Peripheral vasculopathy in rats induced by humic acids

Adult male Wistar rats averaging 380 g in weight were injected with single doses (100 mg kg^?1 body weight) of humic acids intraperitoneally. ‘Black tails’ were noticed 12 days later. In severely affected regions, thrombosis with mild to moderate acute necrotizing vasculitis was seen in most of the blood vessels located in the dermis, subcutaneous fat and muscle. These blood vessels were markedly dilated. Frequently, colonies of a shortrod bacterium of varying sizes were observed in necrotic tissues. There was mild to moderate edema and minimal accumulation of mast cells and macrophages in the dermis. Acute fat necrosis with fibrin deposition was also seen in the subcutaneous fat. Based on the pathological findings, we diagnosed that the tails of Wistar rats which had been injected with humic acids had severe multifocal thrombosis and mild to moderate, multifocal, acute necrotizing vasculitis with bacterial colonization, mild dermatitis, and fat necrosis. The relevance of these observations to the discussions on arsenic as an etiological factor of human Blackfoot disease is examined.     

Accurate quantification of PAHs in water in the presence of dissolved humic acids using isotope dilution mass spectrometry

The effect of dissolved humic acids on the recovery of PAHs from water samples has been investigated using a commercially available humic acid preparation as colloid model and a mixture containing the 16 EPA PAHs. The presence of humic acid reduced the extraction efficiency down to between 10 and 75%. An analytical protocol was therefore developed for the accurate determination of PAHs in the presence of humic acids based on isotope dilution mass spectrometry. The procedure compensates for losses due to sorption of PAHs and can be used for the determination of the total PAH concentration in water, i.e. dissolved PAHs plus PAHs adsorbed on colloids. To obtain reliable estimates it is essential to allow a certain time for equilibration between the isotope spike and the aqueous matrix which may vary between 5 and 24 h, in correlation with the water solubility of PAHs. The protocol allows one to recover the 16 PAHs studied at 94 to 105%. The expanded uncertainty of the measurements was 5–7% for all PAHs. Liquid–liquid extraction and solid-phase extraction in combination with the developed isotope dilution protocol performed equally well for the quantification of PAHs from water samples rich in colloidal material.      

Evaluation of copper(II)-binding ability of humic acids in peat using sulphopropyl-Sephadex C-25 cation exchanger

A method for the determination of copper(II) complexes with humic acids was developed by batch operation with the cation exchanger sulphopropyl-Sephadex C-25 (C-25). The copper-binding ability (conditional stability constants and copper-complexing capacities) of humic acids which were extracted from peat in Hokkaido was evaluated. A solution containing copper(II) ions and humic acids was shaken with the C-25 exchanger. The copper-humic acid complexes remained in the supernatant and the uncomplexed free copper ion was retained on the C-25. The copper-humic acid complexes were determined by flame atomic absorption spectrometry. The copper-binding ability of nitrilotriacetic acid (NTA) as a model ligand was similarly determined with a Scatchard plot. The conditional stability constant obtained at pH 4.5 was in good agreement with the reported value. The copper-binding abilities of the humic acids from peat were estimated using a Scatchard plot adopting a two-site model. The functional groups in the humic acids which contribute to the complexation with copper were investigated by conductimetric and pH titration, and the relationship between the copper-binding sites and functional groups in the humic acids was investigated.     

Fourier transform infrared study of mercury interaction with carboxyl groups in humic acids

An interaction between humic acid, an organic part of soil and mercury was studied by Fourier transform infrared spectroscopy (FTIR) and by ICP-AES analysis under given pH and concentration conditions. First the spectroscopic model was validated on the interaction of simple molecules representing the structural components of humic acid such as benzoic acid, catechol and salicylic acid with mercury. The interaction of carboxylic parts of humic acid with mercury is very interesting and easily characterised by infrared spectroscopy, an ideal mean for molecular study. Under the salt form (commercial humic acid Fluka TM: FHA), humic acid reacts with mercury in a different way from its acid form (FHA purified noted PFHA) and the Leonardite (LHA). Because of the straightforward exchange between Na⁺, Ca²⁺ and Hg²⁺, fixation of the latter is much more important with the salt form (FHA). However, this reaction is reduced under the acid form (PFHA, LHA) because the exchange with protons is difficult. The effect of this exchange was studied by FTIR showing the intensity decrease of ν_CO (COOH), the carboxylic functional group band of the acid, and the shifting of ν_as (COO⁻), the carboxylate functional group band under given pH and mercury conditions. For the FHA salt form, the characteristic band ν_CO (COOH) represented by a shoulder did not evolute, whereas the corresponding band to ν_as (COO⁻) strongly shifted (40 cm⁻¹) for a maximum Hg²⁺ concentration (1 g l⁻¹). On the other hand, for the acid form (PFHA, LHA), the intense band of ν_CO (COOH) disappeared proportionally to the increase of Hg²⁺concentration and the ν_as (COO⁻) band moved for about 20 cm⁻¹. The same results were reached with pH variations. Our results were confirmed by ICP-AES mercury analysis. This study shows that humic acids react differently according to their chemical and structural state.     

Determination of low-molecular-weight carboxylic acids in aqueous samples by gas chromatography and nitrogen-selective detection of 2-nitrophenylhydrazides

A gas chromatographic method was developed for the determination of low-molecular-weight carboxylic acids in aqueous samples based on a derivatization procedure compatible with aqueous solutions. The technique uses nitrogen-selective detection with a thermionic-specific detector after derivatization of carboxylic acids as 2- nitrophenylhydrazides. The hydrazides were extracted with ethyl acetate prior to injection into the gas chromatograph. The derivatives appear to be stable in ethyl acetate at 0–5° C for long periods and, therefore, can be stored for analysis at a later date. The detection limits of different short-chain acids are in the range 0.8–1.4 pmol per injected sample. The relative standard deviation is less than 10% at the 1 μM level. Examples of the use of the method are given for the determination of carboxylic acids in anoxic marine sediment pore waters, coastal sea water and Black Sea water samples.     

IR study of heterogeneity of OH groups in zeolite HY-splitting of OH and OD bands

We have been studying the problem of heterogeneity of OH groups in zeolites HY for a long time. The heterogeneity was suggested by the shift of the IR band of OH groups restoring upon ammonia desorption and also by the fact that the band of OH groups forming hydrogen bonds was relatively broad (broader than for homogeneous acidic OH). In the present study we present another important argument for heterogeneity: the splitting of the IR band of free OH and OD groups in a zeolite of Si/Al=8.3 dealuminated by (NH₄)₂SiF₆ treatment. Such a splitting is the best seen in low temperature spectra of OD groups. We found less acidic 3640 cm⁻¹ (AlO)(SiO)₂SiO₁HAl(OSi)₃ and more acidic 3625 cm⁻¹ (SiO)₃SiO₁HAl(OSi)₃ groups. The presence of these two kinds of hydroxyls corresponds to the presence of Si(2Al) and Si(1Al), respectively, detected in ²⁹Si MAS NMR spectra. We also found a small amount of strongly acidic 3599 cm⁻¹ hydroxyls interacting with extraframework Al species.     

Ion-exclusion chromatography of carboxylic acids on silica gel modified with aluminium

The modification of silica gel with aluminium by a coating method was effective for the preparation of a silica-based stationary phase, which acted as a cation exchanger under strongly acidic conditions. In order to expand the utility of the laboratory-made aluminium-adsorbing silica gel it was applied as a stationary phase to the ion-exclusion chromatography of various carboxylic acids. Good separations for both aliphatic carboxylic acids and benzenecarboxylic acids with a hydrophobic nature under acidic eluent conditions were achieved in 25 min.     

Surface-enhanced Raman and fluorescence joint analysis of soil humic acids

Surface-enhanced Raman scattering (SERS) and surface-enhanced fluorescence (SEF) combined emissions were used in this work to the analysis of humic acids (HA). This study examined HA structure at different pH and HA concentrations and assessed the structural differences taking place in HA as a result of various amendment trials. Raman and fluorescence emissions behave in opposite ways due to the effect of the metal surface on the aromatic groups responsible for these emissions. The information afforded by these techniques can be successfully employed in the structural and dynamic analysis of these important macromolecules. The surface-enhanced emission (SEE) spectra, that is the sum of the Raman and the fluorescence emissions, were acquired by using both macro- and micro-experimental configurations in order to apply imaging and confocal Raman and fluorescence spectroscopy techniques on the analysis of HA.     

Influence of amide linkages on acidity determinations of humic substances Testing with model-mixtures

A set of experiments, using model compounds, was carried out in order to evaluate the influence of amide linkages on acidity measurements of humic substances (HS). Three benzene-carboxylic acids (2,4-dihydroxybenzoic acid; 3,5-dihydroxybenzoic acid, and phthalic acid) and two peptides (dl-alanyl-dl-alanine, and glycil-l-leucine) were employed to simulate the major acidic functional groups present in humic material. The acidity of the samples was measured, using the Schnitzer and Gupta methods, for each compound separately, as well as for selected mixtures with different compound combinations. General results showed that, in the absence of peptides, phenolic groups with high pK_a values are not detected in the barium hydroxide reaction and the data so obtained do not represent the real concentrations of the acidic groups. For the mixtures containing peptides, the presence of amide linkages distorts the results relative to the total acidity (TA) mainly because under the extreme conditions of the reaction with Ba(OH)₂ hydrolysis of the peptide occurs, consuming extra hydroxyl groups and increasing artificially the phenolic content. Such a condition is minimized by the reduction of the reaction time.     

Bioreduction of hexavalent chromium: Effect of compost-derived humic acids and hematite

Composting can enhance the nutrient elements cycling and reduce carbon dioxide production. However, little information is available regarding the application of compost for the remediation of the contaminated soil. In this study, we assess the response of the redox capacities (electron accepting capacities (EAC) and electron donating capacities (EDC)) of compost-derived humic acids (HAs) to the bioreduction of hexavalent chromium (Cr(VI)), especially in presence of hematite. The result showed that the compost-derived HAs played an important role in the bioreduction of Cr(VI) in presence and absence of hematite under the anoxic, neutral (pH 7) and motionless conditions. Based on the pseudo-first order kinetic model, the rate constants of Cr(VI) reduction increased by 1.36–2.0 times when compost-derived HAs was added. The redox capacity originating from the polysaccharide structure of compost-derived HAs made them effective in the direct Cr(VI) reduction (without MR-1) at pH 7. Meanwhile, the reduction rates were inversely proportional to the composting treatment time. When iron mineral (Fe₂O₃) and compost-derived HAs were both present, the rate constants of Cr(VI) reduction increased by 2.35–5.09, which were higher than the rate of Cr(VI) reduction in HA-only systems, indicating that the hematite played a crucial role in the bioreduction process of Cr(VI). EAC and quinonoid structures played a major role in enhancing the bioreduction of Cr(VI) when iron mineral and compost-derived HAs coexisted in the system. The results can extend the application fields of compost and will provide a new insight for the remediation of Cr(VI)-contaminated soil.     

Derivatized humic acids modified gold electrode: electrochemical characterization and analytical applications

The preparation and application of a humic acids modified gold electrode (HA-CME) have been described. Derivatization of HAs as β-thioesters caused their partial fragmentation and, thus, a consequent separation by size exclusion chromatography (SEC) was necessary.The CME prepared with functionalized HAs having an average MW of 52000 demonstrated to be effective for trace As(III) determination (LOQ = 0.3 μg L⁻¹). This CME has been characterized both by electrochemical techniques and atomic force microscopy (AFM). The HA-CME provided reliable measurements in natural waters at different salinity with no need of desalting the sample. Total inorganic arsenic could be determined after reduction of As(V) with SO₂. Under these conditions, organic arsenic species were not mineralized and did not interfere.     

A study of the interaction between humic acids and acidic metabolites of phenanthrene by capillary electrophoresis

Humic acids (HA) are able to interact with a wide range of pollulants and can influence their solubility, transport and bioavailability. In order to study the interaction between polar aromatic hydrocarbons and these macromolecules, an affinity capillary electrophoretic method, the Hummel-Dreyer (HD) method in its modified version, has been used. Two acidic metabolites of phenanthrene: 1-hydroxy-2-naphthoic acid (1-HNA) and 3,4-dihydroxybenzoic acid (3,4-DBA) were studied. The analysis for the binding studies was carried out by injecting a solution of HA in 25 mM ammonium hydrogen carbonate buffer (pH 9) into an uncoated fused-silica capillary, filled with buffer solutions of 1-HNA or 3,4-DBA in varying and increasing amounts. The results obtained indicate that both compounds bind to HA, as had been confirmed by dialysis experiments and literature data. CE proved to be a useful technique for investigating the link between xenobiotics and environmental macromolecules.     

Determination of carboxylic acids by ion-exclusion chromatography with non-suppressed conductivity and optical detectors

Summary Determination of carboxylic acids using non-suppressed conductivity and UV detections is described. The background conductance of 1-octanesulfonic acid, hexane sulfonic acid and sulfuric acid at varying concentrations was determined. Using 0.2 mM 1-octanesulfonic acid as a mobile phase and an injection volume of 300 l, the detection limits range from 1 M for formic acid to 10 M for butyric acid. The conductivity detector was connected in tandem with a Waters 484 optical detector at 210 nm, which allowed the analysis of carboxylic acids from 0.01 mM to 10 mM. The dependence of the retention of carboxylic acids on the eluent pH is discussed.     

Capillary electrophoretic separation of dicarboxylic acids in atmospheric aerosol particles

2,3-Pyrazinedicarboxylic acid (PZDA), 2,6-pyridinedicarboxylic acid (PDA) and 2,3-pyridinedicarboxylic acid (QUIN) solutions were studied as background electrolytes (BGEs) in the capillary electrophoretic analysis of dicarboxylic acids in aerosol particles with indirect UV detection. The BGEs were selected on the basis of similarity in structure with the analytes so that mobilities would be compatible. Optimised pH values for PZDA, PDA and QUIN solutions were 10.6, 11.0 and 10.2, respectively. Myristyltrimethylammonium hydroxide and myristyltrimethylammonium bromide were added to reverse the electroosmotic flow in the solutions in the direction of anode to enable fast anion detection. Separation was obtained for nine dicarboxylic acids (C₂–C₁₀) differing in the number of CH₂ groups in their skeleton. The electrophoretic mobilities were determined to lie in the range 3.0×10⁻⁴–7.0×10⁻⁴ cm² V⁻¹ s⁻¹. The relative standard deviations (RSDs) for the absolute migration times of the analytes were mostly less than 0.5% (n=6) in PZDA solution. In PDA solution the within-day and day-to-day RSD values for migration were less than 1% and between 2 and 4%, respectively. Peak heights and areas mostly deviated between 1 and 15% in both PZDA and PDA solutions. Detection limits ranged between 1 and 5 mg/l. Methods were applied to the analysis of dicarboxylic acids isolated from aerosol particles.     

Influence of extractant on quality and trace elements content of peat humic acids

Among several extractants used to isolate humic acids (HA) from terrestrial environments, sodium hydroxide (NaOH) and sodium pyrophosphate (Na₄P₂O₇) are the most utilized. In order to evaluate the influence of these different extractant solutions on the HA quality and on their trace elements content, HA were isolated from five Sphagnum-peat samples using three different solutions: (a) 0.5 M NaOH; (b) 0.1 M Na₄P₂O₇; (c) 0.5 M NaOH + 0.1 M Na₄P₂O₇. The obtained HA have been analyzed with respect to ash content, elemental composition, main atomic ratios and characterized by FT-IR and total luminescence (TL) spectroscopies. In addition, both raw peat and HA have been analyzed using X-ray fluorescence in order to determine the Br, Cu, Fe, Ni, Pb and Zn contents.

Results showed that HA extracted with NaOH and NaOH + Na₄P₂O₇ are quite similar with respect to ash, elemental contents and spectroscopic characteristics, while Na₄P₂O₇ solution, which in general reduces the extraction yield, seems to affect the nature of HA, featuring a more complex and aromatic character. With respect to the contents in the corresponding raw peat samples, the HA fractions were richer in Br, Cu and Ni, regardless of the extractant used, and poorer in Fe, Pb and Zn. Further, Br, Cu, Ni and Zn were more concentrated in HA extracted with Na₄P₂O₇ than in those extracted with NaOH and NaOH + Na₄P₂O₇, probably because of the greater affinity of these elements for these more aromatic humic molecules.     

Retention modelling of electrostatic and adsorption effects of aliphatic and aromatic carboxylic acids in ion-exclusion chromatography

The retention mechanism of aliphatic and aromatic carboxylic acids in ion-exclusion chromatography has been investigated with consideration of simultaneous electrostatic repulsion effects and hydrophobic adsorption effects. A mathematical relationship between the retention factor of the analyte and the mobile-phase composition (sulfuric acid concentration and percentage of methanol), the type of analyte (pK_a and hydrophobicity) and some physical characteristics of the stationary phase has been derived. Thirteen carboxylic acids (comprising mono- and divalent, aliphatic and aromatic acids) were chosen and used to acquire retention data on three different cation-exchange stationary phases (in which the sulfonate functional groups are bound to polystyrene–divinylbenzene, polymethacrylate or silica) using 14 mobile-phase compositions of varying pH and percent methanol. These retention data were used to derive the parameters necessary to solve the retention model using non-linear regression. In this way, a quantitative measure of the effects of adsorption phenomena on analyte retention were obtained. The model was then used to optimise the separation of nine carboxylic acids.     

Determination of carboxylic acids in water by gas chromatography using several detectors after flow preconcentration

A novel analytical method is reported that combines continuous solid-phase extraction and gas chromatography for the determination of 22 carboxylic acids in water. The highly polar and hydrophilic analytes were preferentially sorbed on a mixture of LiChrolut EN-Supelclean ENVI-18 (1:1) sorbent column and eluted with methanol; this extraction process did not require derivatisation. The extract was analysed by gas chromatography coupled to a flame ionisation detector as well as a mass spectrometer with electron impact (EI) or positive chemical ionisation modes. The highest sensitivity was achieved when using MS-EI, with good linearity in calibration curves and low detection limits (2-40ngL(-1)) for 50mL of sample. The entire procedure from raw aqueous sample to a ready-to-inject methanol solution of the acids requires less than 15min. Another benefit of this method is the good accuracy (recoveries between 93 and 102%) and precision (relative standard deviation, 3.4-6.2%), which allows the determination of carboxylic acids in environmental water and in real chlorinated and ozonated drinking water.     

Determination of C4-C14 carboxylic acids by capillary zone electrophoresis. Application to the identification of diamide degradation products and partitioning studies.

Capillary zone electrophoresis (CZE) was investigated for the determination of linear saturated carboxylic acid homologues ranging from C₄ to C₁₄. Separation conditions were optimised to overcome the problems of decreasing solubility and decreasing selectivity between successive homologues with increasing chain length. Separations were performed at 20°C, using a 20 kV separation voltage and a pH 8 electrolyte containing 30% methanol. A suitable chromophore (4-aminobenzoate) was added to ensure indirect UV detection of the analytes. Calibration curves and repeatability were established. Minimum detectable concentrations of 3·10⁻⁶ mol l⁻¹ were achieved. Resolution between successive homologues was better than 2. The electrophoretic mobility of each homologue (n=7–14) was assessed and a quasi-linear relationship between the mobility value and 1/n was observed. The quantitative analysis of a diamide degradation solution was performed and compared to potentiometric results. The CZE method was also applied to the determination of C₇–C₁₄ partitioning between an organic medium containing tributylphosphate in n-dodecane and different basic solutions. Their behaviour was established according to the chain length and the pH of the aqueous phase. For C₁₀–C₁₄ compounds, results were validated by comparison with gas chromatographic analysis of the organic phases.     

Characterization of humic acids in sediments from dam reservoirs by pyrolysis-gas chromatography/mass spectrometry using tetramethylammonium hydroxide: Influence of the structural features of humic acids on iron(II) binding capacity

The structural features of humic acids (HAs) isolated from sediments on the bottom of dam reservoirs that can affect their binding capacities for Fe(II) were investigated by pyrolysis-gas chromatography/mass spectroscopy using tetramethylammonium hydroxide (TMAH-py-GC/MS). The binding capacities for Fe(II) increased with increasing O/C molar ratio, suggesting that the oxygen-containing functional group content plays a role in the binding of Fe(II). However, it was not possible to identify specific binding-sites for Fe(II) by TMAH-py-GC/MS analysis. Although C_16:1ω7, iso-C_15:0 and anteiso-C_17:0 fatty acids, which serve as molecular markers of anaerobic microbial activity, were detected in all of the HA samples, the contents of these acids were not correlated with binding capacities for Fe(II). However, the ratio of C_16:0 to C_16:1ω7 fatty acids, which is used as an index of anaerobic bacterial activity, increased with increasing Fe(II) binding capacities of the HAs. It thus appears likely that the activities of anaerobic bacteria on the bottom of dam reservoirs contribute to alterations in the structural features for HAs, and that this process results in increased binding capacities for Fe(II).     

Application of zirconium-modified silica gel as a stationary phase in the ion-exclusion chromatography of carboxylic acids I. Separation of benzenecarboxylic acids with tartaric acid as eluent and with UV-photometric detection

The application of zirconium-modified silica gels (Zr–Silicas) as stationary phases for ion-exclusion chromatography with UV-photometric detection (IEC–PD) for mono-, di-, tri- and tetrabenzenecarboxylic acids (pyromellitic, trimellitic, hemimellitic, o-phthalic, salicylic and benzoic acids) and phenol was carried out using tartaric acid as the eluent. Zr–Silicas were prepared by the reaction of the silanol group on the surface of silica gel with zirconium tetrabutoxide [Zr(OCH₂CH₂CH₂CH₃)₄] in ethanol solution. The effect of the amount of zirconium adsorbed on silica gel on chromatographic behavior of these benzenecarboxylic acids and phenol was investigated. As a result, Zr–Silica adsorbed on 20 mg zirconium g⁻¹ silica gel was the most suitable stationary phase in the IEC–PD for the simultaneous separation of these benzenecarboxylic acids and phenol. Excellent simultaneous separation and highly sensitive UV detection at 254 nm for these benzenecarboxylic acids and phenol were achieved in 20 min by the IEC–PD using the Zr–Silica column (250×4.6 mm I.D.) and a 10 mM tartaric acid at pH 2.5 as eluent.