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Journal of the Chilean Chemical Society

On-line version ISSN 0717-9707

J. Chil. Chem. Soc. vol.54 no.4 Concepción Dec. 2009 

J. Chil. Chem. Soc., 54, N° 4 (2009), págs. 414-416.





1Departamento de Química de Los Materiales, Facultad de Química y Biología, Universidad de Santiago de Chile, Av. Libertador Bernardo O´Higgins, 3363 Estación Central, Santiago, Chile. e-mail:

2Departamento de Química Fundamental, VFPE, Recife-PE, Brasil.


A suitable fow-injection method for the analysis of commercial solutions of hydrogen peroxide is described. A Nafon-coated glassy carbon electrode, modifed by deposition of copper hexacyanoferrate (CuCHF), is employed as an amperometric sensor. The method allows the analysis of 45 samples per hour, with peroxide concentrations in the range of 1.25 -10.0 mM, with relative standard deviations smaller than 1%.

Key words: Nafon-coated copper hexacyanoferrate modifed electrode; FIA system; Hydrogen peroxide quantifcation.



Hydrogen peroxide is present in several biological reactions as main product of enzymatic reactions1,2 and is a very important parameter for treatment and (bio) process monitoring2-4. It is also a key species in the reactions of the troposphere and in reactions such as the catalyzed and uncatalyzed aqueous phase oxidation of SO2 and the UV-enhanced aqueous phase oxidation of organic species5. As an antiseptic, H2O2 can be compared to a preservative, since it quickly acts to kill microorganisms and has no long-term or preserving effect6; it has been used as an antimicrobial agent since the early 1800's, and is well known for its use as a topical skin application in 3% concentration7. In addition, H2O2 has been used in several important areas such as food8,9, and environmental5,10, in the industry11,12 and in clinical evaluations13,14. In food industry, hydrogen peroxide has been used as a disinfectant in milk as early as 1904 and is approved by the FDA for packaging and surface sterilization15. For antimicrobial purposes, hydrogen peroxide is used for milk treating in cheese manufacturing, thermophile microorganism free starch production6,7. It is also used as an oxidizing and reducing agent in wine, dried eggs, corn syrups and as a bleaching agent16.

The quantifcation determination of hydrogen peroxide has been carried out by a variety of analytical methods, based on titration17,18, spectrophotometry19,20, fuorimetric 21,22, chemiluminescent23, spectroscopic24,25 and electrochemical26-28 measurements. Because of its simplicity and versatility, the latter have been more widely used28,29. The use of chemically modifed electrodes based on transition metals has improved their application29,36.

We have previously described glucose biosensors from copper hexacyanoferrates (CuHCF) electrodeposited on glassy carbon electrodes36. Besides being effective for the detection of glucose, these systems could be employed for the amperometric determination of hydrogen peroxide.

In the present communication we describe a simple, fast and low-cost fow-injection procedure for the automated hydrogen peroxide quantifcation in commercial samples based on the use of Nafon-coated CuHCF deposited electrodes.



An Autolab potentiostat-galvanostat (PGSTAT30, Germany) system connected with a computer was employed. The three-compartment electrochemical cell contained a platinum auxiliary electrode, an Ag/AgCl reference electrode and a glassy carbon electrode (diameter 3.0 mm) as working electrode.

Hydrodynamic experiments were performed with a LC4B electrochemical analyzer (Bioanalytical Systems Inc., West Lafayette, IN). Flow-injection experiments were carried out with peristaltic pump, home-made injector-commutator, home-made electrochemical cell, tygon pumping tubes and connectors. For reactors and transmission lines was used a 0.8 mm i.d. polyethylene tubing with wall thickness = 0.3 mm (Fig. 1).

Reagents, solutions and samples

Concentrated solutions of H2O2 (30%), Nafon perfuorinated ion-exchange (5% solution in 90% alcohol) were supplied by Aldrich. All employed reagents were analytically pure and the solutions prepared with doubly distilled water (Millipore-Q).

The solutions used for the electrodeposition of the CuHCF flms onto the glassy carbon electrode were prepared as follows: 0.1 M CuSO4 + 0.5 M H2SO4 (solution, S1); 5 mM K3Fe(CN)6 + 0.1 M KCl (S2). A pH 5,5 buffer solution was prepared as following: 0.025 M 0.05 M K2HPO4 + 0.05 M KH2PO4 + 0.1 M KCl (S3). The last solution was used for conditioning of the CuHCF flm after activation, as well as carrier stream in the fow system (Fig. 1).

The samples used were acquired at local store, and consisted of aqueous solution of H2O2. Before being injected into fow-injection system, the samples were diluted with buffer (S3) between 500 or 2000-fold (v/v).

Hydrogen peroxide standard solutions were prepared from a stock (0.10 M) and diluted with buffer solution S3.


Preparation of the CuCHF-Nafon modifed electrode

Prior to any surface modifcation, the glassy carbon electrode was polished with alumina powder (Al2O3, 1 and 0.25 μm).

The polished electrode was then dipped into solution S1 and a cathodic current of 1 mA was applied for 2 s. The system was left in standby condition for 10 s and then scanned for one cycle between 350 and -50 mV. The deposited flm was then activated by immersing the electrode in solution S2 and applying a constant potential of 1.0 V for 200 s. After washing with water and drying, the modifed electrode was conditioned in solution S3, by applying a potential of -50 mV for 600 s, followed by voltammetric scanning between 350 and -50 mV for 10 cycles. Finally, the electrode was coated with a polymeric flm by allowing two drops of a Nafon solution, at pH 5.5, to evaporate on its surface. The obtained Nafon-coated CuHCF-modifed electrode was stable in solutions with pH values lower than 6. In alkaline media the hexacyanoferrate anions is gradually converted to the corresponding ferrous and then ferric hydroxides. Therefore this media was not considered in the present work. The electrode had a sensitivity of 2.4 and a detection limit of 0.10 mM, quite appropriate for the hydrogen peroxide concentrations employed by us (3-10% (v/v)).

The fow-injection system

Figure 1 depicts the fow-injection system employed in this work. The sample was injected into the system by a 10-cm (ca. 50 μL) loading loop L, and pushed by its carrier stream (solution S3 at 1.0 mL min-1) through conduct C and coil B towards the detector with a fxed potential (-200 mV vs. Ag/ AgCl). The passage of the sample through the electrochemical cell and the contact with the Nafon-coated CuCHF at the tip of the glassy carbon electrode produced a transient current proportional to the hydrogen peroxide content in the sample. After defning the hydrodynamic conditions, the system was applied to analysis of hydrogen peroxide in pharmaceutical formulations (aqueous solutions). Experimental precision was expressed as the relative standard deviation estimated after eleven successive analyses of typical sample (about 6% v/v hydrogen peroxide), and the accuracy was determined by the reference method17.


Figure 2 shows a typical cyclic voltammogram of the CuHCF modifed electrode. One reversible peak arising from the redox process below is observed36,37: Cu32+[Fe(CN)63-]2 + 2K+ + 2e- ↔ K2+Cu32+ [Fe(CN)64-]2. In the presence of hydrogen peroxide, Fe3+ is frst reduced electrochemically to Fe2+ which reacts chemically with the H2O2 resulting in the reduction of H2O2 to H2O and in the regeneration of the catalyst30,31.

An optimum injected volume of 50μL led to a very good linear correlation (R2 = 0.9973) between concentration (1.25 - 10.0 mM) and response current. The infuence of the fow rate on the analytical signal was investigated by varying it between 0.8 and 1.5 mL min-1, keeping the injection volume equal to 50μL. A reduction of the fow rate to values lower than 0.8 mL min-1 led to an increase of the recorded peak intensities, but this effect had the unacceptable cost of reducing the sampling rate to only 20-25 samples per hour. For values higher than 1.5 mL min-1, in spite of the high sampling frequency (>60 samples per hour), the system stability was deteriorated. In fact, standard deviations larger than 6% for a set of 10 samples were observed. Therefore, the optimum fow rate was thus fxed at 1.0 mLmin-1.

The fow-injection analysis was done using standard solutions of hydrogen peroxide in the range of 1.25 to 10.0mM in the buffered (pH 5.5) S3 solutions with an applied potential of -200mV vs. Ag/AgCl in the working electrode. Increasing the injected volume (10-100μL) led to increased signals, as expected. However, above 100μL the signal no longer increased and pronounced losses in linearity between peroxide concentration and response current were observed.

A comparison was made between the results obtained with our method and those of a standard hydrogen peroxide determination based on reference method17. This test method determines the concentration of H2O2 in aqueous solutions particularly used in textile bleaching. A specimen is acidifed with sulfuric acid and titrated with standardized potassium permanganate solution. The concentration of hydrogen peroxide is calculated using the volume and normality of the permanganate solution used17.

Table 1 lists the obtained values with both methods, showing a very good coincidence between them.

It is important to clarify that in cosmetic compositions containing hydrogen peroxide, it is necessary to stabilize this species and a great number of stabilizers for hydrogen peroxide are described, including, among others, certain organic compounds. However, the low interferant-to-analyte ratio and the very high sample dilution involved suggested problems with potentiality interfering species were likely to be minimal. Moreover, the use of Nafon membrane covering the modifed electrode also contributes for eliminating this trouble.

The long-term stability of the modifed electrode was investigated using a 2.5 mM hydrogen peroxide solution in phosphate buffer at pH 5.5 during 2-3 h. Six samples of aqueous hydrogen peroxide at pH 5 were evaluated, employing the optimal conditions described above. Figure 3 illustrates the performance of the sensor refected in the profle of the signals. For higher concentrations (signals c and d) the reproducibility of the signals decreased slightly. However, this drawback did not result in a bad quality of the analysis. With the proposed system, about 40-50 samples could be run per hour, with standard deviations smaller than 1%.


In conclusion, we have described a fast, robust and easily implemented fow-injection method for hydrogen peroxide determination based on an amperometric sensor developed from a Nafon-coated copper hexacyanoferrate-modifed electrode. The modifed electrode presents good performance with a minimum decrease in activity after 2 h. The Nafon coating avoids important interferences. Thus, covering the modifed electrode with this polymeric membrane it is possible to increase selectivity and the applicability of the sensor to different commercial samples in the food and pharmaceutical industry, and in the environmental control.


Support from DICYT-USACH is acknowledged.


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(Received: March 19, 2009 - Accepted: July 29, 2009).

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