ARTICLES

Improvement and discussion of the bicinchoninic acid method for copper determination in bacterial leachates and acid mine drainage

G. Debernardi^†, R. Landaeta^† and C. Carlesi^‡

^† Escuela de Ingeniería Bioquímica, Pontificia Universidad Católica de Valparaíso, Chile
^‡ Escuela de Ingeniería Química, Pontificia Universidad Católica de Valparaíso
Avenida Brasil 2147, 2362804 Valparaíso, Chile. Tel: +56 32 2273728; fax: +56 32 2273807
e-mail: carlos.carlesi@ucv.cl

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Abstract - The spectrophotometric method for the determination of cuprous ions in solution presented by Anwar et al. (2000), has since been analyzed and improved, especially with regard to interference from iron and ion species commonly found in acid mine drainage (AMD). The reduction of cupric ions in the presence of ferrous ions, not considered by Anwar et al., renders this method unfeasible for cuprous ion determination. In the absence ferrous ions, the method is simple, fast, economic, accurate and very reliable for total copper determination, even for copper concentrations as low as 5 mg/L. Iron concentrations > 5 g/L produce less than 3% error in determination of copper concentrations of 50 mg/L. Typical AMD ion concentrations do not interfere, except for aluminum which interferes significantly at ten times (x10) AMD concentration

Keywords: Copper Determination; Acid Mine Drainage; Ore Leaching; Bicinchoninic Acid.

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I. INTRODUCTION

Acidic sulfate-based solutions are the most common leaching agents used in hydrometallurgical processing. It is known that leaching operations with chloride-based solutions have faster kinetics and dissolution extent than sulfate-based solutions for primary sulfides, such as chalcopyrite. However, the high costs involved in construction materials and maintenance due to the high corrosivity of these solutions and the interference caused by chlorides in the solvent extraction process are a drawback that leads to the choice of sulfate-based solutions.

Copper ions released from the mineral to the leaching solution can form copper-chloride complexes of CuCl_X structure in highly concentrated chloride solutions. Some authors (Parker et al., 1981; Crundwell, 1988) have pointed out that those complexes could act as a second redox couple. Cu⁺²Cl_X-1 complexes can oxidize the mineral, releasing cuprous ions in the form of Cu⁺Cl_X, which can in turn be re-oxidized to cupric ion by dissolved oxygen or ferric ions. This copper-based redox couple has a higher redox potential than the Fe⁺³/Fe⁺² couple, thus explaining the higher leaching kinetics observed in chloride leaching media.

Cuprous ion occurrence is under-reported in sulfate-based solutions both in free and in complex form. It is commonly reported that cuprous ions are unstable in solutions of low chloride content due to the high oxidation rate of free cuprous ions by oxygen or ferric ions, both present in leaching operations. However, the existence of free cuprous ions in solutions with high ferrous ion content has recently been reported (Matocha et al., 2005), providing a feasible explanation of reductive chalcopyrite leaching (Hyroyoshi et al., 2001, Third et al., 2000).

Anwar et al. (2000) proposed a simple spectrophotometric method to measure the Cu⁺¹/Cu⁺² ratios in sulfate-based solutions. This method is based on the reaction between bicinchoninic acid (BCA) and cuprous ions, forming a purple complex that is easily measured by spectrophotometry. Nevertherless, laboratory results and a careful analysis of the proposed method question its feasibility in detecting cuprous ions present in leaching solutions.

In this paper we re-analyze and modify the BCA method proposed by Anwar et al., further investigating the interference produced by cations and anions commonly found in acid mine drainage (AMD) in order to enhance the method's performance.

II. MATERIALS AND METHODS

A. Reagents

Bicinchoninic acid (2.2'-Biquinoline-4.4'-dicarboxylic acid, or BCA) was purchased from Sigma. All other reagents used in this study were also of analytical grade. The BCA reagent was prepared dissolving 0.01g reactant in 100 mL of distilled water previously set at pH 12 with NaOH. Phosphate buffer 1 M (pH 6.8) was prepared by mixing 8.7 g of K₂HPO₄ and 6.8 g of KH₂PO₄ in 100 mL distilled water. A stock solution of 100 mg/L cupric ion was prepared dissolving 0.393 g CuSO₄·5H₂O in 1 L distilled water at pH 2 for total copper measurements. For total copper determination, hydroxylamine·HCl (10%) was used to reduce cupric to cuprous ion.

B. Protocol

The original protocol was modified considering the tartrate buffer unnecessary if a phosphate buffer is always present, leading to a final protocol (unless noted otherwise) as follows: Add 100 μL of copper sample (10-100 mg/L) to a 10 mL assay tube, 100 μL of Hydroxylamine·HCl 10% (for total copper measurement only), followed by 500 μL of phosphate buffer 1M. Add 1.5 mL of BCA reagent and fill to 3 mL with distilled water. The samples are the centrifuged for 5 minutes at 2500 x g in order to remove phosphate precipitates and the absorbance is read using 1 cm path length glass cuvettes at 560 nm in a visible spectrophotometer. Absorbance are reported against blanks made with distilled water at pH 2. The average of triplicate measurements is presented.

C. Leaching assays

Chalcopyrite leaching assays were performed in a 9K culture medium (Silverman and Lundgren, 1959) and a new very low jarosite forming medium (Kim et al., 200), with and without the presence of microorganisms, and under the different leaching conditions commonly used in industrial operations. Remnant solutions were analyzed for total copper concentrations via the new BCA method and via atomic absorption spectroscopy.

III. RESULTS AND DISCUSSION

A. Complex formation studies

Bicinchoninic acid forms a strong purple-colored complex with cuprous ion, with maximum absorbance at 560 nm. The final pH of the medium for complex formation must be above 5 in order to avoid the BCA precipitation which occurs at lower pH levels. A mix of acidic samples (pH 2) and BCA reagent prepared at different pH, giving a final reaction pH between 5 and 12, produced no significant variation in absorbance in unbuffered media (no phosphate) throughout an ANOVA analysis (p<0.05).

The relation between BCA/copper for full color development was assessed by increasing the volume of BCA added to the medium from 0.5 to 2.0 mL (Figure 1). For a sample of 100 uL at 100 mg/L copper, full color formation was first detected with 1.25 mL of BCA, equivalent to a BCA/Cu ratio of 2, as indicated by Anwar et al. (2000). In order to ensure full color development, a volume of 1.5 mL BCA was set.

Fig. 1: BCA/Cu ratio required for total complex formation.

A calibration curve was left at room temperature for 24 h in order to analyze complex degradation in time. Another calibration curve was made and incubated at 60°C for 3 minutes in order to establish the influence of temperature on complex formation. ANOVA analysis (p<0.05) revealed no time nor temperature effect over the original calibration curve (data not shown).

Contrary to the method reported by Anwar et al. (2000), tartrate buffer (0.5 M) was not able to fix the reaction pH in the calibration curve. Instead, the addition of phosphate buffer only fixed the reaction pH at 6.8, and did not produce any influence in absorbance measurements (data not shown).

B. Effect of copper ion on light absorbance in the visible spectrum

The relation between total copper and light absorbance was established by sampling the cupric ion stock solution at concentrations from 5 to 200 mg/L, and increasing the volume of BCA reagent added when linearity was lost (Fig. 2). The relation was found to be linear in all the concentration ranges tested depending on the volume of BCA added, obeying Beer's law with a correlation coefficient of 0.999. The molar absorptivity coefficient was 7220 M^-1·cm^-1 (on the order of that reported by Anwar et al., 2000), i.e. the following linear regression was obtained:

Absorbance = 3.79·C_Cu + 0.0074

where C_Cu represents the copper concentration in g/L.

Fig. 2: Calibration curve for total copper measured by the BCA method with different amounts of BCA volume (mL).

C. Interference by iron ions on cuprous ion determination

Iron ions are the main chemical agent used in traditional leaching operations and are a natural product of ore dissolution. Therefore, high concentrations of this element are expected to be found together with copper ions. Consequently, iron ion interferences must be thoroughly investigated.

Phosphate buffer was found to be both a good buffer for pH stabilization and a good agent for complexing iron ions and precipitating them, avoiding any effects on BCA-copper complex formation. The buffer pH was set at phosphate pKa (6.8), with an initial concentration of 1M.

The effect on total copper measurement of a copper sample of 50 mg/L containing ferric ions or ferrous ions from 1 to 5 g/L is shown in Fig. 3. Ferric ion interference is eliminated by ferric phosphate precipitation and removal by centrifugation, and the difference between both species of iron and the iron-free control in neither case is higher than 3%. This resistance is far superior to the 100 mg/L reported by Anwar et al. (2000).

Fig. 3: Interference of ferric ions (closed symbols) and ferrous ions (open symbols) on the absorbance of a 50 mg/L copper sample.

The amount of phosphate buffer needed for a good absorbance reading was also studied. The volume of phosphate added to a sample of 5 g Fe/L ranged from 100 to 500 μL and the final pH medium was measured (Fig. 4). Though iron phosphate precipitation lowers the buffer capacity of the solution, the absorbance readings were affected by less than 3%, thus a buffer concentration of 1 M was set for the protocol.

Fig 4: Effect of buffer concentration on iron interference and final pH of reaction medium.

D. Interference of other acid mine drainage (AMD) ions on cuprous ion determination.

The use of this method in environmental monitoring and leaching operations requires it to be resilient against interference caused by ions naturally present in AMD. The effects of K⁺, Mg⁺², SO₄^-2 and PO₄^-2 ions was assessed together as components of a 9K medium, and other ions were considered separately as in a typical AMD composition (mg/L): Al 125, Ca 215, Mn 88, Na 72, Zn 155 (Jenke and Diebold, 1983); Ni was also studied at 151 mg/L. These ions were assessed at two concentrations: nominal (1x) and at 10x. They were all also assessed together as an AMD effluent supplemented with 9K medium (Fig. 5).

Fig. 5: Interference of typical AMD ions, 9K medium, and 9K medium + AMD ions in total copper determination (1x: black bars, 10x, white bars.)

The effect of all ions fell inside analytic error, with the exception of Al at 10x which was found to decrease absorbance by 20%, and Na at 10x which enhanced absorbance by 5 %.

E. Feasibility of the BCA reagent for cuprous ion determination

Cuprous ions are known to be unstable in aqueous oxygenated solutions. Higher solubility and stability have been reported in highly concentrated chloride media (Hirato et al., 1987; Parker et al., 1981). Nevertheless, in our experiments and those of Anwar et al. (2000), cuprous ions can apparently exist in a sulfate-based leaching medium.

An explanation for this finding is the presence of high quantities of ferrous ions in solution. It is known that dissolved oxygen and (more strongly) ferric ions oxidize cuprous ions in solution, yielding cupric ions. But the reaction of cupric ion reduction by ferrous ions is less known (Matocha et al., 2005).

Cu⁺² + Fe⁺² ↔ Cu⁺¹ + Fe⁺³ (1)

The existence of cuprous ions in sulfate-based solutions could be related to the chemical equilibrium between species, under a Fe⁺³/Fe⁺² ratios in solution. Unfortunately, the BCA method presented here suffers from interference caused by iron species and thus removes them from solution by phosphate precipitation; this produces preferential precipitation of ferric ions, moving the equilibrium towards cuprous species.

This could be the reason for cuprous ion detection by Anwar et al. without the use of hydroxylamine. In abiotic experiments ferrous ions are only oxidized by air, so a considerable amount is expected to be found in solution. After sampling and application of the BCA method, a sufficient amount of ferrous ions still remain in the reaction media to push equilibrium towards cuprous ion formation. However, in biotic experiments after the microbial population develops, low quantities of ferrous ions are expected because of bacterial oxidation to ferric iron. Thus, minimum amounts of cuprous ions can be expected in those solutions due to chemical equilibrium, and not due to bacterial oxidation as indicated by Anwar et al. (2000).

Various attempts were made to modify the methodology to effectively detect cuprous ions in acid solutions, all of them unsuccessful.

F. Validation of total copper determinations with atomic absorption spectroscopy

Leaching assays were carried out in media with high and low presence of iron precipitates, with and without the presence of microorganisms, in highly oxidant media (ORP higher than 750 mV SHE), and in reductive conditions for the leaching process (ORP near 600 mV). The differences in total copper measurement between the BCA method and AAS were in neither case as high as 5%.

IV. CONCLUSIONS

A new simple, fast, economic and reliable method for total copper determination in acidic water has been improved. Iron concentrations as high as 5 g/L do not affect measurements beyond 3%. The interference produced by the main cations and anions present in AMD has also been measured. Neither of the considered species affects total copper readings beyond 5%. Only aluminum at 10x typical AMD concentrations affects the measurements significantly. Practical application of this method to leaching shows differences no higher than 5% compared to atomic absorption measurements.

The application of this method for cuprous ion determination in AMD has been analyzed and rejected as presented by Anwar et al. (2000). Further modifications to this method for a reliable application were unsuccessful, nevertheless the capability of the BCA reagent to directly react with cuprous ions and the necessity for a method to measure this copper species make this topic interesting for further research.

REFERENCES
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Received: January 19, 2011.
Accepted: May 16, 2011.
Recommended by Subject Editor Ricardo Gómez.