Copper hexacyanoferate film was prepared electrochemically on carbon paste electrode and was tested for detection of nitric (II) oxide in comparison with unmodified electrodes. Modified electrode could be operated under physiological conditions (pH 7.5, 0.1 M phosphate buffer), with an operating potential of ﹣400 mV (vs. Ag/AgCl) in hydrodynamic amperometry. The amperometric response of the electrode showed good linearity up to 250 μM with a detection limit (3σ) of 8.32 μM. The relative standard deviation for the repeatability of measurements for 100 μM nitric (II) oxide was 4.1% (n = 10 measurements) and the corresponding reproducibility was 14% (n = 5 electrodes). The effect of investigated interferences (nitrite and nitrate ion) was not fatal and could be eliminated using the standard addition method. The modified electrode also seems promising to detect NO in car exhaust fumes.
Carbon paste electrode (CPE) is a very useful material on electrochemical analysis, because of its biocompatibility for in vivo measurements [
The determination of NO is not a simple task because of its high reactivity; the half-life of nitric oxide in physiological conditions is around 5 seconds; therefore the required analytical methods for its spatial detection must have rapid response time [
The different modifiers were investigated on their ability to decrease the overpotential of nitric oxide on different electrode materials, such as nickel phtalocyanine, which has a catalytic effect on the NO oxidation [
Carbon paste electrode was modified in different ways, for example, to increase its catalytic effect on oxidation of nitric (II) oxide using chromium (III) oxide [
The copper hexacyanoferrate electrocatalytic effects were confirmed for hydrogen peroxide [
All used chemicals were analytical reagent grade. Sulfanilamide, N-(1-naphtyl)-ethylene diamine, copper sulphate pentahydrate, potassium hexacyanoferrate (III), Nafion® (5% m:m solution in lower aliphaic alcohols) were obtained from Sigma. Phosphate buffer solution (PBS) was prepared by mixing aqueous solutions of sodium dihydrogen phosphate (0.1 mol/L) and disodium hydrogen phosphate (0.1 mol/L) until the required pH was achieved. A nitric oxide stock solution was prepared by bubbling NO produced by the reaction of a saturated aqueous solution of sodium nitrite with 2 M sulfuric acid (Equation (1)), through a 4 M potassium hydroxide solution and finally collecting it in phosphate buffer solution (0.1 M, pH 7.5).
First, all the apparatus for NO gas production was deaerated with nitrogen gas for 30 minutes. The standard stock solution was freshly prepared before the use. The concentration of NO in the stock solution was determined using sulfanilamide and N-(1-naphtyl)-ethylenediamine as described in [
The potentiostat Autolab PSTAT 10 with software GPES version 4.9 and a potentiostat PalmSens with software PSTrace were used for cyclic voltammetry and hydrodynamic amperometry. The electrochemical cell consisted of a carbon paste electrode as the working electrode, an Ag/AgCl/3M KCl reference electrode (Metrohm 6.0733.100), and a platinum wire as the counter electrode. Nitrogen was used for degassing the solutions. A magnetic stirrer provided convection of the solution. All potentials mentioned in this paper are referred to the Ag/AgCl reference electrode. The flow injection system was assembled from a potentiostat as the detector, a high performance liquid chromatographic pump (1100 ICI instruments), a sample injection valve (5020 Rheodyne, Cotati, CA, USA), and a thin layer electrochemical detector (LC 4C, BAS, West Lafayette, Indiana, USA) with a flow through cell (spacer thickness 0.19 mm; CC-5, BAS).
Unmodified carbon paste was prepared by mixing 1.000 g graphite powder and 360 μL paraffin oil (Uvasol®, 0.84 - 0.89 kg/L,) in an agate mortar by gently stirring with a pestle until uniformity and proper compactness was obtained.
Modified carbon paste electrode was prepared by electrodeposition of copper at −0.40 V v.s. Ag/AgCl (3 M) for 60 s, after that electrode was immersed on potassium hexacyanoferrate solution 1 mM and scanned in cyclic voltammetry for 20 cycles until no difference was mention on peak currents. The prepared film electrode of copper-hexacyanoferrate was stabilized on phosphate buffer 0.1 M pH 7.5 with 5 scans and electrode was stored on phosphate buffer.
Cyclic voltammograms were scanned between 900 and −700 mV with a scan rate of 50 mV/s, unless stated otherwise. Hydrodynamic amperometric measurements were made at operating potentials of −400 mV, −300 mV, −200 mV, −100 mV and 0 mV if not mentioned otherwise; NO stock solution (0.50 - 4.00 mL) was added per step.
The copper electro deposition parameters are very important, because they effect on electrode response. Copper film (Cu-film) was prepared with potentiostatic method in 10 mM copper solution, using carbon paste electrode, as a working electrode. To find best time of Cu-film deposition (
Carbon paste electrode modified with Cu-film electrode was tested on cyclic voltammetry in phosphate buffer solution 0.1 M (pH 7.5). In
Cyclic voltammogram with scan rate of 5 mV/s of Cu-film electrode is shown in
two steps of copper (II) reduction, Cu(II) ® Cu(I) and Cu(I)®Cu˚, peak potentials at −0.10 V respectively −0.20 V. These peaks may be shown because of complex compound formation in electrode surface with phosphate ion in solution.
Copper film electrode was covered with hexacyanoferrate using cyclic volatmmetry. The copper hexacyanoferrate film was prepared at 0.1 M potassium nitrate solution containing 1 mM potassium hexacyanoferrate. Numbers of cyclic voltammetric scans were around 20, until no significant difference was noticed of hexacyanoferrate redox peaks between the last two scans.
The reduction and oxidation peaks at potential 0.50 V and 0.80 V are peaks of redox reactions of iron hexacyanoferrate FeIIIóFeII. The peak potential at −0.40 and 0.0 V corresponds to the conversion of copper from its oxidation state +2 and 0.
Based on cyclic voltammetric scans (
Carbon paste electrode modified with copper hexacyanoferrate film was tested on 0.1 M phosphate buffer solution pH 7.5 for its response to nitric (II) oxide (
The modified electrode with copper hexacyanoferrate film was tested for its response to different concentration of nitric oxide solutions. The modified electrode shows linearity up to concentration 250 µM, with sensitivity 5.696 nA/µM and correlation factor 0.994 (
The detection limit (3σ) calculated from standard deviation in hydrodynamic amperometry for 43.7 µM solution of NO was 8.32 µM (SD = 15.8, average 219.4 nA).
The main interferent compounds may come from nitrite and nitrate ions, which are also the reaction products of NO oxidation, and other compounds that are present in real samples. To avoid interferences we used sulfonil polyfluorotetraethylene (Nafion®) membrane. This anionic membrane contains negatively charged sulfonyl
groups which prevent penetration of negatively charged ions to electrode surface. The neutralized Nafion solution was diluted with alcohol, and the electrode surface was covered with 10 µL.
Based on the results shown in
Carbon paste electrode modified with copper hexacyanoferrate as an electrocatalytic mediator exhibit improved performance for the determination of nitric oxide, compared to unmodified electrodes, because the modifier lowers the over-potential and increases the sensitivity for the electrochemical reduction of the analyte. The reaction mechanism involves the reaction of copper Cu˚(I/II) and iron of hexacyanoferrate ion Fe(II/III). The suggested reaction mechanism assumes the chemical oxidation of copper Cu˚