Development of an automated system for isolation and purification of humic substances

Characterization of humic substances (HS) in environmental samples generally involves labor-intensive and time-consuming isolation and purification procedures. In this paper, the development of an automated system for HS isolation and purification is described. The novelty of the developed system lies in the way the multiple liquids and columns used in the isolation/purification procedure are handled in both forward and back-elution mode by solenoid valves. The automated procedure significantly reduces the total throughput time needed, from 6–7 days to 48 h, and the amount of labor to obtain purified HS for further characterization. Chemical characterization of purified HS showed that results were in good agreement with previously published values for HS from a variety of sources, including the IHSS standard HS collection. It was also shown that the general properties of HS were consistent among the different source materials (soil, waste, aquatic) used in this study. The developed system greatly facilitates isolation and characterization of HS and reduces the risk of potential (time-dependent) alteration of HS properties in the manual procedure. Figure Photograph of the rear (left) and front (right) of the automated system for the isolation and purification of humic substances Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Determination of molecular formulas of natural organic matter molecules by (ultra-) high-resolution mass spectrometry: Status and needs

Electrospray ionization (ESI) combined with ultra-high-resolution mass spectrometry on a Fourier transform ion cyclotron resonance mass spectrometer has been shown to be a very powerful tool for the analysis of fulvic and humic acids and of natural organic matter (NOM) at the molecular level. With this technique thousands of ions can be separated from each other and their m/z ratio determined with sufficient accuracy to allow molecular formula calculation. Organic biogeochemistry, water chemistry, and atmospheric chemistry greatly benefit from this technique. Methodical aspects concerning the application of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) to NOM isolated from surface water, groundwater, marine waters, and soils as well as from secondary organic aerosol in the atmospheric are reviewed. Enrichment of NOM and its chromatographic separation as well as possible influences of the ionization process on the appearance of the mass spectra are discussed. These steps of the analytical process require more systematic investigations. A basic drawback, however, is the lack of well defined single reference compounds of NOM or fulvic acids. Approaches of molecular formula calculation from the mass spectrometric data are reviewed and available graphical presentation methods are summarized. Finally, unsolved issues that limit the quality of data generated by FTICR-MS analysis of NOM are elaborated. It is concluded that further development in NOM enrichment and chromatographic separation is required and that tools for data analysis, data comparison and data visualization ought to be improved to make full use of FTICR-MS in NOM analysis.     

Nuclear magnetic resonance relaxometry as a spectroscopic probe of the coordination sphere of a paramagnetic metal bound to a humic acid mixture

Protons on water molecules are strongly affected by paramagnetic ions. Since the acid-base properties of water facilitate rapid proton exchange, a single proton nuclear magnetic resonance (NMR) signal is seen in aqueous solutions of paramagnetic ions. Proton relaxation times are significantly affected by paramagnetic species and the readily detectable single signal serves as a powerful amplifier of the information contained concerning the protons in the paramagnetic environment. Where water molecules coordinated to free paramagnetic ions and to metal complexes of ligands that form non-labile (on the NMR time scale) complexes, the effects on water in the two environments can be distinguished. This can provide information on the nature of the ligand binding sites. The example of Cu²⁺ bound to the Laurentian humic acid mixture reported here using convenient low field NMR relaxometers shows that the information can enrich our understanding of complexation and speciation in the presence of complex mixture ligands characteristic of natural water systems. In this case, the data underline the role of aggregation and conformation in defining the complexation sites.     

H NMR study of hydrogen bonding and molecular dynamics of 5CB confined to molecular sieves

It has been found that the main mechanism of ¹H protons spin–lattice relaxation of bulk 5CB at 200 MHz is its intramolecular motion, namely, the reorientation of CH₂ and CH₃ groups of its alkyl chain. Activation parameters of such motions have been estimated.

Drastic decrease in proton spin–lattice relaxation times at the nematic-to-isotropic phase transition can be explained by the activation of molecular translational diffusion and reorientations around long and short molecular axes of bulk 5CB.

Our NMR analysis revealed the slowing-down of molecular dynamics of confined 5CB molecules and their fragments. This can be explained by the interaction of some part of 5CB molecules with the surface active Si(Al)–OH centers of MCM matrix via hydrogen bonds of Si(Al)–OHN≡C-type.     

Interaction of atrazine with Laurentian humic acid

Ultrafiltration fractionation and liquid chromatography have been applied to study the binding and hydrolysis of polar herbicide atrazine on a stoichiometrically well characterized Laurentian humic acid. The main advantage of this method over gas chromatography is the simultaneous determination of both free and bound atrazine, hydroxyatrazine and copper(II) ion with satisfactory accuracy and precision. Atrazine binding requires extensive carboxylate site protonation but the binding sites represent only a very small fraction of total carboxylate of humic acid. The results show that binding of atrazine is not competitive with binding of the hydrolysis product hydroxyatrazine, the binding capacity is reduced at higher ionic strength or by cation competition for carboxylate and the atrazine binding constant and free energy of binding can be fitted by a single value at all pH values. The differences between atrazine binding by fulvic acid and humic acid can be ascribed to the structure difference, one being a flexible linear polymer and the other a three-dimensional colloidal gel particle.     

Coupling of chromatography and NMR part 5: Analysis of high-boiling gas-chromatographic fractions by on-line nuclear magnetic resonance

Summary A procedure is described for recording¹H NMR spectra of flowing gaseous GC fractions boiling as high as 200°C. The line width of the signals in the NMR spectra is smaller than 2.2Hz.Part 4: J. Buddrus, H. Herzog, Anal. Chem.55, 1611 (1983).     

Polysilicato-iron for improved NOM removal and membrane performance

The natural organic matter (NOM) removal efficiency of polysilicato-iron (PSI) coagulants and the fouling potential of PSI pretreated waters have been studied using two microfiltration (MF) membrane types: polyvinylidene fluoride (PVDF-2) and polypropylene (PP). The results showed that PSI coagulant with a Si/Fe ratio of 1 (PSI-1) was the most effective, compared to conventional coagulants, in removing dissolved organic carbon (DOC) and in improving the fouling potential. A relative flux of unity through PVDF-2 membrane was achieved for both water sources pretreated with PSI-1.

Aluminium-based coagulants, particularly aluminium chlorohydrate (ACH), worked best at lower coagulant dose. Increasing the coagulant dose to improve DOC removal led to increased membrane fouling, possibly due to increased level of unsettleable flocs and pore blocking. For PSI with larger floc size, the advantage of increased DOC removal was not overridden by the adverse effect of pore blocking. In addition, the residual neutral fraction in the waters and/or the presence of a filter cake on the membrane surfaces seemed to have a limiting effect on the fouling rates through both PP and PVDF-2 membranes to the extent that similar rates were obtained, despite substantial differences in DOC removal.

In contrast, these limiting factors did not influence the fouling potential of PSI-1 treated waters through the PVDF-2 membrane, as suggested by the relative flux of unity for both water sources. It is suggested that the oxide deposits on the PVDF-2 membrane may act as a ‘screening layer’, acting as pre-filtration by the filter cake. This layer may be effectively removed by backwashing, together with deposited NOM, throughout the experiment to maintain the flux at unity. The hydrophobic nature of the PP membrane may discourage the deposition of the oxides, thus minimising the positive effects of the oxides in the system. The high removal of hydrophobic fractions by PSI-1 may also lead to less association between residual NOM and less binding to the membranes, particularly on the PVDF-2 membrane.     

Quantitative determination of formic acid in apple juices by H NMR spectrometry

A quantitative determination method of formic acid in apple juices is proposed by means of the proton nuclear magnetic resonance (¹H NMR) technique. Formic acid gives a singlet signal at the 8.2-8.4 ppm interval of the spectrum, and its area is used to determine the concentration of the acid. 1,3,5-Benzenetricarboxylic acid is added to the juice as an internal standard. Since the chemical shift of both species varies with the pH, ascorbic acid is also added to adjust it at 2.74 and to avoid the overlapping of the signals. Recoveries between 95 and 109% are obtained when the standard addition method is applied to the juices of five different cider apple varieties. The coefficient of variation obtained is 3.9% for intra-day repeatability (n = 5), and 4.6% for inter-day repeatability (n = 10). The limit of detection is 1.49 mg/l, calculated from “3S_y/x + intercept”. The described method is direct and no previous derivatization is needed.     

A rapid and automated low resolution NMR method to analyze oil quality in intact oilseeds

Oilseeds with modified fatty acid profiles have been the genetic alternative for high quality vegetable oil for food and biodiesel applications. They can provide stable, functional oils for the food industry, without the hydrogenation process that produces trans-fatty acid, which has been linked to cardiovascular disease. High yield and high quality oilseeds are also necessary for the success of biodiesel programs, as polyunsatured or saturated fatty acid oil produces biofuel with undesirable properties. In this paper, a rapid and automated low resolution NMR method to select intact oilseeds with a modified fatty acid profile is introduced, based on ¹H transverse relaxation time (T₂). The T₂ weighted NMR signal, obtained by a CPMG pulse sequence and processed by chemometric methods was able to determine the oil quality in intact seeds by its fatty composition, cetane number, iodine value and kinematic viscosity with a correlation coefficient r > 0.9. The automated system has the potential to analyze more than 1000 samples per hour and is a powerful tool to speed up the selection of high quality oilseeds for food and biodiesel applications.     

Characterization of fulvic acids by liquid chromatography-quadrupole time-of-flight mass spectrometry

Fulvic acid standards from Suwannee River, Pony Lake, Elliot Soil, Waskish Peat, and Nordic Reservoir were characterized by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) operating in negative electrospray ionization mode. The method employed a commercially available stationary phase that resulted in a distinctive chromatographic peak for each of the fulvic acid samples that differed in width and retention time at peak maximum. The QTOF-MS, operating in TOF mode, revealed that the unique chromatographic peak shapes were the result of the relative fraction of hydrogen and oxygen contained in various fulvic acid components. Those species that contained larger amount of hydrogen displayed a larger mass defect and were retained longer on the LC column, indicating reduced polarity. This is supported by a reduction in the degree of fragmentation related to polar functional groups as the mass defect and retention time increased. Lastly, the analysis of even and odd mass (at m/z 1 greater) ion intensity ratios revealed a correlation to the percent nitrogen of the various standards.     

Spectroscopic approaches to the study of the interaction of aluminum with humic substances

Aluminum ion (Al³⁺) in the ‘free’ (aquo) state is becoming increasingly prevalent in environmental waters, especially fresh waters, as a consequence of acid rain and other environmental processes. As Al³⁺ ion is known to affect markedly a wide range of biological systems, and since the presence of Al³⁺ in humans has been linked to a number of human diseases, it is important to understand the speciation of Al³⁺ ion in natural waters. Since some of the most important complexation agents for Al³⁺ in both fresh and sea waters are members of the complex humic substances group, it is important to understand the manner in which Al³⁺ interacts with this class of molecules, especially since binding of Al³⁺ to these molecules can effectively increase the bioavailability of this toxic metal ion to biological systems. The objective of this review is to present the current state of our understanding of aqueous aluminum complexation with the most acidic members (and therefore the most likely candidates for serving as Al³⁺ complexing agents) of the humic substances group, the fulvic acids. Much of the current knowledge has been revealed by comprehensive fluorescence titration analyses. Some additional information has come from other experimental approaches, including infrared spectroscopy, nuclear magnetic resonance spectroscopy, and a variety of electrochemical approaches. In this review, we also report on the results of our recent fluorescence and IR spectroscopy survey of the interaction of metals from of all three Nieboer and Richardson categories of environmental metals (Class A, Class B and Intermediate Class) with the fulvic acid sub-group of the humic substances. This has proven helpful in understanding some of the unique spectral behaviors of the Al³⁺-fulvic acid complex vis-a-vis fulvic acid complexes with many other metal ions. The results of our fluorescence and IR experiments with the model compounds, such as salicylic and phthalic acids, have allowed confirmation of the important roles played by both salicylic acid-like sites and phthalic acid-like sites in the unique complexation of Al³⁺ to humic substances, and help to explain some of the observed spectroscopic changes associated with Al³⁺ ion complexation to humic material. From the current work, it seems clear that major sources of the deviation in spectral properties between Al³⁺ and many other metal ions (across all three Nieboer and Richardson categories) are the unusually high value of its charge density and relatively low propensity for involvement in covalent bonding interactions (i.e. a very high ionic index combined with a relatively low covalent index in the Nieboer and Richardson classification of environmental metals), as well as affinity for certain functional groups.     

RNA unrestrained molecular dynamics ensemble improves agreement with experimental NMR data compared to single static structure: a test case

Nuclear magnetic resonance (NMR) provides structural and dynamic information reflecting an average, often non-linear, of multiple solution-state conformations. Therefore, a single optimized structure derived from NMR refinement may be misleading if the NMR data actually result from averaging of distinct conformers. It is hypothesized that a conformational ensemble generated by a valid molecular dynamics (MD) simulation should be able to improve agreement with the NMR data set compared with the single optimized starting structure. Using a model system consisting of two sequence-related self-complementary ribonucleotide octamers for which NMR data was available, 0.3 ns particle mesh Ewald MD simulations were performed in the AMBER force field in the presence of explicit water and counterions. Agreement of the averaged properties of the molecular dynamics ensembles with NMR data such as homonuclear proton nuclear Overhauser effect (NOE)-based distance constraints, homonuclear proton and heteronuclear ¹H–³¹P coupling constant (J) data, and qualitative NMR information on hydrogen bond occupancy, was systematically assessed. Despite the short length of the simulation, the ensemble generated from it agreed with the NMR experimental constraints more completely than the single optimized NMR structure. This suggests that short unrestrained MD simulations may be of utility in interpreting NMR results. As expected, a 0.5 ns simulation utilizing a distance dependent dielectric did not improve agreement with the NMR data, consistent with its inferior exploration of conformational space as assessed by 2-D RMSD plots. Thus, ability to rapidly improve agreement with NMR constraints may be a sensitive diagnostic of the MD methods themselves.     

Determination of dissolved organic matter based on UV-light induced reduction of ionic silver to metallic nanoparticles by humic and fulvic acids

We describe a novel solution-based method for the determination of dissolved organic matter (DOM) relying on the formation of silver nanoparticles (AgNPs) via photo-stimulated reduction of silver ions by humic and fulvic acids. The method is based on natural driven formation of nanoscale materials yielding a direct relationship between DOM concentration and AgNPs formation. The aqueous dispersion of the formed AgNPs show strong and uniform UV–Vis absorption bands between 450 and 550 nm irrespectively of DOM nature and properties (i.e. humic or fulvic acids). The ensuing chromatic shift accompanying the appearance of the new absorption bands is easily conceivable by a simple spectrophotometer and the bare eye, holding great promise for the on-site, instrumental-free screening of DOM levels. Under the optimum experimental conditions the determination of DOM was successfully demonstrated to various water samples with high sensitivity (<1.0 mg L⁻¹), satisfactory recoveries (87.5–123.5%) and reproducibility (5.87–6.73%).     

NMR探测苯丙氨酸转移核糖核酸中镧离子的键合

用ＮＭＲ谱研究了溶液中镧离子对苯丙氨酸转移核糖核酸（简称ｔＲＮＡＰｈｅ）结构和构象变化的影响。研究表明Ｌａ３＋对ｔＲＮＡＰｈｅ亚胺质子范围的１ＨＮＭＲ谱具有特殊的影响，位于扩大二氢尿嘧啶螺旋（Ｄ螺旋）的端梢三级碱基对Ｇ１５·Ｃ４８明显受Ｌａ３＋的影响（向低场位移０３５）；堆积在Ｇ１５·Ｇ４８上的Ｕ８·Ａ１４碱基对在存有１～２个Ｍｇ２＋时亦受Ｌａ３＋的影响。酵母ｔＲＮＡＰｈｅ中可能受到Ｌａ３＋影响的另一亚胺质子为Ｇ１９·Ｃ５６三级碱基对，由于Ｇ１９·Ｃ５６的亚胺质子共振位于高度叠加的１２６～１２２之间。该碱基对有助于Ｄ环对ＴΨＣ环的联接。Ｌａ３＋引起ｔＲＮＡ分子构象的变化并且导致一些谱峰向高场或低场位移。     

Analogues of DIOP. Synthesis, reactivity, and NMR behaviour

The (4R,5R)-4,5-Bis(diphenylphosphinomethyl)-2-(-naphthoxy)-1,3,2-dioxaphospholane (1) and (4R,5R)-4,5-bis(diphenylphosphinomethyl)-2-dimethylamino-2-oxo-1,3,2-dioxaphospholane (2), analogues of the chiral bisphosphine ligand DIOP, have been synthesized. Both compounds have a second phosphorus functionality in the backbone of the chiral bisphosphine. That the reactivities of the various phosphorus atoms in 1 are different was shown for the reaction with BH₃. The complexation behaviour of 1 towards Rh^I was studied with the aid of ³¹P NMR spectroscope.     

Development of a new analytical approach based on cellulose membrane and chelator for differentiation of labile and inert metal species in aquatic systems

A new procedure was developed in this study, based on a system equipped with a cellulose membrane and a tetraethylenepentamine hexaacetate chelator (MD-TEPHA) for in situ characterization of the lability of metal species in aquatic systems. To this end, the DM-TEPHA system was prepared by adding TEPHA chelator to cellulose bags pre-purified with 1.0 mol L⁻¹ of HCl and NaOH solutions. After the MD-TEPHA system was sealed, it was examined in the laboratory to evaluate the influence of complexation time (0-24 h), pH (3.0, 4.0, 5.0, 6.0 and 7.0), metal ions (Cu, Cd, Fe, Mn and Ni) and concentration of organic matter (15, 30 and 60 mg L⁻¹) on the relative lability of metal species by TEPHA chelator. The results showed that Fe and Cu metals were complexed more slowly by TEPHA chelator in the MD-TEPHA system than were Cd, Ni and Mn in all pH used. It was also found that the pH strongly influences the process of metal complexation by the MD-TEPHA system. At all the pH levels, Cd, Mn and Ni showed greater complexation with TEPHA chelator (recovery of about 95-75%) than did Cu and Fe metals. Time also affects the lability of metal species complexed by aquatic humic substances (AHS); while Cd, Ni and Mn showed a faster kinetics, reaching equilibrium after about 100 min, and Cu and Fe approached equilibrium after 400 min. Increasing the AHS concentration decreases the lability of metal species by shifting the equilibrium to AHS-metal complexes. Our results indicate that the system under study offers an interesting alternative that can be applied to in situ experiments for differentiation of labile and inert metal species in aquatic systems.     

On-Line Coupling of Separation Techniques to NMR

The hyphenation of chromatographic separation techniques with NMR spectroscopy is one of the most powerful and time-saving methods for the separation and structural elucidation of unknown compounds and molecular compositions of mixtures. Most of the routinely used NMR flow-cells have detection volumes between 40–180 μL for conventional separations with analytical columns, and the newest designs employ detection volumes in the order of 200 nL for capillary separations. The low flow rates used in capillary chromatography permit the use of deuterated solvents. Unequivocal structural assignment of unknown chromatographic peaks is possible by two-dimensional stopped-flow capillary HPLC-NMR experiments.     

NMR studies of the reversible and regioselective lactonization of α-2,8-linked trisialic acid in aqueous acid

The pH dependent lactonization of trisialic acid was examined using NMR and CE techniques. The combined studies indicate that lactonization is a dynamic, pH dependent process that occurs with preferential lactonization between the carbonyl of the central residue and the reducing end C-9 hydroxyl group.     

3个新呋喃黄酮的NMR研究

从厚果难血藤（Millettia pachycarpa Benth)根中分离得到3个呋喃黄酮类化合物,经NMR谱等分析研究,确定了它们的结构为：4H－呋喃并[2,3-h][1]苯并吡喃－4－酮,3－甲氧基－2－（3,4,5－三甲氧基苯基）（I）、4H－呋喃并[2,3-h][1]苯并吡喃－4－酮,3－甲氧基－2（3－甲氧苯基）（Ⅱ）、10,11二甲氧基－[2]苯并吡喃[4,3-b]-呋喃并[2,3-h][1]苯并吡喃－6（8H）－酮（Ⅲ）,皆未见文献报道,分别命名为厚果鸡血藤丙素（pachycarin C)、厚果难血藤丁素（pachycarin D)和厚果鸡血藤戊素（pachycarin E)。     

Software-assisted serum metabolite quantification using NMR

The goal of metabolomics is to analyze a whole metabolome under a given set of conditions, and accurate and reliable quantitation of metabolites is crucial. Absolute concentration is more valuable than relative concentration; however, the most commonly used method in NMR-based serum metabolic profiling, bin-based and full data point peak quantification, provides relative concentration levels of metabolites and are not reliable when metabolite peaks overlap in a spectrum. In this study, we present the software-assisted serum metabolite quantification (SASMeQ) method, which allows us to identify and quantify metabolites in NMR spectra using Chenomx software. This software uses the ERETIC2 utility from TopSpin to add a digitally synthesized peak to a spectrum. The SASMeQ method will advance NMR-based serum metabolic profiling by providing an accurate and reliable method for absolute quantification that is superior to bin-based quantification.