratory personnel and superior laboratory
equipment. Moreover, they might not be always available in all routine analytical
laboratories. Many of the reported methods have some short comings such as the availability
of sophisticated instruments, time-consuming operation, tedious work, multi-step procedures
for pre-concentration of the analyte and low selectivity with higher interference problems.
The reported methods [9-12] have demerits with respect to their high cost and the instruments
used in day-to-day analysis. GF-AAS [13] often lacks its sensitivity due to the sublimation at
high temperature. The other reported sophisticated and time consuming method is catalytic
kinetic effect of copper on the oxidation of cysteine by hexacyanoferrate(III) [17]. In
contrast, spectrophotometry is well-known by its quick analysis ability, low cost and
simplicity in its operation. Due to these advantages, a number of spectrophotometric methods
have been developed to determine trace amount of copper(II).
Recently many advanced solvent extraction techniques has been developed for the
determination of metal ions. Some potentially applied method includes solid phase extraction
4

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(SPE) [14-16,18-25] and dispersive liquid-liquid microextraction (DLLME) [26,27], single
drop microextraction (SDME) [28], solidified floating organic drop microextraction
SFODME) [29], hollow fiber liquid-phase microextraction (HF-LPME) [30], cloud point
(
extraction (CPE) [31] and classical liquid-liquid extraction (LLE) methods. But these
methods also have their disadvantages. In SPE and DLLME microextraction methods, the
determination methods are more complex, time consuming and involve multiple steps for
pre-concentration of analyte which leads to occurrence of errors in the determination.
SFODME has the restricted choice of extracting solvents because only a small number of
organic solvents have melting points nearer to room temperature. Another problem with this
technique is the overlapping of solvent peaks with those of the analytes in the
chromatograms. SDME is limited by rate of stirring which causes dislodgment of drop. HF-
LPME technique requires pre-conditioning of the membrane and problem with memory
effects when membranes are reused. In CPE method, the extracted analyte cannot be injected
directly as the surfactants (rich phase) has more viscosity. Due to these disadvantages, the
better approach is to use the classical liquid-liquid extraction (LLE) methods which are
simple, quick, selective, economical and sensitive as well.
It is well-known that sulphur containing ligands and some biomolecules [32,33] form
strong complexes with copper(II), as copper(II) belongs to the class of soft acids, whereas
ligands containing donating sulphur atom belongs to soft bases. Soft acids always prefer to
bind the soft bases [34]. The thiol and phenyl thiosemicarbazones are important sulphur and
nitrogen that contain chromogenic reagents where copper(II) forms stable complexes with
them and can be extracted into suitable organic solvents. The literature survey revealed that
only a few thiol and phenyl thiosemicarbazones were employed for extractive determination
of copper(II) [35-38]. A comparison of a few selected spectrophotometric determination
methods of copper(II), their spectral characteristics and drawbacks are summarized in Table 1
5

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[
1,3,39-47], but these reported reagents to have disadvantages, such as maximum absorbance
in UV region [3], some require the use of surfactants for full colour development [39,43],
synergic extraction [42], heating of aqueous phase [43], longer shaking time [45] and they are
less sensitive [46]. In the present paper, we have introduced a novel chromogenic reagent, 4-
(4’-chlorobenzylideneimino)-3-methyl-5-mercapto-1,2,4-triazole [CBIMMT] and have
developed a new, simple, rapid, selective and reproducible extractive spectrophotometric
method for the determination of copper(II) in synthetic mixtures, copper(II) complexes,
water samples, vegetable samples, food samples, fertilizer samples and separation from
binary mixtures. The chromogenic reagent CBIMMT in chloroform instantaneously forms
yellow coloured complex with copper(II). An extracted Cu(II)-CBIMMT complex in
chloroform shows the maximum absorbance in visible region. The present method does not
require the heating of aqueous phase, the use of surfactant and any synergent for complete
complexation and no use of any sophisticated instruments.
2
. Experimental
2
.1 Apparatus
All absorbance measurements are taken with Elico digital UV-VIS spectrophotometer
model-SL-159) by using 1cm quartz cells. All the pH measurements are made with
(
Equiptronics digital pH meter (model EQ-615). Calibrated glass wares are used throughout
the experimental procedure. Glass wares are cleaned by soaking them in acidified solutions of
potassium dichromate which was followed by washing them with soap water and rinsing two
times with distilled water.
2
.2 Standard copper(II) solution, buffer solution and solution of various cations and anions
-1
Standard stock solution of copper(II) with the concentration of 1.0 mg mL is
prepared by dissolving a 3.928 g portion of copper sulphate pentahydrate (CuSO .5H O, s.d.
4
2
fine chemicals) in water. The stock solution is then standardized by well-known established
6

ACCEPTED MANUSCRIPT
method [48]. The working solution of copper(II) is prepared by suitable dilution of a standard
stock solution with doubly distilled water. The acetate buffer of pH 4.2 is prepared by mixing
-1
-1
appropriate volume of 0.2 mol L acetic acid and 0.2 mol L sodium acetate solutions [49]
and pH of this buffer solution is confirmed by using a pH meter. All chemicals and various
organic solvents used are of analytical reagent grade and are used without any further
purification. The solutions of various cations and anions are prepared by dissolving
calculated quantities of corresponding salts in either water or dilute hydrochloric acid.
Various synthetic mixtures containing copper(II) are prepared by combination with different
metal ions in fixed composition. Doubly distilled water was used throughout all the
experiments.
2
2
.3 Synthesis and characterization of 4-(4’-chlorobenzylideneimino)-3-methyl-5-mercapto-1,
,4-triazole [CBIMMT]
The chromogenic reagent, CBIMMT is synthesized in the laboratory by simple
-1
condensation reaction of 3-methyl-5-mercapto-1,2,4-triazole (2.6 g, 0.02mol L ) with 4-
-1
chlorobenzaldehyde (2.8 g, 0.02mol L )[50]. A reaction mixture of 3-methyl-5-mercapto-1,
2
, 4-triazole and 4-chlorobenzaldehyde in ethyl alcohol, containing about 3 drops of glacial
acetic acid is refluxed for about 4 h (scheme 1).
H C
3
N
CHO
H3C
N
N
NH
S
H C
N
3
+
N
H
N
N
SH
N
+
Alcohol
N
N
SH
Cl
CH
Cl
CH
H N
2
Cl
-Chlorobenzaldehyde
3
-Methyl-4-amino-5-
4
4
-
(4'-Chlorobenzylidineimino)-3-methyl
5-mercapto-1,2,4-triazole (CBIMMT)
mercapto-1,2,4-triazole
Scheme – 1 Synthesis of CBIMMT.
The product obtained after reaction is filtered and recrystallized from hot ethyl
alcohol. On cooling, faint yellowish needles of CBIMMT are obtained. The purity of the
7

ACCEPTED MANUSCRIPT
CBIMMT is checked by melting point and by thin layer chromatographic method. The
structure is confirmed by IR and NMR spectra. A reagent solution which has concentration of
-1
0
.025 mol L is prepared by dissolving 0.631 g of CBIMMT in chloroform and is diluted up
to 100 mL in a calibrated volumetric flask. The fresh reagent solution is used it is whenever
required.
2
2
.4 Environmental and synthetic samples preparations
.4.1 Determination of copper(II) in various binary and ternary synthetic mixtures
-1
The binary and ternary synthetic mixtures containing 10 µg mL of copper(II) with
known amounts of other associated metal ions at higher concentrations are prepared in
laboratory, and the recovery experiments are performed. The interfering metal ions are
masked with suitable masking agents and the copper(II) is extracted by using recommended
procedure.
2
.4.2 Water samples
Various water samples from different locations such as municipal potable water,
borewell water, lake water (Ganesh lake, Barshi, Solapur district, M S, India) and dam water
Ujani dam, Solapur district, MS, India) are collected in clean and dry polythene bottles. All
(
water samples are filtered through Whatman filter paper No.40. Then 1000 mL of each
filtered water sample is taken and evaporated nearly it dries on hot plate with a mixture of 2
mL of concentrated H
further 5 mL of concentrated HNO
until the solution becomes much colourless. The resultant solution is cooled and NH
2
SO
4
and 5 mL of concentrated HNO
is added and heated up to dense white fumes continue or
OH is
3
. After dryness, it is cooled and
3
4
added for neutralization of acid in presence of 1 mL 0.01% tartarate solution. The solution
was then filtered and transferred to 100 mL volumetric flask and diluted up to the mark with
distilled water. The procedure is carried out to determine copper(II) content as per
recommended procedure.
8

ACCEPTED MANUSCRIPT
2
.4.3 Food samples
The copper(II) content from various food samples are determined by the present
method. Food samples are collected from the local market of Barshi city (Solapur district,
Maharashtra) and crushed in a mortar. A known amount (5 g) of dried food sample is taken
into a silica crucible. This is followed by strong heating to oxidize organic matter at about
8
0oC for 2 h. Then the sample is dissolved in 5 mL of aqua regia solution and evaporated on
hot plate near to dryness. The residue is treated with concentrated HClO and subjected to
4
moist dryness. The resultant residue is boiled with water, subjected to filtration by the use of
ordinary filter paper and washed with hot distilled water. The filtrate is taken in 25 mL
volumetric flask and diluted up to the mark with distilled water. From this solution an aliquot
of 5 mL is taken and the content of copper(II) is determined by the present method and
further it is confirmed by atomic absorption spectrophotometer.
2
.4.4 Leafy vegetables samples
The proposed method is also used for the determination of copper(II) from some
green leafy vegetable samples collected from nearby vegetable market of Barshi city (Solapur
district, Maharashtra). 25 g of green leafy vegetable is taken, cleaned and dried in an oven for
protection from contamination of minerals. After drying the samples are crushed in a mortar.
A calculated weight (5 g) of ash of each sample is taken in a dry silica crucible and is
o
subjected to strong heating for oxidation of organic matter at about 80 C for 2 h. To the
-1
resultant ash, 15 mL of 1 mol L hydrochloric acid is added and heated up to dryness on a
hot plate. Then the contents are boiled with water and filtered through ordinary filter paper
and washed with hot water. The filtrate is collected in 25 mL volumetric flask and diluted
with distilled water up to the mark. 5 mL aliquot from this solution is taken and the content of
copper(II) is determined by the recommended procedure and are further confirmed by atomic
absorption spectrophotometer.
9

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2
.4.5 Micronutrient fertilizer samples
Further the present method is also applied for the determination of copper(II) in
various micronutrient fertilizer samples. The samples are collected from a fertilizer market.
Known amount of sample (3 mL) is evaporated on hot plate near to moist dryness with 5 mL
concentrated HNO and 10 mL concentrated HCl mixture. This procedure is repeated two to
3
three times and heated up to dense white fumes till the solution becomes clear. The resultant
solution is cooled and filtered by Whatman filter paper No. 1. Then it is transferred to 100
mL volumetric flask and diluted up to the mark with distilled water. From this, aliquot of 1
mL is taken and determined by the recommended procedure. These results are compared with
the results obtained by using atomic absorption spectrophotometer.
2
.4.6 Copper(II) from complexes
The application of the present procedure are also studied to determine the copper(II)
in various synthetic complex compounds. Copper(II) complexes with salicylaldoxime, 4-
amino-5-mercapto-3-methyl-1,2,4-triazole [51,52], 4-(2’-nitrorobenzylideneimino)-3-methyl-
5
-mercapto-1,2,4-triazole [53] and ammonia are prepared in a laboratory and purified as per
the reported procedure [54]. The calculated quantity of the complex is carefully decomposed
with aqua regia and evaporated up to moist dryness. The residue is then dissolved in distilled
water and made up to 100 mL with distilled water. The stock solution is then standardized
gravimetrically by the salicylaldoximate method and volumetrically by the iodometric
method [54]. Aliquots of this solution are used for the determination of copper(II) as per the
recommended procedure.
2
.4.7 Separation of copper(II) from binary mixtures
The separation of copper(II) from other commonly associated metal ions like Pd(II),
Se(IV), Te(IV), Bi(III), Co(II), Au(III), Fe(III), Ag(I) and Ni(II) using 4-(4’-
chlorobezylideneimino)-3- methyl-5- mercapro-1, 2, 4-triazole is achieved. Among these, all
1
0

ACCEPTED MANUSCRIPT
the added metal ions remain quantitatively in the aqueous phase from which they are
determined by spectrophotometry using reported standard methods [50, 55-62]. The metal
ions such as Pd(II), Au(III), Ag(I) and Ni(II) are interfered in the determination method due
to their co-extraction with copper(II). These metal ions are separated from copper(II) by
masking them with suitable masking agent. These masked metal ions from the aqueous phase
are de-masked with HClO
methods.
4
and determined spectrophotometrically by using the standard
2
.5 Recommended procedure
An aliquot of the sample solution containing 25-175 µg mL of copper(II) solution is
-1
diluted with acetate buffer having pH 4.2 solutions up to the mark in 25 mL calibrated
volumetric flask. The solution is then transferred into 125 mL separatory funnel, followed by
-1
the addition of 10 mL 0.025 mol L CBIMMT in chloroform. The two phases are allowed to
equilibrate for 1 min. The yellow-coloured organic extract is collected over anhydrous
sodium sulphate to remove the traces of water if present. The organic extract is then
transferred into 10 mL calibrated volumetric flask and further diluted up to the mark with the
chloroform, if required. The absorbance of the extracted complex is measured at λmax 414 nm
against reagent blank prepared in the similar manner without copper(II). A calibration curve
is obtained and the unknown amount of copper(II) is determined from the same.
2
.6 Optimization of the experimental variables
To determine the optimum conditions for maximum extraction recovery of copper(II),
several analytical parameters are assessed, using a 1 mL standard solution containing 100 µg
of copper(II) ions. A univariate optimization procedure is then undertaken; i.e. varying one
parameter at a time while keeping the others constant.
1
1

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3
. Results and discussions
3
.1 Absorption spectra and spectral characteristics of the complex
In this work, UV–VIS spectrophotometry is used to accomplish the determination. In
order to carry out the quantification analysis, the maximum wavelength of absorption is to be
found out. Copper(II) forms yellow coloured complex with 4-(4’-chlorobezylideneimino)-3-
methyl-5-mercapro-1,2,4-triazole and extracted into chloroform at pH 4.2. The yellow
coloured complex in organic phase shows maximum absorbance at λmax 414 nm and is stable
for more than 48 h. The absorbance of reagent at 414 nm wavelength is found to be too low
(Fig. 1). For the effective extraction of copper(II), the optimum conditions are established by
studying various parameters, such as effect of pH, a reagent concentration, the choice of
organic solvent, an equilibration time. The spectral characteristics of Cu(II)-CBIMMT
complex in chloroform are mentioned in the table 2.
3
.2 Effect of pH
In the extraction of metal ions, the extraction efficiencies of metal complex are
closely related to the pH of the system. Therefore, we have investigated the effect pH on the
development of coloured complex of Cu(II)-CBIMMT by varying the pH of copper(II)
solution ranging from 1 to 10 before an addition of the organic phase. The pH of copper(II)
-1
-1
solution is adjusted by 0.1 mol L HCl and 0.1 mol L NaOH solutions. The effect of pH on
the absorbance values of extracted Cu(II)-CBIMMT complex are measured at λmax 414 nm
against reagent blank prepared in similar manner without taking copper(II). The effect of pH
on the formation of Cu(II)-CBIMMT complex is also investigated by using series of acetate
buffer solutions. It is observed that the absorbance of Cu(II)-CBIMMT complex obtained
with buffer solution is in accordance with that one obtained with pH, which is adjusted by
HCl and NaOH solutions. Fig. 2 shows the maximum absorbance of Cu(II)-CBIMMT
1
2

ACCEPTED MANUSCRIPT
complex is observed in the pH range of 3.8 to 4.4. Therefore, acetate buffer of pH 4.2 is
selected conveniently for all the further studies.
3
.3 Effect of organic solvents
Solvent plays a crucial intrinsic role in solvation of copper(II) species with ligand. For
this purpose, the influence of various organic solvents are examined for the effective
-1
extraction of copper(II) with CBIMMT. Solutions of CBIMMT (0.025 mol L ) is prepared in
various organic solvents and the effect of solvents on the absorbance value of Cu(II)-
CBIMMT complex at 414 nm is examined. It is also observed that the absorbance values at
λmax 414 nm increased in the order ethyl acetate < MIBK < dichloromethane < isoamyl
alcohol < 1, 2-dichloroethane < carbon tetrachloride < benzene < chloroform. Among all
these organic solvents, chloroform is selected for all further experiments as it showed
maximum absorbance at 414 nm as compared to other solvents (Fig. 3).
3
.4 Effect of CBIMMT concentration
It is necessary to study the effect of reagent concentration, as the concentration of
ligand has a direct effect on the complexation reaction and extraction as well. The effect of
reagent concentration is investigated on the formation of complex species of copper(II) at pH
-
1
4
.2 by measuring absorbance at 414 nm of solution containing 10 µg mL of copper(II). The
reagents in chloroform are added to the copper(II) solution maintained at pH 4.2 by using
-1
acetate buffer. It is observed that 0.008 mol L of CBIMMT in chloroform was sufficient for
complete complexation of copper(II) (Fig. 4). Further it is observed that there is no adverse
effect of excess of reagent. In the present investigation, to ensure the complete complexation
-1
of copper(II), we recommend 0.025 mol L of CBIMMT in chloroform for a further study.
3
.5 Stability of Cu(II)-CBIMMT complex
The absorbance values of the Cu(II)-CBIMMT complex is measured at different time
intervals at λmax 414 nm to ascertain the stability of the complex at room temperature. It is
1
3

ACCEPTED MANUSCRIPT
observed that the colour of the Cu(II)-CBIMMT complex remains almost constant for more
than 48 h.
3
.6 Effect of shaking time
Equilibration of organic phase with aqueous phase plays a very important role in
extraction procedures. Minimum extraction time is often necessary for investigation of trace
amounts of metal ions with high efficiency. Here, the results indicate that the optimum
shaking time of 1 min is determined for Cu(II)-CBIMMT complex by varying the shaking
time from 0.5 to 15 min for full colour development as is shown in Fig 5. It is found that, the
colour formation was quite quick. Prolonged shaking has no any adverse effect on the
absorbance of Cu(II)-CBIMMT complex. Hence, shaking time of 1min is selected for
equilibration in subsequent experiments. Complexation reaction of copper(II) with 0.025 mol
-1
L of CBIMMT in chloroform is rapid and the colour developed is found to be stable for
more than 48 h.
3
.7 Analytical figures of merit
The performance of the present method was evaluated for the quantification of
copper(II). Under optimal experimental conditions, the system follows Beer’s law (Fig. 6) up
-1
to 17.5 µg mL of copper(II). Optimum concentration range of copper(II) is determined by
Ringbom’s plot. The curve is sigmoid with linear segment at intermediate corresponds to 5.0
to 17.5 µg mL-1 concentration values as for copper(II). The limit of detection and
quantification defined for copper(II) as LOD = 3.3 x σ/k, where k = slope of calibration plot
-1
and σ = standard deviation and LOQ = 10 x σ/k are 0.6 and 1.8 µg mL , respectively. The
-1
relative standard deviation (% R.S.D.) for five replicates measurements of 10 µg mL of
copper(II) was 0.41%. The regression equation can be expressed as y = 0.050x - 0.007, with
2
the correlation coefficient R =0.999. The regression equation shows value of intercept is
closer to the zero indicating good sensitivity. The molar absorptivity for the Cu(II)-CBIMMT
1
4

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4
complex in chloroform as calculated from Beer’s law over the range studied is 0.3381× 10 L
-1
-1
-2
mol cm and the Sandell’s sensitivity is 0.01996 µg cm . The enrichment factor of the
present method is 2.49. These results indicate that the developed method has good level of
precision and hence is found suitable for the analysis of the copper(II) from various samples.
The precision and accuracy data of the method is shown in the Table 2. The values of molar
absorptivity and Sandell’s sensitivity indicate that the method is sensitive [63].
3
.8 Stoichiometry of the Complex
The extraction of copper(II) by chelating extractant has been investigated at pH 4.2.
In order to apply liquid-liquid extraction system for the separation of copper(II) from
environmental samples and real samples, it is important to investigate the composition of
complex. Job’s method of continuous variation, the mole ratio method and log-log method
are applied to ascertain the stoichiometry composition of the Cu(II)-CBIMMT complex.
3
.8.1 Job’s continuous variation method
-1
-1
Equimolar (0.001573 mol L , 0.001179 mol L ) solutions of copper(II) and reagent,
CBIMMT is prepared. Copper(II) solution is taken and added to this acetate buffer having
pH 4.2 in a total volume of 25 mL in volumetric flask and transferred it into 125 mL
separatory funnel. The reagent, CBIMMT was mixed in complementary proportions,
containing varying amount of copper(II) and is shaken for 1 min in a separatory funnel and
then it is extracted. The absorbance of an organic extract is measured at 414 nm against the
reagent blank prepared in similar way without taking copper(II). The plot of absorbance
versus mole fraction (M/M+L) (Fig. 8) indicates that the stoichiometry of complex is 1:2
(copper(II) : CBIMMT).
3
.8.2 Mole ratio method
Different aliquots of copper(II) solution with the concentration 0.001573 mol L-1
having pH 4.2 with acetate buffer is taken. These are equilibrated with 5 mL CBIMMT
1
5

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−
1
having equimolar concentration i.e. 0.001573 mol L . To each of these solutions, the
required volume (5 mL) of chloroform is added so as to keep the final volume of organic
phase to 10 mL. The absorbance of the organic phase obtained is recorded at 414 nm. The
-1
same procedure was followed for 0.001179 mol L for both copper(II) and CBIMMT. The
graph of absorbance versus the mole ratio, M/L shows the stoichiometry of complex is 1:2
(
copper(II) : CBIMMT) (Fig. 9).
.8.3 Log-log plot method
In this method, amount of copper(II) is kept constant and acetate buffer of pH 5.6 is
added to made the volume 25 mL. The 10 mL of reagent having various concentrations
3
-1
-1
(
0.0001mol L to 0.025 mol L ) is added to it and equilibrated for 1 min as per
recommended procedure mentioned above. The absorbance of yellow coloured organic
phases is recorded at 414 nm against reagent blank. The composition of extracted Cu(II)-
CBIMMT complex is confirmed by plotting graph of Log D[Cu(II)] against Log C[CBIMMT]
(Fig.10). The plot is linear with the slope 1.6 at pH 5.6, confirming the composition of
extracted Cu(II)-CBIMMT complex is 1:2.
On the basis of the results obtained, it can be concluded that, the extracted coloured
complex is formed by combining the two ions of reagent and one ion of copper(II), resulted
into formation of 1:2 neutral species. Based on this investigation, the plausible mechanism
for the formation of Cu(II) complex is given in scheme 2.
N
H C
3
N
N
H C
3
N
N
N
N
S
pH 4.2
Cl
+
N
SH
2+
Cu
Cl
2
+
Cu
+ 2H
Cl
S
N
N
N
CH
3
N
0
.025 M CBIMMT in Chloroform
Extracted copper(II) chelate
Scheme - 2 Mechanism for formation of the Cu(II)-CBIMMT complex.
1
6

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4
. Analytical applications:
Effect of diverse ions
In order to check the selectivity of the method, excess of potentially interfering ions
relevant to industrial effluent and present in the environment have been studied by measuring
absorbance of the Cu(II)-CBIMMT complex in chloroform. The effect of foreign ions on the
determination of copper(II) is investigated by adding the known amount of these diverse ion
solutions to a standard copper(II) solution and by comparing the final absorbance with the
standard. The results presented in the Table 3. The tolerance limit of the ions does not cause
deviation of more that ± 2% in absorbance. The interference of various cations is removed by
using suitable masking agents and thus the method becomes more selective.
In order to survey the credibility of the suggested method, it is successfully applied to
the determination of copper(II) in a series of synthetic mixtures of various compositions and
also in a number of environmental samples such as food samples, leafy vegetables and water
samples. The method is also applied to the determination of copper(II) from the real samples
such as complexes and the fertilizers. In the synthetic mixtures analysis, the recovery of
copper(II) is found to be quantitative and the %R.S.D. of the analysis is satisfactory (Table
4
). In view of the unknown composition of environmental water samples, the same equivalent
portions of each such sample are analyzed for copper(II) content; the recoveries in both the
spiked (standard addition calibration used for quantization) and the unspiked samples are in
excellent agreement (Table 5). Data reports that the municipal water, bore well water and the
lake water, the amount of copper(II) lies well within the limit as recommended by WHO [8].
In dam water, the amount of copper is a little more, it may be due to the industrial waste from
the nearby industrial area around the dam. The food samples and the leafy vegetables also
contain copper(II) (Tables 6 and 7). In case of fertilizer samples analysis, the amount of
1
7

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copper(II) found is in excellent agreement with that of given by the manufacturer (Table 8).
Furthermore to check the reliability of the present method, these samples are analyzed by
AAS method for the copper(II) content. Then the method is applied for the analysis of the
copper(II) from its complexes, the % of cooper(II) is in good agreement with that of the
reported methods (Table 9). And finally, the suitability of the method is examined by
applying it to the separation and the determination of the copper(II) from various binary
mixtures. It is found that the recovery of copper(II) and of added metal ions is quantitative as
shown in the Table 10.
5
. Conclusion
This paper proposes a very effective method for rapid micro level single step
extraction and spectrophotometric quantification of copper(II) by using CBIMMT as a
metallochromic ligand and has been proved to be a selective spectrophotometric reagent. As
compared to the other methods, this method is simple, reproducible and it requires less time
for separation and determination of copper(II). Furthermore, we claim that the proposed
method is quick and of low-cost if compared with other spectrophotometric methods. Due to
the following important features, the developed method can be used for micro level
quantification of copper(II) from the real samples.
i)
The recommended procedure requires single step extraction. The yellow
coloured complex remains stable for more than 48 h.
ii)
Complexation reaction takes place at room temperature.
Complexing chromogenic reagent, CBIMMT can be synthesized easily with
high yield and purity.
iii)
iv)
v)
The method has good tolerance towards many potentially interfering ions.
The present method is reliable and the results obtained show that it is in good
agreement with reported methods.
1
8

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Acknowledgement
Authors are highly thankful to Principal Dr. P. R. Thorat for providing necessary facilities.
Authors are also thankful to Dr. V. M. Gurame for valuable suggestions during this work and
Mr. K. K. Sathe for technical and grammatical support.
1
9

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