Determination of molybdenum in biological samples by flame atomic spectrometry after preconcentration on activated carbon

Abstract  

Molybdenum is well known for its toxic effects, although it is also essential for N₂-fixing cyanobacteria and several enzymes. This study proposes a simple and accurate procedure for separation, preconcentration, and determination of trace amounts of molybdenum in biological samples by flame atomic absorption spectrometry. It is based on complexation of Mo by cupferron and sorption onto activated carbon. Effects of parameters such as pH, stirring time, and amounts of activated carbon and cupferron on recovery were examined. The results demonstrated that Mo at pH range of 2.5–3.5 was quantitatively sorbed onto activated carbon as its cupferron complex. The optimum stirring time was found to be 30 min. The relative standard deviation was found to be 12% for 200 cm³ 50 ng/cm³ Mo using 10 replicate preconcentration procedures. The limits of detection and quantification were found to be 1.0 and 3 ng/cm³, respectively, by preconcentration of 200 cm³ initial sample to 2 cm³ final volume. As a result, an enrichment factor of 100-fold was achieved. The proposed preconcentration procedure was applied to determine Mo in biological samples such as vegetables, milk, and animal liver. The molybdenum concentrations were found (as μg/dm³ or μg/kg) in the range of 70–5,500 for plants, 3–124 for milk and milk powder, and 960 for liver samples.     

Chromatographic determination of D-amino acids as native constituents of vegetables and fruits

Summary Free D-amino acids (D-AA) were detected as native constituents in juices of vegetables (cultivars of cabbage, tomato, carrot, garlic) and fruits (organes, clementine, grapefruit, lemon, apples, pear, grapes) using gas chromatography (GC) or high-performance liquid chromatography (LC). For investigation by GC, AA enantiomers were converted into theirN(O)-pentafluoropropionyl 2-propyl esters and resolved on a Chirasil-L-Val capillary column. For determination by LC, precolumn derivatization of AA enantiomers usingo-phthaldialdehyde together with the chiral thiolsN-isobutyryl-L-cysteine orN-isobutyryl-D-cysteine and fluorescence detection of the diastereomeric isoindole derivatives, resolvable on an octadecylsilyl stationary phase, were used. D-Ala (0.6–3.8%) was detected in all freshly pressed plant juices usually in the highest relative amounts. Other D-AA detected were D-Asx (0.1–1.9%), D-Glx (0–1.3%), D-Ser (0–1.7%), D-Arg (0.4–1.2%, in grapes, orange, grapefruit, and clementine) and D-Leu and D-Val (1% in cabbage). Absolute amounts of native D-AA were totally 28–57 mol L^–1 in fruit juices, 14.5 mol L^–1 in a tomato juice and 8.5 mol L^–1 in a carrot juice.Presented in part as lecture at 3rd International Congress on Amino Acids, Peptides and Analogues, August 23–27, 1993, Vienna; and as posters at 31st Scientific Meeting of German Society of Nutrition, Giessen, March 17th and 18th, 1994 [19]; and at Analytica Conference, April 19–22, 1994, Munich [20].     

Multivariate Optimization in Preconcentration Procedure for Manganese Determination in Seawater Samples by FAAS

In the present paper, a preconcentration procedure for manganese determination in seawater samples by flame atomic absorption spectrometry (FAAS) is proposed. It is based on the solid phase extraction of manganese(II) ions as a 4-(2-pyridylazo-resorcinol) (PAR) chelate using activated carbon as sorbent. Optimization of the experimental parameters (pH, activated carbon mass, PAR mass and shaking time) was carried out using a two-level full factorial design (2⁴) and two Doehlert matrix designs. The results of the factorial design, considering the analysis of variance (ANOVA), demonstrate that all these factors and their interactions (pH × PAR mass; pH × activated carbon mass and activated carbon mass × shaking time) are statistically significant. Final optimization was carried out using Doehlert matrix designs considering the results of the factorial design. The validation process evaluated the following parameters: effect of other metal ions, calibration curve, precision, accuracy and robustness. The procedure allows manganese determination in seawater samples with limits of detection (3/S) and quantification (10/S) of 16ngL^–1 and 53ngL^–1 respectively, and a preconcentration factor of 152. The analysis of certified reference materials demonstrated that the proposed procedure can also be used for manganese determination in biological samples. The procedure is not affected by matrix interferences and could be satisfactorily applied in manganese determination in seawater samples. Manganese determination in surface seawater samples collected from the shore of Salvador city, Brazil, was performed, and concentrations ranging from 0.51 to 2.59µgL^–1 were found.     

Square-wave voltammetric determination of cefoperazone in a bacterial culture,pharmaceutical drug,milk, and urine

Use of square-wave voltammetry (SWV) for determination of cefoperazone (CFPZ) in some buffers, bacterial culture, urine, and milk is described. CFPZ provides a specific voltammetric signal which is affected by pH and solution components. Determination of CFPZ in Britton–Robinson buffer, pH 4.4, is sensitive with a low detection limit (about 0.5 nmol L^–1). In a more complex medium (bacterial 2YT medium, pH 7.2) the detection limit was approximately 1.5 mol L^–1. We provide evidence that SWV is a suitable and quick method for CFPZ determination in a culture of living bacteria without separation of biomass. We have found big differences between methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) in cultivation in the presence of CFPZ, depending on time. When CFPZ is cleaved by penicillinase, a new SWV peak b appears at more positive potentials. This peak rises both with increasing concentration of enzyme and with cleavage time while the original CFPZ peak is simultaneously decreasing. We determined the concentration of CFPZ in the drug Pathozone by the standard addition method and achieved good agreement with the declared value of CFPZ in the drug. With a simple pretreatment procedure it is possible to determine CFPZ in milk; for urine no pretreatment was required. Using SWV we could detect CFPZ concentrations as low as 500 nmol L^–1 in bovine milk and human urine.     

Determination of total selenium and selenium distribution in the milk phases in commercial cow’s milk by HG-AAS

A procedure has been developed for determining the selenium in cows milk using hydride generation–atomic absorption spectrometry (HG-AAS) following microwave-assisted acid digestion. The selenium distributions in milk whey, fat and micellar casein phases were studied after separating the different phases by ultracentrifugation and determining the selenium in all of them. The detection limits obtained by HG-AAS for the whole milk, milk whey and micellar casein were 0.074, 0.065 and 0.075 g l^–1, respectively. The accuracy for the whole milk was checked by using a Certified Reference Material CRM 8435 whole milk powder from NIST, and the analytical recoveries for the milk whey and casein micelles were 100.9 and 96.9%, respectively. A mass balance study of the determination of selenium in the different milk phases was carried out, obtaining values of 95.5–100.8%. The total content of selenium was determined in 37 milk samples from 15 different manufacturers, 19 whole milk samples and 18 skimmed milk samples. The selenium levels found were within the 8.5–21 g l^–1 range. The selenium distributions in the different milk phases were studied in 14 whole milk samples, and the highest selenium levels were found in milk whey (47.2–73.6%), while the lowest level was found for the fat phase (4.8–16.2%). A strong correlation was found between the selenium levels in whole milk and the selenium levels in the milk components.     

Indirect flow-injection determination of ascorbic acid by flame atomic absorption spectrometry

A continuous-flow procedure is proposed for the indirect determination of ascorbic acid, based on its reducing properties because of the oxidation of its 1,2-enediol group. Iron(III) was injected into a 1,10-phenanthroline stream, which was mixed with a sample carrier and then with a sodium picrate solution stream. In these conditions the iron(III) was reduced to iron(II) by the ascorbic acid. Thus, the iron(II) formed reacts with 1,10-phenanthroline to form a charged red complex, which with picrate ion forms a stable red-orange uncharged ion-association complex that is adsorbed on-line on a non-ionic polymeric adsorbent (Amberlite XAD-4), proportionally to the ascorbic acid in the sample. The unadsorbed iron was determined by flame atomic absorption spectrometry. The proposed method allows the determination of ascorbic acid in the range 0.5–25 g ml^–1 with a relative standard deviation of 2.9% at a rate of ca. 90 samples h^–1. This method has been applied to the determination of ascorbic acid in pharmaceutical preparations, fruit juices and sweets. The results obtained in the analysis are compared with those provided by the 2,6-dichloroindophenol method.     

Spectrophotometric and titrimetric determination of gallium in minerals and ores using semimethylthymol blue

The composition, overall stability constant and molar absorptivity of the chelate of gallium(III) ion with semimethylthymol blue, SMTB, were determined spectrophotometrically in acetate buffer (pH 4.5–5). A violet Ga(SMTB) chelate was formed with logarithmic overall stability constant of 18.0±0.1 (I=0.1) and molar absorptivity of 4.25×10⁴l mol^–1cm^–1 (_max 580 nm). SMTB is proposed as a new reagent for the spectrophotometric determination of micro-molar amounts of gallium(III). The colour development depends on time, temperature, pH and buffer species. The interference of different cations, anions and organic acids on gallium(III) determination was also investigated. Beer's law was obeyed for 3.5–31.3 gGa(III)/25ml (0.14–1.25 g ml^–1). SMTB was used for the spectrophotometric determination of gallium in different grade minerals and ores and the results were of acceptable error and relative standard deviation. Comparison between the two suggested methods and atomic absorption spectrometry for Ga(III) determination was carried out.     

Flow Analysis of Transition Metals by Thermal Lensing

The conditions for the flow determination of Al(III), Bi(III), Cd(II), Co(II), Cr(III), Cu(II), Fe(III), Mn(II), Nd(III), Ni(II), Pb(II), Pr(III), and Zn(II) by reaction with Xylenol Orange in aqueous solutions at pH 4.5 and the determination of Cd(II), Co(II), Cu(II), Fe(II), Ni(II), Pb(II), and Zn(II) by reaction with 4-(2-thiazolylazo)resorcinol in water–ethanol mixtures (5 : 1) at pH 5.0 using an injected sample volume of 80 L were proposed. The limits of detection were n × 10^–8–n × 10^–7 mol/L; the linearity ranges in the calibration graphs were of about three orders of magnitude; the relative standard deviation was of 3–7%.     

Determination of medecamycin by adsorptive stripping voltammetry with an activated electrode

The adsorptive and electrochemical behaviors of medecamycin were investigated on a glassy carbon electrode (GCE) pretreated by anodic oxidation at +1.8 V for 5 min in 0.025 mol l^–1 NH₃-NH₄Cl (pH 8.6) solution. An adsorptive stripping voltammetric method for the determination of medecamycin at the pretreated glassy carbon electrode has been developed. Medecamycin was accumulated in NH₃-NH₄Cl buffer (pH 9.0) at a potential of –0.7 V (vs. saturated calomel electrode (SCE)) for a certain time, and then determined by second-order differential anodic stripping voltammetry. The second-order differential anodic stripping peak current at +0.72 V was proportional to the concentration of medecamycin in the range 2.0 g ml^–1 to 50.0 g ml^–1. The detection limit (three times the signal-to-noise) was 1.0 g ml^–1 and the relative standard deviation of the results was 3.28% for eight successive determinations of 10.0 g ml^–1 medecamycin. This method has been applied to the direct determination of medecamycin in commercial tablets and spiked urine samples with satisfactory results.     

Determination of ascorbic acid by differential pulse voltammetry

Summary A new approach to the determination of ascorbic acid has been realized with differential pulse voltammetry (DPV) on the carbon paste electrode (CPE). The problems of the reproducibility of the surface of this electrode have been resolved by using always the same surface after its conditioning. The developed method has been applied to the direct determination of vitamin C in pharmaceutical tablets and in different types of fruit juices. Limits of determination are 1.5 ×10^–7 M, 8×10^–7 M and 1×10^–5 M for tablets, dehydrated and liquid juices, respectively.

---

Bestimmung von Ascorbinsäure durch Differential-Puls-Voltammetrie

Zusammenfassung Als neues Verfahren zur Ascorbinsäurebestimmung wird die Differential-Puls-Voltammetrie mit Hilfe der Kohlepasteelektrode vorgestellt. Das Problem der Reproduzierbarkeit der Elektrodenoberfläche wurde dadurch gelöst, daß nach Konditionierung immer dieselbe Oberfläche verwendet wurde. Das Verfahren wurde zur Analyse von pharmazeutischen Tabletten und Fruchtsäften angewendet. Die Bestimmungsgrenzen liegen bei 1,5·10^–7 M, 8·10^–7 M und 1·10^–5 M für Tabletten, dehydratisierte bzw. flüssige Säfte.

     

Raman Spectroscopic Study of Aluminum Silicate Complexes at 20°C in Basic Solutions

Raman spectroscopic measurements were performed at ambient temperature onaqueous silica-bearing solutions (0.005 < m _Si < 0.02; 0 < pH < 14). The spectraare consistent with the formation of monomeric Si(OH)^o ₄, SiO(OH)^– ₃ andSiO₂(OH)^2– ₂ species at acid to neutral, basic, and strongly basic pH, respectively.Raman spectra of aqueous Al-bearing solutions at basic pH confirm thepredominance of the Al(OH)^– ₄ species in a wide concentration range (0.01 < m _Al < 0.1).Raman spectra of basic solutions (12.4 < pH < 14.3), containing both Al andSi, exhibit a strong decrease in intensities of SiO(OH)^– ₃, SiO₂(OH)^2– ₂, andAl(OH)^– ₄ bands in comparison with Al-free Si-bearing and Si-free Al-bearingsolutions of the same metal concentration and pH, suggesting the formation ofsoluble Al—Si complexes. The amounts of complexed Al and Si derived fromthe measurements of the Al and Si band intensities in strongly basic solutions(pH 14) are consistent with the formation, between Al(OH)^– ₄ andSiO₂(OH)^2– ₂, of the single Al—Si dimer SiAlO₃(OH)^3– ₄ according to the reactionSiO₂(OH)^2– ₂ + Al(OH)^– ₄ SiAlO₃(OH)^3– ₄ + H₂OAt lower pH ( 12.5) the changes in band intensities are consistent with theformation of several, likely more polymerized, Al—Si complexes.     

Fluorimetric determination of aluminium in water by sequential injection through column extraction

A fluorimetric procedure for the determination of aluminium with matrix removal in drinking water is proposed. The system is based both on the solid phase extraction of aluminium on a new chelating resin (XAD-4 modified by grafting salicylic acid) and the fluorimetric detection of a complex formed between 8-hydroxyquinoline-5-sulfonic acid (HQS) and Al(III), after elution of the resin by hydrochloric acid. The sorption and elution of aluminium were studied in both competitive and non-competitive conditions, varying pH, flow-rates, volume and concentration of reagents, as well as time contact. The optimised procedure allows determination of Al³⁺ at the sub-ppb level (LOD: 0.2 g L^–1 for 1 ml of sample) within a working range of 0.2–500 g L^–1. The analytical procedure was successfully employed for the determination of aluminium in drinking water during and after flocculation/coagulation treatment processes.     

Determination of phthalate monoesters in human milk,consumer milk,and infant formula by tandem mass spectrometry (LC–MS–MS)

Daily exposure of humans to phthalates may be a health risk because animal experiments have shown these compounds can affect the differentiation and function of the reproductive system. Because milk is the main source of nutrition for infants, knowledge of phthalate levels is important for exposure and risk assessment. Here we describe the development and validation of a quantitative analytical procedure for determination of phthalate metabolites in human milk. The phthalate monoesters investigated were: monomethyl phthalate (mMP), monoethyl phthalate (mEP), mono-n-butyl phthalate (mBP), monobenzyl phthalate (mBzP), mono-(2-ethylhexyl) phthalate (mEHP), and monoisononyl phthalate (mNP). The method is based on liquid extraction with a mixture of ethyl acetate and cyclohexane (95:5) followed by two-step solid-phase extraction (SPE). Detection and quantification of the phthalate monoesters were accomplished by high-pressure liquid chromatography using a Betasil phenyl column (100 mm×2.1 mm×3 m) and triple tandem mass spectrometry (LC–MS–MS). Detection limits were in the range 0.01 to 0.5 g L^–1 and method variation was from 5 to 15%. Analysis of 36 milk samples showed that all these phthalates were present, albeit at different concentrations. Median values (g L^–1) obtained were 0.11 (mMP), 0.95 (mEP), 3.5 (mBP), 0.8 (mBzP), 9.5 (mEHP), and 101 (mNP). We also analysed seven samples of consumer milk and ten samples of infant formula. Only mBP and mEHP were detected in these samples, in the ranges 0.6–3.9 g L^–1 (mBP) and 5.6–9.9 g L^–1 (mEHP).     

Spectrofluorimetric determination of aluminium in silicon and in electronic grade reagents

Summary A sensitive spectrofluorimetric method is based on the complexation of Al with the dye Mordant Black 17 or Calcon (CI 15705). The complex solution in n-propyl alcohol (1.5×10^–4 mol/l) is mixed (5:20) with 1 mol/l acetic acid — sodium acetate buffer (pH 4.8) and maintained at 40°C for 60 min. The fluorescence intensity is measured at Ex=565 nm, Em=610 nm: The calibration curve for Al was found to be linear in the range 0 to 60 ng×ml^–1 with a standard deviation of =1.5 and an Al detection limit of 3 ng×ml^–1. The interferences due to more than 40 ions were investigated; the presence of Fe³⁺, Cr³⁺, Co²⁺, Cu²⁺, Ti⁴⁺, VO²⁺, ZrO²⁺, WO ₄ ^2– , MoO ₄ ^2– , CrO ₄ ^2– and particularly F^–, must be avoided or masked during the determination of Al. The sample of silicon/silicon dioxide was treated with a mixture of conc. HF and HNO₃ (2:1), followed by digestion and distillation at 90°C to eliminate the matrix as fluorosilicic acid. A small residue of AlF₃ was decomposed with HClO₄. In SiO₂ anodized layers (25–250 g) the minimum detection of Al was estimated to be 10¹⁸ At×cm^–3, in monocrystalline silicon (25–250 mg) 10¹⁶ At×cm^–3. The method can also be employed to determine the presence of Al in the residues by distillation of a small quantity (5–10g) of many solvents or reagent solutions. Presented at: IX. Congresso Nazionale — Div Chim Anal (S.C.I.) Ferrara 1990     

Electrochemical behavior of a covalently modified glassy carbon electrode with aspartic acid and its use for voltammetric differentiation of dopamine and ascorbic acid

Aspartic acid was covalently grafted on to a glassy carbon electrode (GCE) by amine cation radical formation in the electrooxidation of the amino-containing compound. X-ray photoelectron spectroscopic (XPS) measurement and cyclic voltammetric experiments proved the aspartic acid was immobilized as a monolayer on the GCE. Electron transfer to Fe(CN)₆^4– in solution of different pH was studied by cyclic voltammetry. Changes in solution pH resulted in the variation of the charge state of the terminal group; surface pK_a values were estimated on the basis of these results. Because of electrostatic interactions between the negatively charged groups on the electrode surface and dopamine (DA) and ascorbic acid (AA), the modified electrode was used for electrochemical differentiation between DA and AA. The peak current for DA at the modified electrode was greatly enhanced and that for AA was significantly reduced, which enabled determination of DA in the presence of AA. The differential pulse voltammetric (DPV) peak current was linearly dependent on DA concentration over the range 1.8×10^–6–4.6×10^–4 mol L^–1 with slope (nA mol^–1 L) and intercept (nA) of 47.6 and 49.2, respectively. The detection limit (3) was 1.2×10^–6 mol L^–1. The high selectivity and sensitivity for dopamine was attributed to charge discrimination and analyte accumulation. The modified electrode has been used for determination of DA in samples, in the presence of AA, with satisfactory results.     

Adsorptive stripping voltammetry of selected cephalosporin antibiotics and their direct determination in urine

Accumulation of selected cephalosporin antibiotics cefazolin, cefotaxime, cefuroxime and ceftriaxone on hanging mercury drop electrode was studied. The effects of supporting electrolyte composition, pH, accumulation potential and time were examined and corresponding analytical parameters were established employing linear sweep (staircase) or differential pulse voltammetric detection. Britton-Robinson buffer of pH 4.0 for ceftriaxone and acetate buffer in the pH range 3.6–5.0 for the other three compounds were found to be the best media. Calibration curves were linear from micromolar down to nanomolar range with limits of detection 2 × 10^–9 moll^–1, 1 × 10^–9 moll^–1, 5 × 10^–10 moll^–1 and 5 × 10^–9 moll^–1 for cefazolin, cefotaxime, cefuroxime and ceftriaxone respectively, applying the accumulation times of 5 to 14 min. Direct and simple determination of cefazolin as a model compound in urine was established with no other manipulation of urine sample than dilution 1 + 500 with buffer and subsequent adsorptive stripping voltammetric determination. The detection limit of the method is 7 gml^–1 (1.5 × 10^–5 moll^–1) of cefazolin in urine, and the relative standard deviation of 9 measurements was found to be 2.8% at 23 gml^–1 of cefazolin in urine. Similar values were found for other three compounds as well. These values are comparable or even better than those obtained by high performance liquid Chromatographic methods and approve the suitability of the proposed method for urine level monitoring in clinical practice and pharmacological studies.     

TIRF-based biosensor for sensitive detection of progesterone in milk based on ultra-sensitive progesterone detection in water

We report on recent advances of our immunoassay for the hormone progesterone in cows milk. Detection is based on total internal reflectance fluorescence (TIRF), the binding-inhibition assay with an immobilized progesterone derivative, and a commercially available monoclonal antibody to progesterone as biological recognition element. The fully automated River Analyzer (RIANA) biosensor for unattended, cost-effective, and continuous monitoring of environmental pollution therefore was adapted for sensitive determination of progesterone in milk. First, the sensitivity and robustness of the existing progesterone assay for water analysis were improved, resulting in a detection limit (LOD) of only 0.2 pg mL^–1 and a quantification limit (LOQ) of only 2.0 pg mL^–1. These extraordinary results are the lowest detection and quantification limits for progesterone determination using biosensors yet reported in the literature. Second, the accurate indicator of ovulation was calibrated and detected in three different types of milk (UHT milk, fresh milk, and raw milk). For commercial milk and randomly procured raw milk nominal levels of progesterone are typically in the range 5–15 ng mL^–1. Limits of detection (LOD) achieved for added progesterone (i.e. spiked samples) were between 45.5 and 56.1 pg mL^–1 depending on milk type. Having in mind the 1:10 dilution factor, these results are still a success. For the first time a commercially available antibody was incorporated into an immunoassay for progesterone detection in bovine milk, giving a detection limit below 1 ng mL^–1 for a fully automated biosensor. Thus the outstanding progress made with this biosensor in environmental monitoring and water analysis has now been successfully adapted to milk analysis for use in the field of reproduction management.Dedicated to the memory of Wilhelm Fresenius.     

Separation of fluoride ion by extraction with triphenyltin chloride. Application for NAA of fluorine by means of 14-MeV neutrons

The separation of fluoride by extraction with toluene solution of triphenyltin chloride has been studied. Quantitative isolation of fluoride from solutions with a wide acidity range (pH 4.0–11.5) has been established. It is suggested that interferences by Ca, Mg, Fe, and Al can be avoided by masking these elements using sulfate and hydroxyde ions. Interference by phosphate ions can be overcome in a similar fashion. The halogenated species can be masked by mercury nitrate. Detection limit for fluorine determination is about 3 g for a neutron generator flux of 2·11¹¹ n·cm^–1·s^–1. A method for fluorine assay in water using a neutron generator with a detection limit of 1 ppm has been developed.     

Application of a Carbon Nanotube Modified Electrode in Anodic Stripping Voltammetry for Determination of Trace Amounts of 6-Benzylaminopurine

Carbon nanotubes were modified on the surface of a glassy carbon electrode (GC) and initially applied in semi-derivative anode stripping voltammetry for the determination of 6-benzylaminopurine. The experiments demonstrated that the presence of the carbon nanotube greatly increased the current of the oxidation peak of benzylaminopurine. Cyclic voltammetry (CV) and semi-derivative voltammetry were used in a comparative investigation into the electrochemical oxidation of benzylaminopurine with the modified electrode. Studies on the effect of pH on the peak current and potential were carried out over the pH range of 9.013.0 with the NH₃–NH₄Cl buffer solution. A pH of 10.0 was chosen as the optimum pH. Other experimental parameters, such as film thickness, accumulation potential, temperature etc. were optimized. The anodic peak current was found to be linearly related to the concentration of benzylaminopurine over the range of 4.0×10^–8 to 1.0×10^–5mol·L^–1 with a detection limit of 5.0×10^–9mol·L^–1 in an accumulation time of 5min. Interferences of some inorganic and organic species on the response have been studied.     

Amperometric nitrite sensor based on hemoglobin/colloidal gold nanoparticles immobilized on a glassy carbon electrode by a titania sol-gel film

A novel amperometric nitrite sensor was developed based on the immobilization of hemoglobin/colloidal gold nanoparticles on a glassy carbon electrode by a titania sol-gel film. The sensor shows a pair of well-defined and nearly reversible cyclic voltammogram peaks for Hb Fe(III)/Fe(II) with a formal potential (E°) of –0.370 V, and the peak-to-peak separation at 100 mV s^–1 was 66 mV vs. Ag/AgCl (3.0 M KCl) in a pH 6.9 phosphate buffer solution. The formal potential of the Hb Fe(III)/Fe(II) couple shifted linearly with pH with a slope of –50.0 mV/pH, indicating that electron transfer accompanies single-proton transportation. The sensor exhibited an excellent electrocatalytic response to the reduction of nitrite. The reduction overpotential was 0.45 V below that obtained at a colloidal gold nanoparticles/TiO₂ sol-gel film-modified GCE. The linear range for nitrite determination for the sensor was 4.0×10^–6 to 3.5×10^–4 M, with a detection limit of 1.2×10^–6 M. The stability, repeatability and selectivity of the sensor were also evaluated.