A Rapid Spectrophotometric Method for the Determination of Copper in Real, Environmental, Biological and Soil Samples Using 1-(2-pyridylazo)-2-naphthol

Keywords: Spectrophotometry; 1-(2-pyridylazo)-2-napthol; copper determination; Environmental; biological and soil samples.

Introduction

Copper is an essential trace nutrient to all high plants and animals. In animals, including human it is found in primarily in the bloodstream, and in copper-based pigments. However, insufficient amounts; copper can be poisonous and even fatal to organisms. Copper also has a significant presence as a decorative metal art. It can also be used as an anti-germ surface that can add to the anti-bacterial and antimicrobial feature of buildings such as hospitals [1]. Copper has a high electrical and thermal conductivity, second only to silver among pure metals at room temperature [2]. On the other hand, toxic rule of the metal ion is well recognized [3]. Increasing accumulation of copper(II) in the environment through numerous industrial sources, poses danger to public health. Hence; there is a great need to develop, simple, sensitive, selective and inexpensive methods for the determination of copper in environmental, biological, soil, and industrial samples for continuous monitoring to establish the levels of Cu(II) in environmental and biological matrices.

1-(2-pyridylazo)-2-naphthol

Spectrophotometry is essentially a trace-analysis technique and is one of the most powerful tools in chemical analysis. 1-(2-pyridylazo)-2-naphthol has not previously been used for the spectrophotometric determination of copper. This paper reports on its use in a very sensitive, highly specific spectrophotometric method for the trace determination of copper. This method is far more selective, non-extractive, simple and rapid than all of the existing spectrophotometric methods [4-18]. The method is based on the reaction of non-absorbent PAN in a slightly acidic solution (0.02-0.4 mol L^-1) with copper(II) to produce a highly absorbent red chelate product followed by a direct measurement of the absorbance in an aqueous solution with suitable masking, the reaction can be made highly selective and the reagent blank solution do not show any absorbance.

Experimental Section

Instrumentation

GBC (Australia) (Model:Cintral-6) double beam UV/VIS the recording spectrophotometer and Jenway (England, U.K) (Model - 30100) pH meter with a combination of electrodes were used for the measurements of absorbance and pH respectively. Chem. Tech. Anal. (U.K.) (Model-ALPHA 4) atomic absorption spectrophotometer equipped with a microcomputer controlled air-acetylene flame at 324.7nm was used for comparing the results. (Experimental conditions were: Slit width, 2 nm; lamp current, 3 mA; wavelength, 324.7; flow rate of carrier gases are- air, 6.5 L min.^-1; acetylene, 2 L min.^-1; sample volume, 10 µL.)

Reagent and solutions

All of the chemicals used were of analytical reagent grade or the highest purity available. Doubly distilled deionized water, which is non-absorbent under ultraviolet radiation, was used throughout. Glass vessels were cleaned by soaking in acidified solution of KMnO₄ or K₂Cr₂O₇ followed by washing with concentrated HNO₃ and rinsed several times with deionized water. Stock solutions and environmental water samples (1000-mL each) were kept in polypropylene bottles containing 1-mL of concentrated HNO₃. More rigorous contamination control was applied when the copper levels in the specimens were low.

Sample collection and preservation

Water: Water samples were collected in polythene bottles from shallow tube wells, tap-well, river, sea and drain of different places of Bangladesh. After collection, HNO₃ (1 mL L^-1)was added as preservative.

Blood and Urine: Blood and urine samples were collected in polypropylene bottles from effected persons of Chittagong Medical College Hospital, Bangladesh. Immediately after collection they were stored in a salt-ice mixture and latter, at the laboratory, were kept at-20°C.

Soil: Soil (surface) samples were collected from different locations in Bangladesh. Samples were dried in air and homogenized with a mortar.

PAN solution 4x10^-3 mol L^-1

This solution was prepared by dissolving the requisite amount of 1 - (2-Pyridrylazo)-2-naphthol (Aldrich A.C.S) is a known volume of distilled deionized water. More dilute solution of the reagent was prepared as required.

Copper(II) standard solution 1.57 × 10^-2 mol L^-1

A 100-mL amount of stock solution (1 mg mL^-1) of Cu(II)was prepared by dissolving 392.9 mg of copper sulfate pentahydrate (CuSO₄. 5 H₂O) in doubly distilled deionized water. Aliquots of this solution were standardized by iodometric titration. Working standard solution was prepared by suitable dilutions of the stock solution.

Copper(I) standard solution 1.57 × 10^-2 mol L^-1

A 100-mL amount of stock solution (1 mg mL^-1) of Cu(I) was prepared by dissolving 155.7 mg of cuprous chloride (CuCl) in doubly distilled deionized water. Aliquots of this solution were standardized by iodometric titration. Working standard solution was prepared by suitable dilutions of stock solution.

Other solutions

Solutions of a large number of inorganic ions and complexing agents were prepared from their Analar grade or equivalent grade water salts (or the oxides and carbonates in hydrochloric acid), those of niobium, tantalum, titanium, zirconium and hafnium were specially prepared from their corresponding oxides (Specupure, Johnson Matthey) according to the recommended procedures [19]. In the case of insoluble substances, special dissolution methods were adopted [20].

Procedure

A volume of 0.1-1.0-mL of neutral aqueous solution containing 0.1-70 µg of copper (II) in a 10-mL volumetric flask was mixed with a 1:5 to 1:100 fold molar excess of 1-(2-pyridylazo)-2-napthol (PAN) reagent solution (preferably 1-mL of 4 x 10^-3 mol L^-1) followed by the addition of 0.1 – 2.0-mL of 0.2 mol L^-1 sulfuric acid. The mixture was diluted to the mark with deionized water. After 1 min the absorbance was measured at 560 nm against a corresponding reagent blank. The copper content in an unknown sample was determined using a concurrently prepared calibration graph.

Result and Discussion

Fig 1 · A and B absorbance spectra of Cu^II-PAN system and
the reagent blank (λ_max = 427 nm) in aqueous solutions

Factors Affecting the Absorbance

Absorption spectra
The absorption spectra of a copper (II)-PAN system in 0.2 mol L^-1 sulfuric acid medium was recorded using the spectrophotometer. The absorption spectra of the copper (II)-PAN is a asymmetric curve with maximum absorbance at 560 nm and an average molar absorption coefficient of 2.03 x 10⁴ L mol^-1 cm^-1 (Fig 1).

The reagent did not show any absorbance. In all instances measurements were made at 560 nm against a reagent blank. The reaction mechanism of the present method is as reported earlier [21].

Fig 2 · Effect of solvent on the absorbance of Cu^II-PAN system

Effect of solvent

Because PAN is partially soluble in water, an organic solvent was used for the system of the various solvents (acetone, benzene, carbon tetrachloride, chloroform, 1-butanol, isobutyl methyl ketone, methanol, ethanol and 1, 4-dioxane) studied, 1, 4-dioxane was found to be the best solvent for the system. Different volumes (0-7-mL) of 1, 4-dioxane was added to fixed metal ion concentration and the absorbance were measured according to the general procedure. It was observed that at 1 mg L^-1 Cu(II)-chelate metal, 2-6-mL 1, 4-dioxane produced a constant absorbance of the Cu-chelate (Fig 2). For all subsequent measurements, 2-mL of 1, 4-dioxane was added.

Fig 3 · Effect of the acidity on the absorbance of Cu^II-PAN system

Effect of acidity

On the various acids (nitric, sulfuric, hydrochloric and phosphoric) studied, sulfuric acid was found to be the best acid for the system. The variation of the absorbance was noted after the addition of 0.05-5.0-mL of 0.2 mol L^-1 sulfuric acid to every 10-mL of test solution. The maximum and constant absorbance was obtained in the presence of 0.1-2-mL of 0.2 mol L^-1 sulfuric acid at room temperature (25±5)°C. Outside this range of acidity, the absorbance decreased (Fig 3). For all subsequent measurements 1-mL of 0.2 mol L^-1 sulfuric acid was added.

Fig 4 · Effect of the time on the absorbance of Cu^II-PAN system

Effect of time

The reaction is very fast. A constant maximum absorbance was obtained just after dilution to volume and remained strictly constant for over 72 h (Fig 4), a longer period of time was not studied.

Fig 5 · Effect of reagent (PAN:Cu^II molar concentration ratio)
on the absorbance of Cu^II-PAN system

Effect of reagent concentration

Different molar excesses of PAN were added to a fixed metal ion concentration and the absorbance was measured according to the general procedure. It was observed that a 1 mg L^-1 of Cu metal, the reagent molar ratio of 1:5 to 1:100 produced a constant absorbance of Cu - chelate (Fig 5) for all subsequent measurements, 1-mL of 4 × 10^-3 mol L^-1 PAN reagent was added.

Fig 8 · Calibration graph C: 1-12 mg L^-1 of cu(II)

Calibration graph (Beer’s law and sensitivity)

The well known equation for a spectrophotometric analysis in a very dilute solution was derived from Beer’s law. The effect of the metal concentration was studied over 0.01-100 mg L^-1 distributed in four different sets (0.01 -0.1, 0.1-1.0, 1.0-10, 10-100 mg L^-1) for convenience of the measurement. The absorbance was linear for 0.01-7.0 mg L^-1 at 560 nm. Of the three calibration graphs one showing the limit of the linearity is given in Fig 8.

The next two are straight-line graphs passing through the origin (Fig 6 & 7).

Fig 7 · Calibration graph B: 0.1-1.0 mg L^-1 of cu(II)

Fig 6 · Calibration graph A: 0.01-0.1 mg L^-1 of cu(II)

The molar absorption co-efficient and the Sandell’s sensitivity [22] were found to be 2.03 x 10⁴ L mol^-1 cm^-1 and 9 ng mL^-1 of Cu(II). The results of optimization are shown in Table 1.

Precision and accuracy

The precision of the present method was evaluated by determining different concentration of Cu (each analyzed at least five times). The relative standard deviation (n=5) was 0-2% for 0.1-70 µg of copper in 10-mL indicating that this method is highly precise and reproducible. The detection limit (3s of the blank) and Sandell’s sensitivity (concentration for 0.001 absorbance unit) for copper(II) were found to be 4 ng mL^-1 and 9 ng mL^-1, respectively. The results for total copper(II) were in good agreement with the certified values (Table 4).

The reliability of our Cu-chelate procedure was tested by recovery studies. The average percentage recovery obtained for the addition of a copper(II) spike to some environmental water samples was quantitative, as shown in Table 5.

The method was also tested by analyzing several synthetic mixtures containing copper(II) and diverse ions (Table 3).

The result of biological samples analyses by the spectrophotometric method were in excellent agreement with those obtained by AAS Table 6.

The results of speciation of Cu(I) and Cu(II) in mixture were highly reproducible Table 8. Hence, the precision and accuracy of the method were found to be excellent.

Effect of foreign ions

The effect of over 40 ions and complexing agents on the determination of only 1 mg L^-1 of Cu(II) was studied. The criterion for interference [23] was an absorbance value varying by more than 5% from the expected value for Cu(II) alone. As can be seen, a large number of ions have no significant effect on the determination of copper. The some interference was from Co(II), V(V), Fe(II), Fe(III), Ni(II) ions. Interferences from these ions are preferably due to complex formation with PAN. The greater tolerance limits for these ions can be achieved by using several masking methods. In order to eliminate the interference of V(V), Fe(II), Co(II), Ni(II) ions tartrate, citrate, dimethylglyoxime, 1, 10- phenanthroline can be used as masking agents. A 10-fold excess of Ni(II) could be masked with dimethylglyoxime and 10-fold Fe(II) could be masked with 1,10 phenanthroline, Pb²⁺ could be masked with SO₄^2-and Mo(VI) could be masked with tartrate.

As stated above the proper masking agents may be added by while aiming at different interfering ions according to the actual composition of the sample. For this reason, the reliability of the proposed method is greatly improved and the practically is increased particularly the copper amounts in complex sample may be determined by using the proposed method. Moreover, the tolerance limit of NO₃¯, SO₄^2-, and PO₄^3- are especially high which is advantageous with respect to the digestion of samples. During interference studies, if a precipitate was formed, it was removed by centrifugation. The quantities of these diverse ions mentioned were the actual amounts added and not the tolerance limits. However, for those ions whose tolerance limits have been studied, their tolerance ratios are mentioned in Table 2.

Composition of the absorbent complex

Job’s method [24] of continuous variation and the molar-ratio method were applied to ascertain the stoichiometric composition of the complex. A Cu:PAN (1:1) complex indicated by the method

Applications

The present method was successfully applied to the determination of copper(II) in series of synthetic mixtures of various compositions (Table 3) and also in number of real samples, e.g. several standards alloys and steels (Table 4). The method was also extended to the determination of copper in a number of environmental water samples, biological and soil samples. In view of the unknown composition of environmental water samples. The same equivalent portions of each sample was analyzed for copper content, recoveries in both ‘spiked’ was (added to the samples before the mineralization and dissolution) and the ‘unspiked’ conditions are in good agreement (Table 5). The results of biological analyses by spectrophotometric method were found to be in excellent agreement with those obtained by AAS (Table 6). The results of soil samples analysis by the spectrophotometric method are shown in Table 7. The speciation of Cu(I) and Cu(II) in mixtures are in Table 8.

Determination of copper in some synthetic mixtures

Several synthetic mixtures of varying compositions containing copper(II) and diverse ions of known concentrations were determined by the present method. The results were found to be highly reproducible. The results are given in Table 3.

Determination of copper in some alloys, steels and brass (Certified Reference Materials)

A 0.1g amount of an alloy or steel or brass sample containing 60.8 - 70.61% of copper was accurately weighed and placed in a 50-mL Erlenmeyer flask. To it, 10-mL of concentrated HNO₃ and 1-mL of concentrated H₂SO₄ were carefully added and then covered with a watch-glass until the brisk reaction subsides. The solution was heated and simmered gently after the addition of another 5-mL of concentrated HNO₃ until all carbides were decomposed. The solution was carefully evaporated to dense white fumes to drive off the oxides of nitrogen and then cooled to room temperature (25±5)°C. After suitable dilution with deionized water, the contents of the Erlenmeyer flask were warmed to dissolve the soluble salts. The solution was then cooled and neutralized with a dilute NH₄OH solution in the presence of 1-2-mL of 0.01% (w/v) tartrate solution. The resulting solution filtered, if necessary, through Whatman no. 40 filter paper into a 25-mL calibrated flask. The residue (silica and tungstic acid) was washed with a small volume ( 5-mL) of hot (1:99) sulfuric acid, followed by water, the filtration and washing were collected in the same calibrated flask and the volume was made up to the mark with deionized water.

A suitable aliquot (1-2-mL) of the above solution was taken into a 10-mL calibrated flask and the copper content was determined as described under procedure using citrate or fluoride as masking agent. The results are given in Table 4. The certified copper value in alloys, steels and brass were obtained.

Determination of copper in some environmental water samples

Each filtered (with Whatman No. 40) environmental water sample (1000-mL) was evaporated nearly to dryness with a mixture of 2-mL of concentrated H₂SO₄ and 5-mL of concentrated HNO₃ to sulfur trioxide fumes. After cooling additions of 5-mL of concentrated HNO₃ was repeated and heating to a dense fume continued or until the solution became colorless. The solution was then cooled and neutralized with dilute NH₄OH in the presence of 1-2-mL of a 0.01% (w/v) tartrate solution. The resulting solution was then filtered and quantitatively transferred into a 25-mL calibrated flask and made up to the mark with deionized water.

An aliquot (1-2-mL) of this preconcentrated water sample was pipetted into a 10-mL calibrated flask and the copper content was determined as described under the procedure using citrate or fluoride as a masking agent. The results of analyses of environmental water samples from various sources for copper are given in Table 5.

Most spectrophotometric methods for the determination of copper in natural, and sea water require the preconcentration of copper [25]. The concentration of copper in natural and sea water is a few µg L^-1 in developed countries. The mean concentration of copper in natural found in U.S. drinking water is greater than 20 µg L^-1 [26].

Determination of copper in some biological samples

Regarding human blood (3-mL) and urine (20-mL), transferred into a 25-mL beaker. The sample was then ashed in a Muffle furnace at 500°C for a 4 h in the presence of 1-mL concentrated nitric acid following a method recommended by Stahr [27]. Then at the following content of each beaker were cooled at room temperature, 1.5-mL of concentrated hydrochloric acid to each beaker and warmed slightly. The content of each beaker was filtered and neutralized with dilute ammonia in the presence of 1-2-mL of 0.01% (w/v) tartrate solution, transferred quantitatively into a 10-mL calibrated flask and made up to the mark with deionized water.

A suitable aliquot (1-2-mL) of the final solution was pipetted into a 10-mL calibrated flask and the copper content was determined as described under the procedure using a fluoride or thiocyanate solution as masking agent. The results of the biological analyses by the spectrophotometric method were found to be in excellent agreement with those obtained by AAS. The results are in given in Table 5.

A deficiency of copper causes diseases such as anemia while excess of it causes “Jaundice and Wilson’s disease” [28]. An excess of copper can contribute to many symptoms: depression, spaciness, paranoia, alternating moods, anxiety, panic, fearfulness, schizophrenia, phobias, etc [29].

Determination of copper in some soil samples

An air-dried homogenized soil sample (100g) was accurately weighed and placed in a 100-mL micro-Kjeldahl flask. The sample was digested in the presence of an oxidizing agent following a method recommended by Jackson [30]. The content of flask was filtrated through Whatman No. 40 filter paper into a 25-mL calibrated flask, and neutralized with dilute ammonia in the presence of 1-2-mL of a 0.01% (w/v) tartrate solution. It was then diluted up to the mark with deionized water. A suitable aliquots (1-2-mL) were transferred into a 10-mL calibrated flask and the copper content was determined as described under procedure using fluoride or thiocyanide solution as a masking agent. The results are given in Table 7.

Determination of cu(I) and cu(II) speciation in mixture

Suitable aliquots (1-2-mL) of copper (I+II) mixtures (preferably 1:1 and 1:3) were taken in 25-mL conical flasks. A few drops of 1 mol L^-1 sulfuric acid and 1-3-mL of 1% (w/v) potassium permanganate solution were added to oxidize the monovalent copper, 5-mL of water was added to the mixtures and heated on a steam bath for 10-15 min. with occasional gentle shaking and the cooled to room temperature. Then, 3-4 drops of freshly prepared sodium azide solution (2.5% w/v) were added and gently heated with a further addition of 2-3-mL of water if necessary for 5 min. to drive off the azide, cooled to room temperature. The reaction mixture was neutralized with dilute ammonia and transferred quantitatively into a 10-mL volumetric flask; 1-mL of 4 × 10^-3 mol L^-1 PAN reagent solution was then added, followed by the addition of 1-mL of 0.2 mol L^-1 sulfuric acid was made unto the mark with deionized water. The absorbance was measured after 1 min. at 560 nm against reagent blank. The total copper content was calculated with the help of a calibration graph.

An equal aliquot of the above mentioned copper (I+II) mixture was taken into a 25- mL beaker. A 1-mL volume of 0.05 % (w/v) thiocyanide (SCN^-) was added to mask copper(I) and was neutralized with dilute NH₄OH. The content of the beaker was transferred into a 10-mL volumetric flask. Then, 1-mL of a 0.2 mol L^-1 sulfuric acid solution was added followed by the addition of 1-mL of 4 × 10^-3 mol L^-1 PAN and made up to the volume with deionized water. After 1 min. the absorbance was measured against a reagent blank as before. The copper concentration was calculated in mg L^-1 or µg mL^-1 with the aid of a calibration graph. This gave a measure of the copper(II) originally present in the mixture. This value was subtracted from that of the total copper to obtain the copper(I) present in the mixture. The results were found to be highly reproducible. The occurrences of such reproducible results are also reported for different oxidation states of copper [31]. The results of a set of determination are given in Table 8.

Conclusions

The proposed method possesses distinct advantages over the existing methods [4-18] and recently published spectrophotometric methods concerning copper [10]. First the determination of copper with the proposed color system can be directly conducted in an aqueous solution without the need for any separations or clean up step. Second, the reaction is instantaneous and the absorbance remains stable for over 72 h. Third, the useful concentration range (0.01-7.0 µg mL^-1) for Beer’s low is widened. Fourth, with suitable masking, the reaction can be made highly selective and reagent blank solutions do not show any absorbance. Finally, the results obtained in this work show that the proposed method is applicable to a variety of copper containing samples, and that the method is simple, selective and accurate. Therefore this method will be successfully applied to the monitoring of small amounts of copper in industrial, environmental, biological and soil samples.

References

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