Development of a non-titration potentiometric method for the determination of acidity in vinegar.

The acidity of a solution is associated with the concentration of Brønsted acids. This work proposes a new non-titrimetric potentiometric method using citrate buffer for the determination of vinegar acidity. The difference between the pH values before and after the addition of a diluted vinegar sample to 10 mmol L-1 citrate buffer (pH 5.5) was related to the acetic acid concentration. The dynamic range of the quadratic analytical curve was from 3.5 to 20 mmol L-1 (R2 = 0.998). The repeatability was 0.8% for acetic acid at 0.01 mol L-1. Comparison with the conventional titration method showed an error between 0.7% and 4.64% (n = 9) for analysis of commercial vinegar samples The behaviour of the system could be explained using the buffering function.

Locust bean gum hydrogels are bioadhesive and improve indole-3-carbinol cutaneous permeation: influence of the polysaccharide concentration

Abstract The locust bean gum (LBG) is a polysaccharide with thickening, stabilizing and gelling properties and it has been used in the preparation of pharmaceutical formulations. Hydrogels (HGs) are obtained from natural or synthetic materials that present interesting properties for skin application. This study aimed to develop HGs from LBG using indole-3-carbinol (I3C) as an asset model for cutaneous application. HGs were prepared by dispersing LBG (2%, 3% and 4% w/v) directly in cold water. The formulations showed content close to 0.5 mg/g (HPLC) and pH ranging from 7.25 to 7.41 (potentiometry). The spreadability factor (parallel plate method) was inversely proportional to LBG concentration. The rheological evaluation (rotational viscometer) demonstrated a non-Newtonian pseudoplastic flow behavior (Ostwald De Weale model), which is interesting for cutaneous application. The HET-CAM evaluation showed the non-irritating characteristic of the formulations. The bioadhesive potential demonstrated bioadhesion in a concentration-dependent manner. Permeation in human skin using Franz cells showed that the highest LBG concentration improved the skin distribution profile with greater I3C amounts in the viable skin layers. The present study demonstrated the feasibility of preparing HGs with LBG and the formulation with the highest polymer concentration was the most promising to transport active ingredients through the skin.

TBISTAT: An open-source, wireless portable, electrochemical impedance spectroscopy capable potentiostat for the point-of-care detection of S100B in plasma samples.

Point-of-Care (POC) testing for biomarker detection demands techniques that are easy to use, readily available, low-cost, and with rapid response times. This paper describes the development of a fully open-source, modular, wireless, battery-powered, smartphone-controlled, low-cost potentiostat capable of conducting electrochemical impedance spectroscopy for the electrochemical detection of the S100B protein captured in an ANTI-S100B functionalized thin-film gold interdigitated electrode platform to support traumatic brain injury diagnosis and treatment. EIS results from the developed potentiostat were validated with a commercial benchtop potentiostat by comparing impedance magnitude and phase values along the EIS frequency range. In addition, an experimental design was performed for detecting S100B in spiked human plasma samples with S100B concentrations of clinical utility, and a calibration curve was found for quantifying S100B detection. No statistically significant differences were found between EIS results from the developed potentiostat and the commercial potentiostat. Statistically significant differences in the changes in charge transfer resistance signal between each tested S100B concentration (p < 0.05) were found, with a limit of detection of 35.73 pg/mL. The modularity of the proposed potentiostat allows easier component changes according to the application demands in power, frequency excitation ranges, wireless communication protocol, signal amplification and transduction, precision, and sampling frequency of ADC, among others, when compared to state-of-the-art open-source EIS potentiostats. In addition, the use of minimal, easy acquirable open-source hardware and software, high-level filtering, accurate ADC, Fast Fourier Transform with low spectral leakage, wireless communication, and the simple user interface provides a framework for facilitating EIS analysis and developing new affordable instrumentation for POC biosensors integrated systems.

Dinuclear copper(II) complexes with derivative triazine ligands as biomimetic models for catechol oxidases and nucleases.

The research reported herein focuses on the synthesis of two new Cu(II) complexes {[Cu2(2-X-4,6-bis(di-2-picolylamino)-1,3,5-triazine], with X = butane-1,4-diamine (2) or N-methylpyrenylbutane-1,4-diamine (3)}, the latter with a pyrene group as a possible DNA intercalating agent. The structure of complex (3) was determined by X-ray crystallography and shows the dinuclear {CuII(µ-OCH3)2CuII} unit in which the CuII···CuII distance of 3.040 Å is similar to that of 2.97 Å previously found for 1, which contains a {CuII(µ-OH)2CuII} structural unit. Complexes (2) and (3) were also characterized in spectroscopic and electrochemical studies, and catecholase-like activity were performed for both complexes. The kinetic parameters obtained for the oxidation of the model substrate 3,5-di-tert-butylcatechol revealed that the insertion of the spacer butane-1,4-diamine and the pyrene group strongly contributes to increasing the catalytic efficiency of these systems. In fact, Kass becomes significantly higher, indicating that these groups influence the interaction between the complex and the substrate. These complexes also show DNA cleavage under mild conditions with moderate reaction times. The rate of cleavage (kcat) indicated that the presence of butane-1,4-diamine and pyrene increased the activity of both complexes. The reaction mechanism seems to have oxidative and hydrolytic features and the effect of DNA groove binding compounds and circular dichroism indicate that all complexes interact with plasmid DNA through the minor groove. High-resolution DNA cleavage assays provide information on the interaction mechanism and for complex (2) a specificity for the unpaired hairpin region containing thymine bases was observed, in contrast to (3).

Electrochemical Detection of dsDNA-Specific Antibodies.

Electrochemical biosensors have shown great promise as useful point-of-care tests since they operate on electronic circuits which can be miniaturized and whose readout process can be easily automated. Here, we describe a method for the electrochemical sensing of antibodies directed against double-stranded DNA (α-dsDNA), which are often present at higher-than-normal levels in the sera of autoimmune disease patients. The method can be easily implemented in any lab and requires little investment in equipment, namely a potentiostat. An artificial reference serum sample containing known amounts of spiked-in α-dsDNA antibodies enables reporting results in absolute scale rather than titer. Once electrodes are modified with DNA and the calibration curves are made (i.e., after the biosensor construction phase), individual measurements in test samples can be obtained in as low as 35 min.

Relationship between the Polymeric Ionization Degree and Powder and Surface Properties in Materials Derived from Poly(maleic anhydride-alt-octadecene).

Polymeric materials derived from poly(maleic anhydride-alt-octadecene)-here referred as PAM-18-have shown interesting properties that make them potential pharmaceutical excipients. In this work, eight polymers derived from PAM-18 were obtained using NaOH and KOH at 1:1; 1:0.75, 1:0.5, and 1:0.25 molar ratios. The resulting products were labeled as PAM-18Na and PAM-18K, respectively. Each polymer was purified by ultrafiltration/lyophilization, and the ionization degree was determined by potentiometric studies, which was related to the zeta potential. The structural characterization was performed using the Fourier transform infrared (FT-IR) espectroscopy, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and X-ray diffraction (XRD) techniques. The physical characterization was carried out by SEM, particle analysis, and humidity loss and gain studies; the surface studies were performed by the sessile drop method. PAM-18Na had ionization degrees of 95%, 63%, 39% and 22%, whereas those for PAM-18K were 99%, 52%, 35% and 20%, respectively. The results also showed that for higher inorganic base amounts used, the polymeric materials obtained possess high ionization degrees, which could form polymeric solutions or hetero-dispersed systems. Likewise, it was observed that for higher proportions of carboxylate groups in the polymeric structure, the capability to retain water is increased and, only can be eliminated by drying at temperatures greater than 160 °C. On the other hand, the modification of PAM-18 to its ionized forms led to the formation of powder materials with low flowability and surfaces that ranged from very hydrophobic to slightly wettable.

Potentiometric perchlorate determination at nanomolar concentrations in vegetables.

In this work, an expeditious method based on the multi-commutated flow-analysis concept with potentiometric detection is proposed to perform determinations of the emergent contaminant perchlorate in vegetable matrices down to nanomolar concentration. To accomplish the task, a tubular shaped potentiometric sensor selective to perchlorate ion was constructed with a PVC membrane containing 12mmol/kg of the polyamine bisnaphthalimidopropyl-4,4'-diaminodiphenylmethane and 2-nitrophenyl phenyl ether 68% (w/w) as plasticizer casted on a conductive epoxy resin. Under optimal flow conditions, the sensor responded linearly in the concentration range of 6.3×10-7-1.0×10-3mol/L perchlorate. In order to extend the determinations to lower concentrations (4.6(±1.3)×10-10mol/L perchlorate), a column packed with 70mg of sodium 2,5,8,11,14-pentaoxa-1-silacyclotetradecane-polymer was coupled to the flow-system thus enabling prior pre-concentration of the perchlorate. The proposed procedure provides a simpler alternative for the determination of perchlorate in foods, nowadays only allowed by sophisticated and expensive equipment and laborious methods.

Label-free potentiometric biosensor based on solid-contact for determination of total phenols in honey and propolis.

We developed a label-free potentiometric biosensor using tyrosinase extracted from Musa acuminata and immobilized by covalent bond on a surface of a solid-contact transducer. The transducer was manufactured containing two layers. The first layer contained a blend of poly(vinyl) chloride carboxylated (PVC-COOH), graphite and potassium permanganate. On this layer, we deposited a second layer containing just a mixture of poly(vinyl chloride) carboxylated and graphite. On the last layer of the transducer, we immobilized the tyrosinase enzyme by reaction with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride. The solid-contact potentiometric biosensor presented at low detection limit of 7.3×10-7M and a linear range to catechol concentration between 9.3×10-7M and 8.3×10-2M. This biosensor was applied to determine the amount of total phenols in different samples of honey and propolis. The results agreed with the Folin-Ciocalteu method.

Parameter space of experimental chaotic circuits with high-precision control parameters.

We report high-resolution measurements that experimentally confirm a spiral cascade structure and a scaling relationship of shrimps in the Chua's circuit. Circuits constructed using this component allow for a comprehensive characterization of the circuit behaviors through high resolution parameter spaces. To illustrate the power of our technological development for the creation and the study of chaotic circuits, we constructed a Chua circuit and study its high resolution parameter space. The reliability and stability of the designed component allowed us to obtain data for long periods of time (∼21 weeks), a data set from which an accurate estimation of Lyapunov exponents for the circuit characterization was possible. Moreover, this data, rigorously characterized by the Lyapunov exponents, allows us to reassure experimentally that the shrimps, stable islands embedded in a domain of chaos in the parameter spaces, can be observed in the laboratory. Finally, we confirm that their sizes decay exponentially with the period of the attractor, a result expected to be found in maps of the quadratic family.

Determinação de fluoreto em baixas concentrações: validação de método com eletrodo íon seletivo para análise da água utilizada na preparação de soluções de diálise / Determination of fluoride at low concentrations: validation of method using ion selective electrode to analyze the water used in the preparation of dialysis solutions

Este trabalho descreve o desenvolvimento e validação de metodologia analítica para determinar a concentração de fluoreto em água empregada para a preparação de soluções de diálise, por meio de potenciometria com eletrodo íon seletivo. Os parâmetros de validação investigados foram: seletividade, homoscedasticidade, linearidade, limite de detecção, limite de quantificação, veracidade de medição e precisão. As condições otimizadas de análise foram: tampão HOAc/-OAc/NaCl/CDTA (pH = 5,0 ± 0,1), na proporção 10:1 (amostra/tampão); concentrações das soluções-padrão da curva analítica: 0,05 a 0,80 mg/L. O método avaliado exibiu parâmetros de validação adequados com limites de detecção e de quantificação, respectivamente, de 0,020 e 0,050 mg/L. Ademais, foi também desenvolvida e validada uma planilha eletrônica para efetuar o monitoramento da qualidade da curva analítica do método que calcula o limite de decisão para 0,20 mg/L...

Fluoride removal in water by a hybrid adsorbent lanthanum-carbon.

Various health problems associated with drinking water containing high fluoride levels, have motivated researchers to develop more efficient adsorbents to remove fluoride from water for beneficial concentrations to human health. The objective of this research was to anchor lanthanum oxyhydroxides on a commercial granular activated carbon (GAC) to remove fluoride from water considering the effect of the solution pH, and the presence of co-existing anions and organic matter. The activated carbon was modified with lanthanum oxyhydroxides by impregnation. SEM and XRD were performed in order to determine the crystal structure and morphology of the La(III) particles anchored on the GAC surface. FT-IR and pK(a)'s distribution were determined in order to elucidate both the possible mechanism of the lanthanum anchorage on the activated carbon surface and the fluoride adsorption mechanism on the modified material. The results showed that lanthanum ions prefer binding to carboxyl and phenolic groups on the activated carbon surface. Potentiometric titrations revealed that the modified carbon (GAC-La) possesses positive charge at a pH lower than 9. The adsorption capacity of the modified GAC increased five times in contrast to an unmodified GAC adsorption capacity at an initial F(-) concentration of 20 mg L(-1). Moreover, the presence of co-existing anions had no effect on the fluoride adsorption capacity at concentrations below 30 mg L(-1), that indicated high F(-) affinity by the modified adsorbent material (GAG-La).

Interaction of myo-inositol hexakisphosphate with biogenic and synthetic polyamines.

Within all the eukaryotic cells myo-inositol phosphates (InsPs) are an important group of biomolecules that are potentially related to signaling functions. The most abundant member of this family in nature is InsP6 (phytate, L(12-) in its fully deprotonated form). The complicated chemical behavior of this molecule demands a great effort to understand its function in the cell medium. In this work we follow our earlier studies on the interaction of InsP6 with metal cations by inclusion of polyamines (both biogenic and synthetic) as potential agents to produce stable adducts. The stability constants of InsP6-amine adducts and the relevant thermodynamic parameters ΔG°, ΔH°, and ΔS° were determined at 37.0 °C and 0.15 M ionic strength by means of potentiometric titrations and isothermal titration calorimetry (ITC). The biogenic amines studied were 1,4-diaminobutane (putrescine, put), 1,5-diaminopentane (cadaverine, cad), N-(3-aminopropyl)-1,4-diaminobutane (spermidine, spd), N,N'-bis(3-aminopropyl)-1,4-diaminobutane (spermine, spm), and 1-(4-aminobutyl)guanidine (agmatine, agm), while the synthetic models of longer polyamines were 1,19-dimethyl-1,4,7,10,13,16,19-heptaazanonadecane (Me2hexaen), 1,22-dimethyl-1,4,7,10,13,16,19,22-octaazadocosane (Me2heptaen), 1,25-dimethyl-1,4,7,10,13,16,19,22,25-nonaazapentacosane (Me2octaen) and N,N'-bis(3-aminopropyl)-1,3-propanediamine (3,3,3-tet). With the aid of molecular modeling, we also studied the structural aspects of molecular recognition in operation. The final result is a balance between many parameters including charge of the species, flexibility of the amines, H-bonds in the adduct, and desolvation processes.

A new P. putida instrumental toxicity bioassay.

Here, we present a new toxicity bioassay (CO2-TOX), able to detect toxic or inhibitory compounds in water samples, based on the quantification of Pseudomonas putida KT2440 CO2 production. The metabolically produced CO2 was measured continuously and directly in the liquid assay media, with a potentiometric gas electrode. The optimization studies were performed using as a model toxicant 3,5-DCP (3,5-dichlorophenol); later, heavy metals (Pb(2+), Cu(2+), or Zn(2+)) and a metalloid (As(5+)) were assayed. The response to toxics was evident after 15 min of incubation and at relatively low concentrations (e.g., 1.1 mg/L of 3,5-DCP), showing that the CO2-TOX bioassay is fast and sensitive. The EC50 values obtained were 4.93, 0.12, 6.05, 32.17, and 37.81 mg/L for 3,5-DCP, Cu(2+), Zn(2+), As(5+), and Pb(2+), respectively, at neutral pH. Additionally, the effect of the pH of the sample and the use of lyophilized bacteria were also analyzed showing that the bioassay can be implemented in different conditions. Moreover, highly turbid samples and samples with very low oxygen levels were measured successfully with the new instrumental bioassay described here. Finally, simulated samples containing 3,5-DCP or a heavy metal mixture were tested using the proposed bioassay and a standard ISO bioassay, showing that our test is more sensible to the phenol but less sensible to the metal mixtures. Therefore, we propose CO2-TOX as a rapid, sensitive, low-cost, and robust instrumental bioassay that could perform as an industrial wastewater-process monitor among other applications.

Investigation of ß-cyclodextrin-norfloxacin inclusion complexes. Part 1. Preparation, physicochemical and microbiological characterization.

INTRODUCTION: Drugs classified as class IV by the Biopharmaceutical Classification System present significant problems in relation to effective oral administration. In the case of antibiotics, the subsequently high doses required can enhance the emergence of microorganism resistance and lead to a low rate of patient treatment adherence. OBJECTIVE: In an attempt to improve physicochemical properties and microbiological activity of norfloxacin, the aim of this study was to investigate different methods (coevaporation, kneading followed by freeze-drying or spray-drying) to obtain complexes of norfloxacin and different cyclodextrins. METHODS: Guest-host interactions were investigated through a complete physical-chemical characterization and the dissolution profile and microbiological activity were determined. RESULTS: The formation of a complex of norfloxacin and ß-cyclodextrin (1:1), obtained by kneading followed by freeze drying, led to increased drug solubility, which could maximize the oral drug absorption. CONCLUSION: Moreover, the microbiological activity was enhanced by around 23.3%, demonstrating that the complex formed could represent an efficient drug delivery system.

Investigation of ß-cyclodextrin-norfloxacin inclusion complexes. Part 2. Inclusion mode and stability studies.

INTRODUCTION: Norfloxacin (NFX) is a broad spectrum antibiotic with low solubility and permeability, which is unstable on exposure to light and humidity. OBJECTIVE: In this study, the mode of NFX inclusion into ß-cyclodextrin complexes was evaluated and a complete physical, chemical and microbiological stability study of the inclusion complexes was carried out. METHODS: Potentiometric titrations were performed to evaluate changes in the pKa of the NFX molecule due to the formation of an inclusion complex and NMR analysis demonstrated that the NFX molecule is included in the ß-cyclodextrin cavity. RESULTS: Inclusion complexes obtained by kneading followed by freeze-drying showed improved NFX stability compared with the isolated drug or the physical mixture. This method was effective in terms of protecting the drug from photodegradation and also avoiding hydrolysis. Differences between NFX and the complexes could be evidenced by thermal analysis, infrared spectroscopy and x-ray powder diffraction as well as by determining the solubility and drug content. The antimicrobial potency was also preserved on applying the promising method of kneading. CONCLUSION: The satisfactory stability indicates that the NFX/ß-cyclodextrin complexes could be useful as an alternative to the existing NFX drug formulation.

Determination of fluoride at low concentrations: validation of method using ion selective electrode to analyze the water used in the preparation of dialysis solutions / Determinação de fluoreto em baixas concentrações: validação de método com eletrodo íon seletivo para análise da água utilizada na preparação de soluções de diálise

This paper describes the development and validation of an analytical methodology for determining the concentration of fluoride in water used for the preparation of dialysis solutions, by means of potentiometry with ion selective electrode. The investigated validation parameters were: selectivity, homoscedasticity, linearity, limit of detection, limit of quantification, reliability of measurement and precision. The optimized conditions of analysis were: HOAc/-OAc/NaCl/CDTA buffer (pH = 5.0 ± 0.1), in ratio of 10:1 (sample/buffer); concentrations of the standard solutions in the analytical curve: 0.05-0.80 mg/L. The evaluated method exhibited suitable validation parameters with detection and quantification limits equal to 0.020 and 0.050 mg/L, respectively. Also, an electronic spreadsheet was developed and validated for monitoring the quality of analytical curve of the methodology that calculates the decision limit to 0.20 mg/L.(AU)

Este trabalho descreve o desenvolvimento e validação de metodologia analítica para determinar a concentração de fluoreto em água empregada para a preparação de soluções de diálise, por meio de potenciometria com eletrodo íon seletivo. Os parâmetros de validação investigados foram: seletividade, homoscedasticidade, linearidade, limite de detecção, limite de quantificação, veracidade de medição e precisão. As condições otimizadas de análise foram: tampão HOAc/-OAc/NaCl/CDTA (pH = 5,0 ± 0,1), na proporção 10:1 (amostra/tampão); concentrações das soluções-padrão da curva analítica: 0,05 a 0,80 mg/L. O método avaliado exibiu parâmetros de validação adequados com limites de detecção e de quantificação, respectivamente, de 0,020 e 0,050 mg/L. Ademais, foi também desenvolvida e validada uma planilha eletrônica para efetuar o monitoramento da qualidade da curva analítica do método que calcula o limite de decisão para 0,20 mg/L.(AU)

Potentiometric determination of pantoprazole using an ion-selective sensor based on polypyrrole doped films.

The present work reports for the first time the use of polypyrrole (PPy) doped film for development of a potentiometric disposable sensor for determination of pantoprazole (PTZ), a drug used for ulcer treatment. Selective potentiometric response has been found by using a membrane of PPy doped with PTZ anions prepared under galvanostatic conditions at graphite pencil electrode (GPEM/PPy-PTZ) surface. Potentiometric response has been influenced for conditions adopted in polymerization and measurement step. After optimization of experimental (e.g. pH and time of conditioning) and instrumental parameters (e.g. current density and electrical charge) a linear analytical curve from 1.0 × 10(-5) to 1.1 × 10(-2) mol L(-1) with a slope of calibration of the 57.6 mV dec(-1) and limit of detection (LOD) of 6.9 × 10(-6) mol L(-1) was obtained. The determination of the PTZ content in pharmaceutical samples using the proposed methodology and official method recommended by Brazilian Pharmacopeia are in agreement at the 95% confidence level and within an acceptable range of error.

Self-assembled films containing crude extract of avocado as a source of tyrosinase for monophenol detection.

This paper reports on the use of the crude extract of avocado (CEA) fruit (Persea americana) as a source of tyrosinase enzyme. CEA was immobilized via layer by layer (LbL) technique onto indium tin oxide (ITO) substrates and applied in the detection of monophenol using a potentiometric biosensor. Poly(propylene imine) dendrimer of generation 3 (PPI-G3) was used as a counter ion in the layer by layer process due to its highly porous structure and functional groups suitable for enzyme linkage. After the immobilization of the crude CEA as multilayered films, standard samples of monophenol were detected in the 0.25-4.00 mM linear range with approximately 28 mV mM(-1) of sensitivity. This sensitivity is 14 times higher than the values found in the literature for a similar system. The results show that it is possible to obtain efficient and low-cost biosensors for monophenol detection using potentiometric transducers and alternative sources of enzymes without purification.

Measuring the antioxidant capacity of blood plasma using potentiometry.

The use of potentiometry to measure plasma antioxidant capacity to contribute to oxidative stress evaluation is presented. In this assay, plasma (n=60) diluted (0.3 to 1 ml) in phosphate buffer, pH 7.4, NaCl 9%, was submitted to potentiometry. A platinum wire was the working electrode and saturated calomel the reference. The results are presented as the difference between sample and buffer potential (ΔE). ΔE presented a good inverse correlation with added increasing concentrations of ascorbate (2.5-75 µmol/L; R=-0.99), urate (9.0-150 µmol/L; R=-0.99), and bilirubin (0.78-13 µmol/L; R=-0.99). Increase in the antioxidant capacity decreased ΔE. Depletion of the antioxidant capacity by tert-butylhydroperoxide (6.5-50 µmol/L) presented a direct correlation (0.97) with ΔE. Furthermore, ΔE presented an inverse correlation (R=-0.99) with increased antioxidant capacity of plasma (FRAP) induced by the addition of ascorbate (2.5-75 µmol/L). The response of the potentiometric method proved be adequate for measuring the plasma antioxidant depletion induced by acute exhaustive exercise in rats (control, n=15; exercised, n=15). This exercise decreased the concentration of urate (p<0.05), decreased FRAP (p<0.5), increased TBARS (p<0.5), and decreased the potentiometer sensor response (p=6.5×10⁻³). These results demonstrate the adequacy of potentiometry for evaluating the antioxidant capacity of blood plasma samples.

Pb(II) binding to humic substances: an equilibrium and spectroscopic study.

The binding of Pb(II) to humic acids is studied through an approach combining equilibrium and spectroscopic measurements. The methods employed are potentiometric and fluorometric titrations, fluorescence excitation-emission matrices (EEM) and IR spectroscopy. Potentiometric titration curves are analyzed using the NICA equations and an electrostatic model treating the humic particles as an elastic polyelectrolyte network. EEMs are analyzed using parallel factor analysis, decomposing the signal in its independent components and finding their dependence on Pb(II) activity. Potentiometric results are consistent with bimodal affinity distributions for Pb(II) binding, whereas fluorometric titrations are explained by monomodal distributions. EEM analysis is consistent with three independent components in the humic fluorescence response, which are assigned to moieties with different degree of aromaticity. All three components show a similar quenching behavior upon Pb(II) binding, saturating at relatively low Pb(II) concentrations. This is attributed to metal ion induced aggregation of humic molecules, resulting in the interaction between the aromatic groups responsible for fluorescence; this is also consistent with IR spectroscopy results. The observed behavior is interpreted considering that initial metal binding (observed as strongly binding sites), correspond to bi- or multidentate complexation to carboxylate groups, including binding between groups of different humic molecules, promoting aggregation; further metal ions (observed as weakly binding sites) bind to single ligand groups.