Ascorbic acid determination using a carbon paste electrode modified with iron ( III ) ions adsorbed on humic acid

An amperometric sensor was constructed, by using humic acids to immobilize Fe 3+ ions on a carbon paste electrode (CPE-HA-Fe), and used for ascorbic acid (H 2A) determination. The cyclic voltammogram of the electrode showed electrochemical response due to the Fe 3+/Fe2+ couple at E1/2=+0.78 VvsSCE, using 0.5 mol L -1 KCl and 0.2 mol L-1 acetate/0.020 mol L -1 phosphate buffer, at pH = 5.4, as supporting electrolyte. When H 2A is added to the electrolyte solution it is observed an oxidation process. The oxidation current, obtained by chronoamperommetry at +0.87 V vs SCE, is proportional to the concentration, represented by the equation I(μA) = 7.6286 [H2A] (mmol L-1) + 1.9583, r = 0.9996, for concentrations between 0.0 and 1.4 mmol L -1. The electrode showed high stability and was used for H 2A determination in a natural orange juice.


Introduction
Many efforts have been made to the development of new chemically modified electrodes, which can be used as simple and rapid analytical test for determining concentrations of important substances.Different materials, as polymers [1], oxides [2], zeolites [3], inorganic or organic films [4,5], have been used to immobilize different electroactive species on electrode surfaces to use as amperometric sensors.
Humic substances are organic compounds largely distributed in natural soil and water, representing about 80% of the organic matter in these systems [6][7][8].They result of vegetal and animal residues in advanced stages of decomposition and strongly influence the soil properties, as capacity of ions adsorption, pH, color, heat retention, permeability, and others.Humic acids are the fraction of the humic substances that are extracted by dissolution in strongly alkaline medium and precipitated in acid medium.They are formed by molecules of high molecular weigh, in a complex structure, not completely understood, but basically composed by C, H, N and O, with a more or less aromatic character.Humic acids also show a large number of acid sites, carboxylic and phenolic hydroxylic groups, which are responsible for the capacity of adsorption of cations.
Due to this property, humic acids have been used as modifiers in the construction of carbon paste electrodes (CPE-HA) [9][10][11][12][13][14][15].These electrodes were used to voltammetric detection of metal ions.The method is based on metal ion adsorption on the electrode surface at the humic acid sites, application of a negative potential to reduction and scan of potential to positive values.The current obtained due to oxidation is proportional to the metal ion concentration initially present at the solution.Electroanalytical techniques such as cyclic, square wave or anodic stripping voltammetry have been used to characterization and use of these electrodes.
But the use of humic acids as modifiers to immobilize metal cations at carbon paste electrodes to act as electrocatalysts for oxidation or reduction process of other species present in solution, and the use of the electrode as amperometric sensors for this specie, in our knowledge, was not reported in the literature.This is the objective of this work.It was described the construction of a carbon paste electrode modified with humic acids to immobilize iron(III) ions and the use as amperometric sensor to determine ascorbic acid (H 2 A).

Reagents and apparatus
L-ascorbic acid (Reagen), Fe(NO 3 ) 3. 9H 2 O (Vetec), and other chemicals were of analytical reagent grade.All electrochemical measurements were performed on a potentiostat model MQPG-01 (Microquímica).A three electrode system was used, with a platinum sheet and saturated calomel electrode (SCE) as auxiliary and reference electrodes, and a carbon paste electrode with Fe 3+ ions immobilized by use of humic acid (CPE-HA-Fe) as a working electrode.As supporting electrolyte, 0.5 mol L -1 potassium chloride (Reagen) and 0.2 mol L - 1 sodium acetate (Merck)/0.02mol L -1 sodium phosphate (Merck) buffer were used, with the pH controlled by addition of 6 mol L -1 acetic acid (Merck) or NaOH (Synth).All solutions were prepared with distilled and deionized water, in a Quimis system, and the measurements were made in N 2 (White Martins) atmosphere at 25ºC.

Preparation of the carbon paste electrode
Humic acid was extracted from a soil sample collected in a drained bog in Coronel Bicaco city, Rio Grande do Sul state, Brazil (27º42'56" S, 53º42'05" W).The soil sample was air dried and triturated.The organic matter amount was determined by heated sulphocromic solution oxidation and spectrophotometric Cr 3+ determination [16].Humic acid was extracted in accord with the standard method recommended by the International Humic Substances Society (IHSS) [8], using 1 mol L -1 NaOH for dissolution, HCl until pH=1 for precipitation, and purification with HF/HCl 1%.Ash content in the extracted humic acid was determined by calcinations at 700ºC for 5h.
Iron(III) ions were adsorbed on the humic acid by addition of the solid sample (100 mg) to 50 mL of 2x10 -2 mol L -1 Fe(NO 3 ) 3. 9H 2 O solution, at pH=6.5 (adjusted with 6 mol L -1 HNO 3 or NaOH solutions).The mixture was stirred for 10 h, and the solid was filtered, washed and dried on vacuum at 45ºC for 15 h.This material will be represented by AH-Fe.The amount of iron ions adsorbed was determined by calcinations of the solid at 750ºC for 7 h.The residue was dissolved in concentrated HNO 3 , and the volume was completed to 25 mL with 1 mol L -1 HNO 3 .The iron content was determined by atomic absorption spectrometry using a Perkin Elmer spectrometer.
Finally, the carbon paste electrode, CPE-HA-Fe, was constructed by mixing 10 mg of graphite powder (Merck), 5 mg of AH-Fe and one drop of mineral oil (Nujol).

Electrocatalytic determination of L-ascorbic acid
For the electrocatalytic studies, L-ascorbic acid was added to the supporting electrolyte solution at the desired concentrations.To analyze H 2 A in a real sample, a natural juice obtained directly from orange was used.The H 2 A concentration was determined by chronoamperometry using the CPE-HA-Fe electrode and the results were compared with the results obtained by the iodometric method [17].The measurements were repeated forty times.

Soil and humic acid characterization
The soil sample organic matter content was 7.1%, and it was obtained 0.94% of humic acid in the extraction process.Practically no ash was found in the extracted humic acids, indicating that the extraction was efficient.The content of iron ions adsorbed by the humic acid was 0.57 mmol g -1 .

Electrochemical characterization of the CPE-HA-Fe
In Fig. 1a is represented the cyclic voltammogram obtained for the CPE-HA-Fe electrode.It was observed a reversible process at E 1/2 = +0.78V vs SCE, where E 1/2 is the half-wave potential, E 1/2 = (E pa + E pc )/2, and E pa and E pc are anodic and the cathodic peak potentials.The peak separation between the two peak potentials was 0.27V, at a scan rate of 10 mV s -1 .This response may be attributed to the Fe 2+ /Fe 3+ process.No response is observed for the electrode constructed with humic acid with no Fe 3+ ions (CPE-HA) (Fig. 1c).
The half-wave potential at this positive region indicates that the Fe 2+ is the species present at the humic acid sites, indicating that there is reduction of the Fe 3+ ions when adsorbed.In studies reported on literature it was also observed the capacity of humic acids to reduce Fe 3+ ions [18].Fig. 2 shows the dependence of E 1/2 with pH.Decreasing pH, E 1/2 shifts to more positive values.The same dependence was also observed for Fe 3+ /Fe 2+ couple in solution.In cyclic voltammetric experiments using Fe(NO 3 ) 3 1 mmol L -1 at the same solution conditions, it was observed E 1/2 =-0.25 and +0.35 V vs SCE at pH values of 6.5 and 2.0, respectively.It indicates that this dependence of E 1/2 with pH is not due to specific interaction of the ion with the humic acid, and is in accord with the literature, which attributes the easier oxidation of Fe 2+ at higher pH values to the hydroxo complex formed by the Fe 3+ specie [19].Comparing E 1/2 observed for Fe 3+ /Fe 2+ couple in solution and adsorbed in the humic acid it is observed more positive values for the humic acid immobilized ions, indicating that complexation to the carboxylate groups stabilizes the Fe 2+ specie.

Electrocatalytic determination of L-ascorbic acid
When H 2 A is added to the electrolyte solution, it is observed an increase in the anodic and a little decrease in the cathodic peak current in the cyclic voltammogram (Fig. 1).It is due to electron transfer from dissolved H 2 A to oxidized immobilized Fe 3+ ions, indicating electrocatalysis of the H 2 A oxidation.Using CPE-HA electrode significant H 2 A oxidation is observed only at higher potentials (Fig. 1d).
In Fig. 3 is shown the intensity of the current observed for H 2 A oxidation as a function of the applied potential, obtained by chronoamperometry.It is observed that the electrocatalytic process begins at +0.2 V, and the current increases until +0.87 V vs SCE.At more positive potentials the current remains constant.
The current (I), obtained by chronoamperommetry, for H 2 A oxidation at a fixed potential (+0.87 V vs SCE), is shown in Fig. 4. It was obtained a linear relationship with the concentration of dissolved H 2 A in aqueous solution, described by the equation I(?A) = 7.6286 [H 2 A] (mmol L -1 ) + 1.9583, with a linear correlation coefficient r = 0.9996, for H 2 A concentrations between 0.0 and 1.4 mmol L -1 .In accord with this result, this electrode is proposed to be used as a sensor for ascorbic acid in aqueous solution.
Recovery test was performed using a natural orange juice, and the result was very good.The H 2 A concentration obtained, using chronoamperommetry, by the CPE-HA-Fe electrode was 42.1±1.8mg/100 mL, and by the use of iodometric method [17] was 42.9±2.2mg/100mL.
The CPE-HA-Fe electrode showed high stability.It was used continuously for 52 h and no decrease in the peak currents was observed.After 52 h there is a gradual decrease of the current, and after 180 h the response is not observed anymore, and is necessary renovation of the surface.immobilize Fe 3+ ions on a carbon paste electrode.The metal ion maintained the electrochemical response and the capacity of electron transfer, demonstrated by the electrocatalysis of the ascorbic acid oxidation.The high stability of the electrode and linearity of the current with the concentration of the ascorbic acid permitted the use of the electrode as an amperometric sensor.
Humic acid is an abundant material on the nature and may be obtained at low cost.This study demonstrates that humic acid is a suitable material for immobilizing metal ions on electrodes.Other electrodes may be constructed using other metal ions to be used as sensors for other species.

Figure 1 .
Figure 1.Cyclic voltammogram of the CPE-HA-Fe electrode in absence (a) and presence of 2 mmol L -1 ascorbic acid (b), and of the CPE-HA electrode in absence (c) and presence of 2 mmol L -1 ascorbic acid (d); pH = 5.4; scan rate of 10 mV s -1 .

Figure 2 .
Figure 2. Half-wave potentials (E 1/2 ) obtained, by cyclic voltammetry at scan rate of 25 mV s -1 , for the CPE-HA-Fe electrode at different pH values.

Figure 3 .
Figure 3. Current due to ascorbic acid oxidation at CPE-HA-Fe electrode, as a function of the applied potential, obtained by chronoamperometry, in 2 mmol L -1 ascorbic acid at pH = 5.4.

Figure 4 .
Figure 4. Current due to ascorbic acid oxidation at CPE-HA-Fe electrode, as a function of ascorbic acid concentration, obtained by chronoamperometry at +0.87 V vs SCE; pH = 5.4.