Analytical pyrolysis and computational chemistry of aquatic humic substances and dissolved organic matter

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<ul><li><p>Journal of Analytical and Applied Pyrolysis49 (1999) 385415</p><p>Analytical pyrolysis and computationalchemistry of aquatic humic substances and</p><p>dissolved organic matter</p><p>H.-R. Schulten *Institute of Soil Science, Uni6ersity of Rostock, Justus-6on-Liebig-Weg 6, 18059 Rostock, Germany</p><p>Received 10 July 1998; accepted 12 December 1998</p><p>Abstract</p><p>Humic acids (HA), fulvic acids (FA), non-humic substances (NHS) and dissolved organicmatter (DOM) in a bog lake water are investigated by analytical pyrolysis. The appliedthermal methods are direct, in-source pyrolysis-field ionization mass spectrometry in the highelectric field (Py-FIMS), and Curie-point pyrolysis-gas chromatography:mass spectrometry(Py-GC:MS) in combination with library searches. Based on the identified building blocksand together with complimentary analytical data, proposals for a general concept of thebasic molecular structures of humic macromolecules in water are put forward. Computa-tional chemistry is utilized for structural modeling and geometry optimization of DOM.Molecular mechanics calculations are performed to evaluate the conformation of structural,three-dimensional models and to determine the total energy and the partial contributionsfrom bond-, angle-, dihedral-, van der Waals-, stretch-bend-, and electrostatic energies.Quantitative structureactivity relationship (QSAR) properties are calculated and allow thecorrelation of molecular structures with properties such as mass, surface area, volume,partial charges (electronegativity), polarizability, refractivity, hydrophobicity, and hydrationenergy. The principal aim and long-term strategy are to develop step by step improvementsof the presented model structures of organic matter in water which explain the molecularcomposition as well as their ecological meaning, dynamic character, and structurepropertyrelationships in natural and contaminated aquatic and terrestrial systems. In a first integratedapproach, the dissociation and association processes of humic substances are simulated atnanochemistry level and are proposed as concepts for future collaboration incorporatingresults of additional chemical, biological, spectroscopic and microscopic methods. 1999Elsevier Science B.V. All rights reserved.</p><p>* Tel.: 49-381-4982137; fax: 49-381-4982159; e-mail: hans-rolfschulten@agrarfak.uni-rostock.de.</p><p>0165-2370:99:$ - see front matter 1999 Elsevier Science B.V. All rights reserved.PII: S0165 -2370 (98 )00137 -5</p></li><li><p>386 H.-R. Schulten : J. Anal. Appl. Pyrolysis 49 (1999) 385415</p><p>Keywords: Association:dissociation processes; Dissolved organic matter; Fulvic, humic acids; Modeling;Molecular mechanics; Nanochemistry; Pyrolysis-field ionization mass spectrometry; Pyrolysis-GC:MS;Surface-water; QSAR</p><p>1. Introduction</p><p>Humic substances and soil organic matter are essential bases for life on earth,and the study of their structures is the aim of a fast growing, interdisciplinary,scientific community. The soil organic matter (SOM) refers to the sum-total of allcarbon-containing substances in soils. It influences plant growth through its effectson the physical, chemical, and biological properties of soils. SOM consists of amixture of plant and animal residues in various stages of decomposition, ofsubstances synthesized microbiologically and:or chemically from breakdown prod-ucts, and of the bodies of live and dead microorganisms, small animals, and theirdecomposition remains. To simplify this chemically very complex and physicallyheterogeneous system, SOM is usually subdivided into non-humic and humicstructures. Non-humic substances include those with still recognizable chemicalcharacteristics (e.g. carbohydrates, proteins, fats, waxes, etc.). The major part ofSOM, however, consists of humic substances. These are amorphous, dark-colored,partly aromatic, polyelectrolyte-like materials which range in molecular weightsfrom a few hundred to several thousand [1,2]. The latter macromolecular structuresbetween 1 nm and 1 mm in size can be regarded as colloids and above 1 mm ashumic particles.</p><p>Similar humic substances can be isolated from aquatic systems by reverseosmosis, ultrafiltration and adsorption:desorption from resins. The water-solublepart obtained by filtration B0.45 mm can be determined as dissolved organiccarbon (DOC). Since the applied methods of analytical pyrolysis are well-suited forinvestigations of structural and molecular properties of humic substances, in thefollowing we refer to the sum-total of all carbon-containing substances in filtered,freeze-dried water as dissolved organic matter (DOM). Comprehensive surveys onthe isolation, characterization and properties of DOM and humic substances inwater and SOM in soils have been published and underline the structural complex-ity and environmental relevance of these materials [310].</p><p>Among the analytical methods of aquatic and terrestrial humic substances thecombination of pyrolysis and field ionization mass spectrometry (Py-FIMS) andCurie-point pyrolysis-gas chromatography:mass spectrometry (Py-GC:MS) wasfound to be a powerful tool to produce structural information about the molecularbuilding blocks of SOM and DOM. The advantages of the methodology aresensitivity, specificity and speed. Moreover, future applications and basic researchin soil science utilizing results of pyrolysis-field ionization mass spectrometrytogether with other modern spectroscopic methods, particularly in an integratedapproach, appear promising [11].</p><p>The aim of this work is to clarify whether analytical pyrolysis, molecularmodeling, and computational chemistry can contribute to a better understanding of</p></li><li><p>387H.-R. Schulten : J. Anal. Appl. Pyrolysis 49 (1999) 385415</p><p>the structure of DOM and aquatic humic substances. Four crucial questions arise:Firstly, is it possible to identify the produced thermal fragments on the basis ofPy-FIMS data banks of well-described standards of biopolymers, humic substances,waters, and soils? Secondly, how far is support available by other analyticalmethods such as Py-GC:MS, W, FTIR, NMR, etc., for an unambigeous assign-ment of structural building blocks? Thirdly, what is the origin of these subunits inhumic substances and DOM and are there possibilities to understand and predictthe processes of DOM, FA, and HA formation? Finally, can 3D molecularstructural concepts for DOM be developed on the basis of accepted analytical datawhich allow the visualization and simulation of DOM? What are the results ofcomputational chemistry and nanochemistry?</p><p>2. Experimental</p><p>2.1. Materials</p><p>Standard reference samples of the priority research program Refractory OrganicAcids in Water of the German Research Association (Deutsche Forschungsgemein-schaft; DFG) were investigated. Refractory organic acids in water were opera-tionally defined as organic acids of different origin prepared according to themethods recommended by the International Humic Substances Society (IHSS). Theisolation and purlfication by the XAD procedures [12,13] and characteristic data ofaquatic and terrestrial organic substances have been reported [14]. The brown waterof the Hohloh Lake (code no. HO10) was sampled in Feb. 1995 and the HA andFA fractions were prepared in the following month.</p><p>2.2. Curie-point pyrolysis-gas chromatography:mass spectrometry (Py-GC:MS)</p><p>The experimental set-up for Py-GC:MS has been described previously [8,15].Samples of freeze-dried water, humic substances, sediments and soils are pyrolyzedin a type 0316 Curie-point pyrolyzer (Fischer, 53340 Meckenheim, Germany). Thematerials were not pretreated except drying and, if necessary, milling. The finalpyrolysis temperatures employed were 300C, 500C and 700C, respectively.However, the data reported for aquatic materials in this study were all obtained at500C final temperature. The total heating time was varied between 3 and 9.9 s.Following split injection (split ratio 1:3; flow rate 1 ml 20 s1) the pyrolysisproducts were separated on a gas chromatograph (Varian 3700, 64289 Darmstadt,Germany), equipped with a 30 m capillary column (DB5), coated with 0.25 mm filmthickness and an inner diameter of 0.32 mm. The starting temperature for the gaschromatographic temperature program was 40C, and the end temperature 250C,with a heating rate of 10 K min1. The gas chromatograph was connected to athermionic nitrogen-specific detector and a double-focusing Finnigan MAT 212mass spectrometer. Conditions for mass spectrometric detection in the electronionization mode are3 kV accelerating voltage, 70 eV electron energy, 2.2 kV</p></li><li><p>388 H.-R. Schulten : J. Anal. Appl. Pyrolysis 49 (1999) 385415</p><p>multiplier voltage, 1.1 s (mass decade)1 scan speed and a recorded mass rangebetween m:z 50 and 500. A detailed description of the principle, potential andlimitations of Py-GC:MS of humic fractions and soils has been presented [15].</p><p>2.3. Pyrolysis-field ionization mass spectrometry (Py-FIMS)</p><p>For temperature-resolved Py-FIMS, 15 mg of freeze-dried water and:or about100 mg of aquatic humic substances such as humic acids (HA), fulvic acids (FA),and non-humic substances (NHS) were thermally degraded in the ion source of aMAT 731 (Finnigan, 28127 Bremen, Germany) modified high performance (AMDIntectra GmbH, 27243 Harpstedt, Germany) mass spectrometer. The instrumentalset-up for the field ionization (FI) ion source has been reported [16].</p><p>The samples were weighed before and after Py-FIMS (error90.01 mg) todetermine the pyrolysis residue and the produced volatile matter and to comparethem with the corresponding results from off-line pyrolysis. A heatable:coolabledirect introduction system with electronic temperature-programming, adjusted atthe 8 kV potential of the ion source and a field ionization emitter, were used. Theslotted cathode plate serving as counter electrode was set at 6 kV potential.Thus, at 2 mm distance between the emitter tips and the cathode, a total potentialdifference of 14 kV is applied, resulting in an extremely high electric field strength,essential for soft ionization. All samples were heated in high vacuum (1.3104</p><p>Pa) from 100 to 700C at a heating rate of approximately 10 K min1. Dependingon the volatility and thermal stability of the sample materials, 4060 magneticscans of the gaseous, ionized pyrolyzate components were recorded in the massrange 151000 mass units.</p><p>In general, at least three replicates were run for each sample. The total ionintensities (TII) of the single spectra were normalized to 1 mg sample weight,averaged for replicate runs, and plotted against the pyrolysis temperature, produc-ing Py-FIMS thermograms. For the selection of biomarkers and quantitativeevaluations, in particular freeze-dried water and aquatic humic substances, detaileddescriptions of the method for SOM in soil particle-size fractions and whole soilshave been published [17]. It should be noted that the marker signals have beenselected mainly for humic fractions and topsoils and are valid only for investiga-tions using in-source field ionization. Moreover, careful examination of the data hasto consider: (a) only series of ions for one compounds class; (b) the appropriatetemperature interval of volatilization; (c) the thermograms of biomarker signalsshould display gaussian-shape convolutions; and finally, the crucial point is thecorroboration of the results by independent analytical methods. In our work mainlyPy-GC:MS results in connection with chemical, biological and physical data wereexamined.</p><p>2.4. Structural modeling and geometry optimization</p><p>Since practically all pyrolysis data of aquatic humic substances and DOM havebeen published as two-dimensional plots, it is of interest to illustrate the potential</p></li><li><p>389H.-R. Schulten : J. Anal. Appl. Pyrolysis 49 (1999) 385415</p><p>of available, relatively low cost software and personal computer (PC) equipmentwhich allow threedimensional (3D) displays and computer-aided design for chemi-cal structures and model reactions. Especially the possibilities for molecular model-ing and geometry optimizations of complex macromolecules, which are often thetarget of analytical pyrolysis, virtually open up a new dimension. This is demon-strated below for molecular visualization and simulation of aquatic humic sub-stances and DOM. One of the essential aspects of applying computationalchemistry to geometry optimization, determination of partial energies, associationand dissociation processes, trapping and binding of biological and anthropogenicmolecules, chemical properties, etc., is that the available spatial dimensions coverthe range of atomic-, functional group- and whole colloid-structures at nm level(Nanochemistry).</p><p>The previously proposed terrestrial HA [18,19], SOM [20,21] and soil [22,23]model structures were obtained using the HyperChem software [24] (release 5.02;Microsoft Windows 95) for all model construction, chemical interaction studies,molecular mechanics and molecular dynamics calculations. The original Hyper-Chem outputs in Angstrom and kcal have been converted to in nm and kJ,respectively. Quantitative structureactivity relationship (QSAR) properties weredetermined using the ChemPlus software (Hypercube Inc., release 2.0). Theemployed IBM-compatible PC consisted of the processor Pentium Pro 200:256K,Intel-board Venus ATX P-Pro, in combination with 128 MB random accessmemory, Diamond Stealth Video 25 000 2MB, PCI graphic card, Iiyama 17%% colormonitor, 2.1 GB hard disk, and peripheral hardware.</p><p>The molecular mechanics calculations were performed using the HyperChemMM force field developed for organic molecules. Their atoms are treated asNewtonian particles interacting through a potential energy function. The potentialenergies depend on bond lengths and angles, torsion angles, and non-bondedinteractions which include van der Waals forces, electrostatic interactions, andhydrogen bonds. In these calculations, the forces on atoms are functions of atomicposition [24].</p><p>In this contribution we used molecular mechanics and an all atom force fielddeveloped for organic molecules to compute a potential energy surface. This surfacerepresents the energy of a molecular system with N atoms as a function of the 3NCartesian coordinates. In order to optimize the geometry (minimize the totalenergy) of a chemical conformation, the number of computing cycles required fora gradient calculation is approximately proportional to the number of atoms N,and the time per cycle is proportional to N2. This gradient is the derivative of theenergy with respect to all Cartesian coordinates. Moreover, the size and quality ofthe initial structure, the computer capacity and the termination conditions of themodeling calculations are crucial. In general, the convergence limit is either thenumber of calculations cycles (the default number is 15 times the number of atoms)or a gradient for the molecular system (default of 0.419 kJ (0.1 nm)1 mol1). Forthe humic colloids and DOM a gradient \1 kJ (0.1 nm)1 mol1 was chosen andthus within reasonable calculation times (N\1000 g mol1, B100 h) local...</p></li></ul>

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