Cobalt(II) Hematoporphyrin IX Immobilized in a Cellulose Acetate Niobium(V) Oxide Composite Membrane: Preparation and Oxygen Reduction Study

A Hematoporfirina IX [ácido (8,13,-bis(1-hidroxietil)-3,7,12,17-tetrametil-21-H-porfirina2,18-dipropionico)] foi absorvida na superfície da membrana composta orgânica/inorgânica Cel/Nb2O5. A porfirina é fortemente aderida à superfície do Nb2O5 pela interação com o grupo carboxílico --COOH da porfirina e formação da ligação --COO--Nb. A metalação do anel porfirínico é feita mergulhando-se a membrana na solução de Co(II) por algumas horas. As bandas de absorção eletrônica Q indicaram a mudança de simetria de D2h para D4h pela metalação do anel porfirínico. A análise química demonstrou que a metalação ocorreu com rendimento de práticamente 100%. O oxigênio foi reduzido na superfície do eletrodo feito com o material a um potential de pico de -390 mV em solução de KCl 1 M a pH 1, a uma temperatura de 298 K à pressão ambiente. Supõe-se que o processo de redução ocorre segundo o mecanismo de transferência de dois elétrons. Foi obtida uma resposta linear da corrente de pico catódico para concentrações do oxigenio dissolvido entre 1,5 e 15 ppm.


Introduction
Niobium pentoxide has been investigated with respect to its surface acid strength, ion exchange capacity, and use as specific sorbent for many metal ions [1][2][3] .Since the bulk oxide is normally obtained as a fine untractable powder with poor mechanical resistance, it has been dispersed on a surface of silica gel 4 .However, the mineral matrix is rigid and for some applications where molding of the material is required it is not convenient.In a recent work, the obtention and characterization of the composite membrane celulose/oxide, Cel/Nb2O5, with high degree dispersion and homogeinity of the oxide particles was described 5 .The Lewis acid sites of the dispersed oxide were shown to be reasonably stable.It is known that these Lewis acid sites can immobilize an organic acid enabling the chemical species be tightly anchored to the metal oxide by the --COO--Nb bond formation and thus, avoiding leaching from the surface as the material is immersed in a solvent 6 .
In this work the immobilization procedures of hematoporhyrin IX [8,13,-bis(1-hydroxyethyl)-3,7,12,17tetramethyl-21H-porphyrin-2,28-dipropionic acid], H2(HMP), in cellulose/niobium(V) oxide composite membrane and further metallation with cobalt (II) ion are described.The nature of the cobalt ion was firstly investigated, since it has been described that the metal is readily oxidized in air 7 .A membrane of the resulting material was adhered on a platinum electrode surface and the study of electrocatalytic reduction of dissolved oxygen by the electroactive species is also reported in this work.

Experimental
Preparations About 10 g of cellulose acetate (Aldrich, 2.5 degree of acetylation) was added to a mixture containing 31 mL of glacial acetic acid and 71 mL of acetone.The mixture was vigorously stirred for 2 h and allowed to rest for 24 h after which a viscous syroup was formed.Freshly sublimed NbCl5 (2 g) was added into 10 g of the celullose acetate syrup and the mixture stirred under a nitrogen atmosphere until complete dissolution of the metal halide which occured with evolution of gaseous HCl.The reaction flask was purged with nitrogen until almost all entrapped HCl was eliminated and could not be detected in the syrup.The test for Cl -was carried out by adding AgNO3 solution into a sample taken from the syrup and diluted with 0.1 M HNO3 solution.This viscous syroup is named as solution A.
Films of the composite membranes were prepared by spreading the solution A over a glass plate and the thickness was controlled to 200 µm by using a spacer.The film was allowed to rest for 2 h open to room atmosphere in order to evaporate most of the solvents.To promote the metal hydrolysis, the glass plate was immersed in distilled water and the film was detached from the surface.About 0.054 g (10 -4 mol) of H2(HMP) (Sigma) was dissolved in 50 mL of ethanol/water solution (9:1 v/v).The membrane of Cel/Nb2O5 with approximately 3 cm 2 was immersed in this solution and after 4 h it was washed and stored in bidistilled water protected against light.To metallate this membrane, it was immersed in 0.1 M CoCl2 aqueous solution for 5 h with occasional shaking.The membrane was washed with water and dried at 333 K.

Chemical analyses
The quantity of incorporated metal oxide in the matrix was determined by igniting the material to 1173 K for 1 h and weighing the residue as Nb2O5.
In order to determine the amount of metallated hematoporphyrin incorporated in Cel/Nb2O5, 0.1 g of the membrane was immersed in concentrated sulphuric acid and gently heated until dissolution.To analyze the metal, the solution was carefully diluted to 100 mL and neutral-ized with sodium bicarbonate aqueous solution (10% m/v) and measured on a atomic absorption spectrometer.The amount of porphyrin molecule was determined by nitrogen determination using the Kjeldhal method.The results of the elemental analyses can be summarized as follow: N = 152 ± 3 µmol g -1 (corresponding to 38 µmol g -1 of Hematoporhyrin); Co = 39.5 ± 0.8 µmol g -1 .The results indicate that porphyrin ring metallation was 100%.

Spectra measurements
The UV-Visible spectra of Cel/Nb2O5/HMP thin films, metallated and unmetallated, were obtained on a Beckman DU 640 spectrophotometer.
Electron spin resonance spectrum of the sample was obtained at 77 K, frequency modulated at 9.45 gHz.The equipment used was a Bruker 106 electron spin resonance spectrometer.

Electrochemical measurements
The electrode, consisting of a glass tube with a platinum disk fused at one end was immersed in the solution A, in which LiCl was previously dissolved (0.1 g per gram of solution A).The objective of adding this electrolyte in solution A was to obtain a porous membrane adhered on the platinum surface 5 .The solvent was evaporated in air and the film adhered electrode was washed with water.Adsorption of the porphyrin on the film was made by dipping the electrode in 10 -4 M H2(HMP) ethanol/water (9:1 v/v) solution, as described above.The metallation procedure was the same as that described above.The electrode was stored in a 1 M KCl solution.

Cyclic voltammetry study
The cyclic voltammetry studies were carried out in a cell with three electrodes: the saturated calomel electrode was the reference, the counter electrode was a platinum wire and the electrode prepared as above was the working electrode.The measurements were carried out on a PAR 273 A model instrument.

Dissolved oxygen
To measure the electrode response to dissolved oxygen, the cell was filled with 1 M KCl solution at pH 1 and saturated with pure oxygen at 298 ± 0.5 K. To change the concentration of the oxygen, argon was passed through the solution and the content of oxygen in each case determined by the Winkler method 8 .

Results and discussion
Organic compounds having carboxylate functional groups are immobilized on niobium oxide surface by a --COO--Nb bond formation 6 .Figure 1 illustrates the HMP and Co(HMP) immobilized on Cel/Nb2O5.The pKa of the protonated imino nitrogens of the porphyrin ring are 3.0 and 6.1 in water 9 and thus, insertion of the metal ion in the porphyrin ring is not difficult.The absorption spectra are shown in Fig. 2. The H2(HMP) absorbs intensely at λmax 350 nm (Soret band, corresponding to the strongly allowed transition from a1u to eg energy levels) and more weakly between 470 and 630 nm (Q bands, correponding to the transition from a2u to eg energy levels 10 ) (Fig. 2A).Upon metallation of the porphyrin ring system, a change in the spectrum (Fig. 2B) is observed.For H2(HMP) the Q bands (λmax in nm) are observed at 470, 530, 570 and 620 and, for Co(HMP) at 520 and 560.The decrease in the number of bands is assigned to the symmetry increase of the metal-lated complex relative to the free immobilized porhyrin (Fig. 1) i.e., the symmetry changed from D2h in Cel/Nb2O5/HMP to D4h in Cel/Nb2O5/Co(HMP).As the chemical analyses showed above, almost all porhyrin rings were metallated.The Soret band is broadened in the unmetallated ring due to dimerization of the porphyrin rings and it becomes narrower upon metal incorporation 9 .

ESR spectrum
It has been reported that the complex of cobalt (II) with hematoporhyrin is readly oxidized in air 7 .In order to certify that in the present case such oxidation can also occur, the esr spectrum of the Cel/Nb2O5/Co(HMP) was obtained at 77 K.The spectrum is shown in Fig. 3.
Under magnetic field below 3000 gauss, the g values are 2.573 and 2.315 which correspond to a Co II species and the signal observed between 3200 and 3500 gauss (inserted figure in detail) is an indication of the existence of molecular oxygen from air coordinated to Co II 11,12 .The calculated parameters are: g|| = 1.973 and g⊥ = 2.042; hyperfine coupling constants A|| = 14.24 x 10 -4 cm -1 and A⊥ = 15.83x 10 -4 cm -1 .

Electrochemical properties
Figure 4 shows the pulse differential voltammetry of the metallated complex between -800 and 1000 mV under oxygen (Fig. 4A) and argon (Fig. 4B) atmospheres.Under oxygen atmosphere the anodic waves show the peak maxima of higher intensity at -390 mV and of a weaker peak at -190 mV (Fig. 4A) and, under argon atmosphere the intensity of these peaks are reversed (Fig. 4B).The observed peaks correspond to the Co I → Co II oxidation process of both complexes, the more negative peak being due to the oxygen axially coordinated complex species and the less negative peak to the uncoordinated complex species.Sweeping the potential to the more positive potential region, a single peak with maximum at 823 mV due to Co II → Co III oxidation process is observed.Reversing the sweeping direction, from the more positive to more negative potentials, similar results for Co II → Co I reduction process under oxygen (Fig. 4C) and argon (Fig. 4D) atmospheres are observed.The peak corresponding to Co III → Co II process was not observed.
The catalytic reduction of dissolved oxygen is very dependent on the solution pH in which the measurements are carried out [13][14][15][16] .In the present case, the oxygen reduction at the electrode surface was studied in 1 M KCl solution and pHs between 1 and 6 at temperature of 298 K. Figure 5 shows the plot of current peak maxima against the solution pH.The current intensities with the maximum value at pH 1 exponentially decreased as the pH of the solutions was increased up to pH 6.
In a typical cyclic voltammetry experiment, the cyclic voltammogram curves of Cel/Nb2O5/Co(HMP) in the presence of oxygen (Fig. 6A) and when the solution was purged with argon (Fig. 6B) are shown.The peak that remains at about -450 mV (Fig. 6B) is presumably due to the oxygen molecule coordinated to Co(II) 11,12 which is difficult to eliminate during purging of the system with argon.Fig. 6C shows the curve obtained for the electrode made with Cel/Nb2O5 in the presence of oxygen (blank test).In this case, a peak with small current intensity is observed at -400 mV, the origin of which has not clear explanation.However, it disappeared on purging the electrochemical cell with argon.Anyway, the observed peak at -390 mV (Fig. 6A) is considerably enhanced in the presence of O2 and it is assigned to the oxygen electrocatalytic reduction at the modified electrode surface.On the Pt electrode surface under similar conditions, oxygen is reduced at nearly -570 mV 17 .
The stability of the electrode was tested by cycling the potential between -200 and -500 mV many times.Leaching of the electroactive species was not observed and the intensity of the peak current remained constant during the experiment.In another experiment, in order to test electrode response to dissolved oxygen, the electrochemical cell was alternately purged with pure argon and pure oxygen at a fixed potential of -390 mV and constant temperature of 298 K and pH 1. Bubbling oxygen into the cell (O2 in , Fig. 7) or argon (Ar in , Fig. 7) the current intensities increased  for O2 in and decreased for Ar in alternately in five sucessive cycles, the maxima and the minima remaining practically constant.Furthermore, the electrode response towards dissolved oxygen concentrations was also tested.The experiment was carried out measuring the cathodic peak current of the cyclic voltammograms for a given dissolved oxygen concentration in the cell.Figure 8 shows the cathodic peak current intensity against dissolved oxygen concentration, where a linear correlation between 1.5 and 15 ppm was observed.
Although the untreated cellulose acetate membranes normally present high rejection coefficient for electrolytes, the efficient electrochemical process at the electrode surface-solution interface is related with the facility under which the membrane is wet when immersed in the electrolyte solution 18 and also the presence of Nb2O5 in the matrix which increases its hydrophilic character.
The probable mechanism of oxygen reduction is that of a two electron transfer process 13 as described for a similar system in an homogeneous solution, according to the the following suggested mechanism 14,15 : [Co(II)O2] + e - [Co(I)]O2 [ The electroreduction of O2 by Co(II) porphyrin starts with oxygen coordination to the metal ion (Eq. 1) 19 followed by further electrochemical reduction at the electrode surface (Eq.2).The transference of two electrons by cobalt(I) ion to O2 occurs at the third stage of the reaction (Eq.3).As the last stage, Co(III) is electrochemically reduced to Co(II) and the cycle starts with coordenative adsorption of O2.

Conclusions
Cobalt(II) ion is quantitatively incorporated to the hematoporphyrin IX ring immobilized on the Cel/Nb2O5 surface.The complex is not leached from the surface even at high supporting electrolyte solution since it is immobilized   into the matrix surface by the --COO--Nb chemical bond.The cobalt-porphyrin acts as the active site oxygen reduction, presumably by a two-electron transfer mechanism.
Considering the relatively good chemical stability, the linear response of the peak current against the dissolved oxygen concentration at atmospheric pressure and ambient temperature, this metalloporphyrin-modified electrode system appears to be potentially useful for developing a new oxygen sensor.

Figure 1 .
Figure 1.Metallation of the Hematoporphrin IX immobilized on Cel/Nb2O5 with Co(II).The symmetry of the ring changes from D2h to D4h on metallation.

Figure 2 .
Figure 2. UV-Visible spectra of the membranes: A) Cel/Nb2O5/HMP, B) Cel /Nb2O5/Co(HMP).Between 450 and 650 nm the spectra were expanded since the intesities of Q bands are very weak in relation to the Soret bands.

Figure 8 .
Figure 8. Cathodic peak current intensities against concentration of dissolved oxygen concentration.Experimental conditions: 298 K, 1 M KCl solution at pH 1, ambient pressure, and electrode of Cel/Nb2O5/Co(HMP)

Figure 7 .
Figure 7. Cathodic peak currents in 1 M KCl solution at pH 1 for alternated cycles in presence of oxygen (O2 in ) and purged with argon (Ar in ) measured at -390 mV.

Figure 6 . 1 M
Figure 6.Cyclic voltammograms of Cel/Nb2O5/Co(HMP) electrode in 1 M KCl solution at pH 1 and scan rate of 20 mV s -1 : A) in dissolved oxygen solution, B) in solution purged with argon.C) unmetallated Cel/Nb2O5/HMP in dissolved oxygen solution measured in similar conditions.