Amperometric detection of phenolic compounds by polypyrrole-based composite carbon paste electrodes

This contribution describes new composite carbon paste electrodes (CPEs) for the determination of phenolic compounds. The composite CPEs were prepared by in situ generation of polypyrrole (PPy) within a paste containing the enzyme polyphenol oxidase (PPO). The best paste composition (enzyme/pyrrole monomer/carbon particles/Nujol) was determined for a model enzyme, glucose oxidase (GOx) according to the enzymatic activity of the resulting electrodes and to the enzyme leakage from the paste during storage in phosphate buffer. The in situ electrogenerated PPy improves the enzyme immobilisation within the paste since practically no enzyme was lost in solution after 72 h of immersion. Moreover, the enzyme activity remains particularly stable under storage since the biocomposite structure conserves 80% of its activity after 1 month of storage. Following the optimisation of the paste composition, PPO-based carbon paste biosensors were prepared and presented excellent analytical properties toward catechol detection with a sensitivity of 4.7 A M(-1) cm(-2) and a response time lower than 20 s. The resulting biosensors were applied to the determination of polyphenolic compounds (e.g., epicatechin and ferulic acid).     

Dopamine and Glucose Sensors Based on Glassy Carbon Electrodes Modified with Melanic Polymers

This work deals with the study of polymers electrogenerated from different catechols at glassy carbon electrodes and the analytical applications of the resulting modified electrodes for dopamine quantification and glucose biosensing. The electropolymerization was performed from a 3.0×10^?3 M catechol solution (catechol, dopamine, norepinephrine, epinephrine or L ‐dopa in a 0.050 M phosphate buffer pH 7.40) by applying 1.00 V for 60 min. The properties of the polymers are very dependent on the nature of the catechol, L ‐dopa being the best. Glassy carbon electrodes modified with melanic polymers electrogenerated from L ‐dopa and norepinephrine were found to be suitable for dopamine determinations in flow systems, although the behavior was highly dependent on the nature of the monomer. Detection limits of 5.0 nM dopamine and interferences of 9.0 and 2.6% for 5.0×10^?4 M ascorbic acid and 5.0×10^?5 M dopac, respectively, were obtained at the glassy carbon electrode modified with a melanin‐type polymer generated from L ‐dopa (using 1.0×10^?3 M AA in the measurement solution). The advantages of using a melanin‐type polymer generated from dopamine to improve the selectivity of glucose biosensors based on carbon paste electrodes containing Pt and glucose oxidase (GOx) are also discussed. The resulting bioelectrodes combines the high sensitivity of metallized electrodes with the selectivity given by the polymeric layer. They exhibit excellent performance for glucose with a rapid response (around 10 seconds per sample), a wide linear range (up to 2.5×10^?2 M glucose), low detection limits (143 μM) and a highly reproducible response (R.S.D of 4.9%). The bioelectrodes are highly stable and almost free from the interference of large excess of easily oxidizable compounds found in biological fluids, such as ascorbic acid (AA), uric acid (UA) and acetaminophen.     

Simultaneous Determination of Epinephrine and Dopamine by Using Candida tropicalis Yeast Cells Immobilized in a Carbon Paste Electrode Modified with Single Wall Carbon Nanotube

A new electrochemical microbial biosensor system based on Candida tropicalis was developed for the fast detecting of dopamine and epinephrine. Candida tropicalis was immobilized in a carbon paste electrode (CPE) with single wall carbon nanotube (SWCNT). Immobilized cells were used as a origin of the polyphenol oxidase (PPO) to develop voltammetric epinephrine and dopamine biosensor. Voltammetric determination of phenolic compounds such as epinephrine and dopamine a simple technique which is available. Direct oxidation of phenols can be used, but the oxidation potentials of this compounds are similar and they can not be detected distinctively. Another possibility is the use of biosensors based on the polyphenol oxidase (tyrosinase) enzyme that oxidizes the phenolic compounds into their related quinones. By this way, phenolic compounds are epinephrine and dopamine which were used in this study as well detected at different potentials. In this study differential pulse voltammetry and amperometry techniques were used for the determination of dopamine and epinephrine. The effect of varying the amounts of SWCNT and the response of microorganism to epinephrine was investigated to find the optimum composition of the sensor. The effects of pH and temperature were also examined. Increases in biosensor responses obtained by amperometric measurements were linearly related to dopamine concentrations between 0.025 and 0.25 mM and epinephrine concentrations between 0.01 and 0.1 mM. Limits of detection of the biosensor for dopamine and epinephrine were calculated to be 0.008 and 0.0023 mM, respectively. Finally, proposed system was applied to epinephrine and dopamine analysis in pharmaceutical drugs and synthetic serum and the results were compared with LC MS MS method.     

Selective Determination of Catechol in the Presence of Hydroquinone at Bare Indium Tin Oxide Electrodes via Peak‐Potential Separation and Redox Cycling by Hydrazine

The electrochemical, selective determination of catechol (CT) in the presence of hydroquinone (HQ) is not readily achieved, because the formal potentials of two phenolic compounds are very close. Here, we have developed a simple electrochemical method for the selective determination of CT by using bare indium tin oxide electrodes and employing CT redox cycling by hydrazine. The cyclic voltammetry of CT and HQ was investigated in Tris buffer (pH 9.0), phosphate buffered saline buffer (pH 7.4), and acetate buffer (pH 4.5). Especially in Tris buffer, the anodic peak potential of CT is much lower than that of HQ, resulting in a large difference between two peak potentials (ca. 0.4 V). The difference allows the selective determination of CT in the presence of excess HQ. The anodic current of CT is amplified using CT redox cycling by hydrazine, which also helps to stabilize CT and HQ in Tris buffer for several hours. The detection limits of CT in Tris buffer containing 0.1 mM HQ are 1 μM and 10 μM in the presence and absence of hydrazine, respectively.     

Microchip device for rapid screening and fingerprint identification of phenolic pollutants

A new microchip protocol has been developed for rapid measurements of the ‘total’ content of phenolic compounds, as well as for a detailed fingerprint identification of the ‘individual’ ones. The protocol involves the use of a microchip flow-injection analysis for fast screening and early detection of phenols and switching to the separation (fingerprint) mode once such compounds are detected. This is readily accomplished by exchanging the run buffers in the separation channel. While operating with an acidic run buffer (pH 5) offers high speed flow-injection measurements of the ‘total’ phenolic content, on chip switching to a basic buffer (pH 8) leads to ionization of the phenolic compounds and to their effective separation and detection. Under optimum conditions, assay rates of about 120 and 18 samples/h can be realized for the ‘total’ and ‘individual’ measurements, respectively. The effect of the buffer pH, switching (washing) time, applied voltages and other relevant variables, is described. The concept is illustrated in connection to amperometric detection and is attractive for a wide range of environmental-monitoring applications.     

The effects of alkyl sulfates on the analysis of phenolic compounds by microchip capillary electrophoresis with pulsed amperometric detection

The effects of different surfactants (sodium 2-ethylhexyl sulfate, sodium decyl sulfate, sodium dodecyl sulfate and sodium tetradecyl sulfate) on the analysis of phenolic compounds by microchip-CE with pulsed amperometric detection were investigated. Using sodium decyl sulfate as a model surfactant, the effects of concentration and pH were examined. Under the optimized conditions, the analysis of six phenolic compounds was performed and compared with control runs performed without surfactant. When these surfactants were present in the run buffer, decreases in the migration time and increases in the run-to-run reproducibility were observed. Systematic improvements in the electrochemical response for the phenolic compounds were also obtained. According to the results presented, surfactants enhance the analyte-electrode interaction and facilitate the electron transfer process. These results should allow a more rational selection of the surfactants based on their electrophoretic and electrochemical effects.     

Optimization of the separation of phenolic compounds by micellar electrokinetic capillary chromatography

A group of phenolic compounds including phenolic aldehydes, acids and flavonoids are separated by micellar electrokinetic chromatography (MECC). The influence of buffer (concentration and pH), concentration of sodium dodecylsulphate (SDS) and applied voltage were studied. To increase the selectivity of the separation and the resolution of the solutes organic solvents are added to the separation buffer, the best results were obtained when methanol was used at lower percentages. An optimized buffer (150 mM boric acid (pH 8.5)-50 mM SDS-5% methanol) provides the optimum separation with regard to resolution and migration time. This method was applied to the determination of these compounds in wine samples with good results.     

Influence of buffer composition on the capillary electrophoretic separation of carbon nanoparticles

Carbon nanoparticles obtained from the flame of an oil lamp were examined by means of capillary electrophoresis. The influence of buffer composition on the separation of the mixture of negatively charged carbon nanoparticles was studied by varying buffer selection, pH, and concentration. The electrophoretic pattern was affected by both the co- and counter-ion in the buffer solution, influencing selectivity and peak shape. The capillary electrophoretic separations at different pH revealed species with large electrophoretic mobilities under a wide range of pH. The mobility of selected species in the mixture of nanoparticles showed a strong dependence upon the solution ionic strength. The mobility of these nanoparticles as a function of ionic strength was compared to classical electrokinetic theory, suggesting that under the experimental conditions utilized, the species are small, highly charged particles with appreciable zeta potentials, even at low pH.     

Quinoprotein glucose dehydrogenase modified thick-film electrodes for the amperometric detection of phenolic compounds in flow injection analysis

The use of thick-film electrodes as basic transducers for highly sensitive amperometric biosensors using PQQ (pyrroloquinoline quinone) dependent glucose dehydrogenase (GDH) with short response times is described. The enzyme is embedded in a polyurethane matrix on top of a platinum based thick film electrode and its ability to reduce oxidized phenolic compounds is exploited. The electrochemical amplification is based on the oxidation of the analyte on the surface of the electrode followed by its enzymatic reduction. Different parameters of the glucose dehydrogenase electrode system using dopamine as a model analyte were optimized, e.g., membrane thickness, pH value, buffer system, flow rate and storage conditions. Using optimized parameters the sensitivity and detection limits for various phenolic compounds were evaluated. The comparison of electrodes from the identical as well as from different batches shows the ability to produce a number of well reproducible sensors showing remarkably small differences with respect to parameters as sensitivity, response times and measuring range.     

Characterization of graphite electrodes modified with laccase from Trametes versicolor and their use for bioelectrochemical monitoring of phenolic compounds in flow injection analysis

Spectrographic graphite electrodes were modified through adsorption with laccase from Trametes versicolor. The laccase-modified graphite electrode was used as the working electrode in an amperometric flow-through cell for monitoring phenolic compounds in a single line flow injection system. The experimental conditions for bioelectrochemical determination of catechol were studied and optimized. The relative standard deviation of the biosensor for catechol (10 μM, n=12) was 1.0% and the reproducibility for six laccase-modified graphite electrodes, prepared and used different days was about 11%. The optimal conditions for the biosensor operation were: 0.1 M citrate buffer solution ( at pH 5.0), flow rate of 0.51 ml min⁻¹ and a working potential of −50 mV versus Ag|AgCl. At these conditions the responses of the biosensor for various phenolic compounds were recorded and the sensor characteristics were calculated and compared with those known for biosensors based on laccase from Coriolus hirsutus, cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium and horseradish peroxidase (HRP).     

Electrochemical detection of phenolic estrogenic compounds at carbon nanotube-modified electrodes

The use of a carbon nanotube-modified glassy carbon electrode (CNT-GCE) for the LC-EC detection of phenolic compounds with estrogenic activity is reported. Cyclic voltammograms for phenolic endocrine disruptors and estrogenic hormones showed, in general, an enhancement of their electrochemical oxidation responses at CNT-GCE attributable to the electrocatalytic effect caused by CNTs. Hydrodynamic voltammograms obtained under flow injection conditions lead to the selection of +700 mV as the potential value to be applied for the amperometric detection of the phenolic estrogenic compounds, this value being remarkably less positive than those reported in the literature using other electrode materials. Successive injections of these compounds demonstrated that no electrode surface fouling occurred. A mobile phase consisting of a 50:50 (v/v) acetonitrile:0.05 mol l⁻¹ phosphate buffer of pH 7.0 was selected for the chromatographic separation of mixtures of these compounds, with detection limits ranging between 98 and 340 nmol l⁻¹. Good recoveries were obtained in the analysis of underground well water and tap water samples spiked with some phenolic estrogenic compounds at a 14 nmol l⁻¹ concentration level.     

Application of microchip-CE electrophoresis to follow the degradation of phenolic acids by aquatic plants

In this paper, we describe the separation and detection of six phenolic acids using an electrophoretic microchip with pulsed amperometric detection (PAD). The selected phenolic acids are particularly important because of their biological activity. The analysis of these compounds is typically performed by chromatography or standard CE coupled with a wide variety of detection modes. However, these methods are slow, labor intensive, involve a multistep solvent extraction, require skilled personnel, or use bulky and expensive instrumentation. In contrast, microchip CE offers the possibility of performing simpler, less expensive, and faster analysis. In addition, integrated devices can be custom-fabricated and incorporated with portable computers to perform on-site analysis. In the present report, the effect of the separation potential, buffer pH and composition, injection time and PAD parameters were studied in an effort to optimize both the separation and detection of these phenolic acids. Using the optimized conditions, the analysis can be performed in less than 3 min, with detection limits ranging from 0.73 microM (0.10 microg/mL) for 4-hydroxyphenylacetic acid to 2.12 microM (0.29 microg/mL) for salicylic acid. In order to demonstrate the capabilities of the device, the degradation of a mixture of these acids by two aquatic plants was followed using the optimized conditions.     

Determination of the hydrolysis rate constants and activation energy of aesculin with capillary electrophoresis end-column amperometric detection

Indirect chemiluminescence (ICL) detection for capillary electrophoresis (CE) of monoamines and catechol using luminol-K3 [Fe(CN)6] system was described. A strong and stable background chemiluminescence (CL) signal can be generated by luminol-K3 [Fe(CN)6] reaction. Based on the principle of that some phenolic compounds may be oxidized in the presence of K3 [Fe(CN)6], quenching effect of catecholamines for luminol-K3[Fe(CN)6] CL reaction results in a quantifiable decrease in the background signal. The conditions for CE separation and the CL detection for four standard catecholamines were systematically investigated using a homemade CE-ICL system. Under the optimum conditions, the detection limits of dopamine (DA), epinephrine (EP), norepinephrine (NE) and catechol (CA) were determined to be 0.18 mciroM 0.39 microM 0.48 microM and 0.09 microM, respectively. It also has been successfully applied to analyze seven pharmaceutical samples and seven human urine samples.     

Quinoprotein glucose dehydrogenase modified thick-film electrodes for the amperometric detection of phenolic compounds in flow injection analysis

The use of thick-film electrodes as basic transducers for highly sensitive amperometric biosensors using PQQ (pyrroloquinoline quinone) dependent glucose dehydrogenase (GDH) with short response times is described. The enzyme is embedded in a polyurethane matrix on top of a platinum based thick film electrode and its ability to reduce oxidized phenolic compounds is exploited. The electrochemical amplification is based on the oxidation of the analyte on the surface of the electrode followed by its enzymatic reduction. Different parameters of the glucose dehydrogenase electrode system using dopamine as a model analyte were optimized, e.g., membrane thickness, pH value, buffer system, flow rate and storage conditions. Using optimized parameters the sensitivity and detection limits for various phenolic compounds were evaluated. The comparison of electrodes from the identical as well as from different batches shows the ability to produce a number of well reproducible sensors showing remarkably small differences with respect to parameters as sensitivity, response times, and measuring range. Received: 15 August 2000 / Revised: 17 October 2000 / Accepted: 24 October 2000     

Determination of phenolic compounds in dietary supplements and tea blends containing Echinacea by liquid chromatography with coulometric electrochemical detection

Analytical methodologies with ultrasonic extraction and liquid chromatography (LC) were developed for the determination of phenolic compounds in dietary supplements containing Echinacea. The phenolic compounds determined by these methods included caftaric acid, chlorogenic acid, cynarin, echinacoside, and cichoric acid. Samples from tablets, capsules, and bags of tea blends were extracted by sonication for < or = 30 min with methanol-water (60 + 40). The extracts were centrifuged and filtered, and the filtrates were diluted and analyzed by LC using a reversed-phase column and coulometric electrochemical (EC) detection. The mobile phase was acetonitrile-ammonium formate buffer, pH 3.5 (15.3 + 84.7) containing tetrabutyl ammonium hydrogen sulfate as an ion-pairing reagent. Extraction conditions (e.g., composition of the extraction solvent and sonication time) were optimized for different types of samples. Intra- and interday analytical variations were determined, and intraday analyses were performed by 2 independent analysts using 2 different LC systems. Results were generally comparable. The LC method with EC detection showed better sensitivity and selectivity when compared with LC with ultraviolet detection, although results were similar for the 2 methods for major compounds, i.e., caftaric acid, echinacoside, and cichoric acid. The identities of these major compounds found in samples were confirmed by LC/electrospray ionization mass spectrometry.     

Catechol reactivity: Synthesis of dopamine derivatives substituted at the 6-position

Dopamine is a ubiquitous neurotransmitter essential in the proper functioning of the human body. In addition to this critical role, the catecholamine core has shown utility as a scaffold for numerous drugs and in other applications, like metal detection and adhesive materials. Substituents at the 6-position of dopamine’s catechol core can modulate its stereoelectronic properties, the acidity of its phenolic hydroxyl groups, and the overall hydrophobicity of the molecule. Herein, we report the synthesis of a series of four novel dopamine analogues substituted at the 6-position of catechol core. The ¹H NMR chemical shift of the aromatic proton meta to the substituent correlated strongly with the Hammett σ_m constant, confirming the electronic properties of substituents.     

Versatile Electrochemical Platform for the Determination of Phenol‐like Compounds Based on Laccases from Different Origins

Simple and fast methods for the monitoring of phenol‐like compounds are relevant in diverse fields ranging from waste management to neurosciences. Laccases are copper‐containing enzymes, which, depending on their origin, are able to oxidize different phenol compounds at different pH conditions. Through adequate laccase immobilization, disposable screen printed electrodes can be used as interphase to build amperometric phenol sensors. In this work three different laccases were studied for the determination of phenol‐like compounds, two of them are isoenzymes from Trametes trogii and the third one from Rhus vernicifera . Their immobilization on screen printed electrodes is presented for the construction of amperometric sensors. The electrode substrate is composed by graphite screen printed electrodes modified with carbon nanotubes and silica microspheres where, depending on the application, one of the three laccases is adsorbed. As each laccase shows an optimum working pH, they were conveniently selected to determine dopamine at physiological pH and catechol at acid pH. Determinations in the micromolar range were possible in both cases. Chronoamperometry shows to be an effective technique for their determinations, simpler than other electrochemical methods already presented in the literature.     

Flow Injection Analysis of Phenolic Compounds with Carbon Paste Electrodes Modified with Tyrosinase Purchased from Different Companies

Abstract

Tyrosinase-modified carbon paste electrodes were prepared using lyophilised powder of the enzyme purchased from different companies. The selectivity of these electrodes for nine phenolic compounds, including six substituted catechols, has been studied. The signals obtained for catechol were always higher than those found for other phenolic compounds. Cyclic voltammetry and flow injection measurements indicated that the response of the tyrosinase-modified carbon paste electrodes was limited by the rate of the enzymatic oxidation of catechols. Different approaches of paste electrode preparation have been studied and compared. Direct mixing of enzyme into the graphite powder doped with the osmium based mediator, resulted in the highest sensitivity for the studied substrates. However, substrate selectivity was found to be dependent on the source of enzyme used for electrode preparation.     

Nitrogen dioxide multiphase chemistry: uptake kinetics on aqueous solutions containing phenolic compounds

The uptake coefficients of NO2 on aqueous solutions containing guaiacol, syringol and catechol were determined over the pH range from 1 to 13 using the wetted wall flowtube technique. The measured uptake coefficients were used to determine the rate coefficients for the reaction of the physically dissolved NO2 with the neutral and deprotonated forms of phenolic compounds listed above. These organic compounds are ubiquitous not only in biomass burning plumes but also in soils, where they form part of the building blocks of humic acids. The NO2 uptake kinetics on solutions containing guaiacol, syringol or catechol were observed to be strongly pH dependent with uptake coefficients increasing from below 10(-7), under acidic conditions, to more than 10(-5) at pH values above 10. This behaviour illustrates the difference of reactivity between the neutral phenolic species and the phenoxide ions. The corresponding second order rate coefficients were typically observed to increase from 10(5) M(-1) s(-1) for the neutral compounds to a minimum of 10(8) M(-1) s(-1) for the phenoxide ions.     

Development of a tyrosinase biosensor based on gold nanoparticles-modified glassy carbon electrodes: Application to the measurement of a bioelectrochemical polyphenols index in wines

The preparation of a tyrosinase biosensor based on the immobilization of the enzyme onto a glassy carbon electrode modified with electrodeposited gold nanoparticles (Tyr-nAu-GCE) is reported. The enzyme immobilized by cross-linking with glutaraldehyde retains a high bioactivity on this electrode material. Under the optimized working variables (a Au electrodeposition potential of −200 mV for 60 s, an enzyme loading of 457 U, a detection potential of −0.10 V and a 0.1 mol l⁻¹ phosphate buffer solution of pH 7.4 as working medium) the biosensor exhibited a rapid response to the changes in the substrate concentration for all the phenolic compounds tested: phenol, catechol, caffeic acid, chlorogenic acid, gallic acid and protocatechualdehyde. A R.S.D. of 3.6% (n = 6) was obtained from the slope values of successive calibration plots for catechol with the same Tyr-nAu-GCE with no need to apply a cleaning procedure to the biosensor. The useful lifetime of one single biosensor was of at least 18 days, and a R.S.D. of 4.8% was obtained for the slope values of catechol calibration plots obtained with five different biosensors. The kinetic constants and the analytical characteristics were calculated for all the phenolic compounds tested. The Tyr-nAu-GCE was applied for the estimation of the phenolic compounds content in red and white wines. A good correlation of the results (r = 0.990) was found when they were plotted versus those obtained by using the spectrophotometric method involving the Folin-Ciocalteau reagent.