Juhani Peuravuori

Molecular size distribution and spectroscopic properties of aquatic humic substances

The number- and weight-averaged molecular weights of different isolated humic fractions and unfractionated organic matter from natural water samples were measured using high-performance size-exclusion chromatography (HPSEC). The chromatograms obtained, with sodium acetate as the mobile phase on a TSK G3000SW high-speed column, provided illustrative information about the nature of the dissolved organic matter that was not available with phosphate buffer eluents. The calibration of the column system merely with protein standards generated misleading information about the molecular weight and size distribution of humic solutes. Instead, the present results support the utilization of polystyrene-sulphonates (PSS) either alone or together with certain other model compounds for the calibration of HPSEC in studying molecular weights of humic solutes. The data obtained with the HPSEC system indicate that the average molecular weight of humic solutes is distinctly lower than is generally assumed. Besides, aquatic humic solutes also contain some very large-sized though minor constituents, leading to a fairly polydisperse mixture. Number-averaged molecular weights obtained using the HPSEC system were 4–5-fold greater than those from vapour-pressure osmometry. Bulk spectroscopic properties such as molar absorptivity at 280 nm and the quotient, E2E3, correlated strongly with the total aromaticity and averaged molecular weights of all the humic solutes. This observation suggests that bulk spectroscopic properties can be applied, as a first approximation, for estimating the size of humic solutes and their aromaticity in natural surface waters.

Characterization, differentiation and classification of aquatic humic matter separated with different sorbents: synchronous scanning fluorescence spectroscopy

Aquatic humic solutes were separated by the non-ionic macroporous XAD-8 and DAX-8 resins and a weakly basic DEAE cellulose anion exchanger from seven different fresh water sources. Synchronous fluorescence spectroscopy was applied for characterization, differentiation and classification of the different humic-solute aggregates. Fluorescence properties verified that humic-solute fractions isolated parallelly with the non-ionic XAD-8 and DAX-8 resins resembled very closely each other speaking strongly for their structural similarities. DAX-8 resin separated ca. 19% more aquatic humic matter than did the analogous XAD-8 resin. It was possible to tentatively differentiate the untreated water samples according to their fluorescent materials. Several distinct classes of chromophores were detected in both DOM and isolated humic fractions by the synchronous technique: lambda(ex)/lambda(em) 280/298, 330/348, 355/373, 400/418, 427/445, 460/478, 492/510 and 516/534 nm.

Isolation and characterization of natural organic matter from lake water: Two different adsorption chromatographic methods

Aquatic humic solutes were isolated from lake water by two different methods: non-ionic sorbing solid (XAD technique) and weakly basic anion exchanger (DEAE cellulose). The DEAE procedure removed from the fresh water sample at natural acidity 80% of the DOC as humic solutes. This was practically equal to the total amount of the three organic acids obtained directly by the XAD-8 resin at preadjusted acidity (pH 2) and of the acidic solutes obtained by a specific anion exchanger in connection with the XAD-8 column. The acidic gross character of the humic solutes obtained by the DEAE procedure was similar to the combined character of the four distinct acidic fractions obtained at pH 2 by different adsorbents. The structural composition of humic solutes obtained by the DEAE isolation procedure appears to be a given combination of the various hydrophobic and hydrophilic acidic solutes obtained at pH 2 by the XAD technique. It is likely the acidic treatments connected to the isolation and fractionation of organic solutes by the XAD technique can be to some extent coincidental, especially from the structural chemistry point of view. However, aquatic humic solutes (especially so-called fulvic and humic acids) modelled by the XAD technique are in some degree real, rather than accidental products of the isolation method.

Comparative study for separation of aquatic humic-type organic constituents by DAX-8, PVP and DEAE sorbing solids and tangential ultrafiltration: elemental composition, size-exclusion chromatography, UV-vis and FT-IR.

Aquatic humic-type solutes were separated in parallel from the same fresh water source by four different procedures: non-ionic polymethyl methacrylate (DAX-8) and functional cross-linked polyvinylpyrrolidone (PVP) resins, functional diethylaminoethyl cellulose (DEAE) and tangential ultrafiltration completed with a weakly basic anion exchange resin (IRA-67). The similarity-dissimilarity between the quantities and qualities of the different humic samples is discussed, especially in the light of the original dissolved organic matter (DOM). During the past two decades, a significant progress has occurred in the aquatic humic research due to the so-called hydrophobic-hydrophilic properties possessed by certain non-ionic sorbing solids. As a result of many coincidences, it may be justifiable to examine critically the prevailing isolation techniques of aquatic humic solutes and to try to update their complicated definitions. For that reason, it is reasonable to summarize the leading principles of different isolation techniques in Section 1 of this article. The results of the present study strongly support the applicability of the PVP resin, alone or completed in sequence with a suitable non-ionic sorbing solid, for isolation of aquatic humic-type solutes from both quantitative and qualitative points of view. In certain cases, the DEAE cellulose gives a useful alternative for conventional sorbing solids in the isolation of the bulk of aquatic humic solutes. The base-catalyzed ester hydrolysis of the HM during the chromatographic isolation of the DOM seems to be relative minor.

Comparisons of sorption of aquatic humic matter by DAX-8 and XAD-8 resins from solid-state 13C NMR spectroscopy's point of view

Aquatic humic solutes were separated in parallel by the non-ionic macroporous DAX-8 and XAD-8 resins from four different fresh water sources. On average, the sorptive power of the DAX-8 resin does not differ systematically from that of the XAD-8 resin. The DAX-8 resin seems to have more precise column characteristics compared with the XAD-8 resin. There was no significant difference between the major elemental compositions of the parallel humic-solute bulks obtained by these two resins. According to the (13)C NMR spectroscopy the content and quality of aliphatic carbons, especially those representing terminal methyl groups or methylene carbons, were the most systematic and powerful discriminating factors between the humic extracts obtained by these two resins. Generally speaking the DAX-8 and XAD-8 resins seem to isolate humic-solute bulks almost equally, although the content of aliphatics is slightly greater for the former, producing mixtures with similar structural compositions for general purposes. The structural composition and quantity of the humic-solute mixture isolable with a weakly basic DEAE-cellulose anion exchange resin differs partially from any humic fraction obtained by non-ionic sorbing solids. The environmental impact was also visible on the quality of the structural fine-chemistry of the different humic isolates obtained both by the DAX-8 and XAD-8 resins.

Characterisation of lake-aquatic humic matter isolated with two different sorbing solid techniques: tetramethylammonium hydroxide treatment and pyrolysis-gas chromatography/mass spectrometry

Tetramethylammonium hydroxide (TMAH) treatment followed up with pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) was applied to characterise the structural composition of lake aquatic humic matter (HM). The HM samples were isolated with two different chromatographic methods: (1) non-ionic sorbing solid (standard XAD technique) with preacidification of a water sample to pH 2; and (2) weakly basic anion exchanger (DEAE, diethylaminoethyl cellulose) without any preacidification. A detailed structural analysis of degradation products was performed and compositional differences between different HM samples were evaluated. The main degradation products, in addition to varying proportions of different nitrogen and sulphur compounds, were methyl derivatives of different phenols, alkylphenols, phenolic acids and aliphatic mono- and dicarboxylic acids, which speak for the importance of ester linkages between the different structural subunits of aquatic HM. Despite all HM samples yielded qualitatively quite similar organic compounds, some fundamental quantitative differences existed. As a general rule, the DEAE isolate and two main XAD fractions: conventional fulvic (FA) and humic (HA) acids, seem to consist of almost similar organic matter dominating by different phenols and aliphatic acids. The other two special XAD fractions appear to belong to different structural categories yielding more alkylbenzenes/styrenes, alkanes and aliphatic monocarboxylic acids. The structural analyses and statistical calculations on the different HM samples support the postulation that the structural composition of the DEAE isolate corresponds to a certain average of the four different acidic XAD fractions.

Sorption of aquatic humic matter by DAX-8 and XAD-8 resins: Comparative study using pyrolysis gas chromatography

Abstract Aquatic humic solutes were separated in parallel by the non-ionic macroporous DAX-8 and XAD-8 resins from four different fresh water sources. The sorptive power of the DAX-8 resin was systematically somewhat greater compared to that of the XAD-8 resin. The DAX-8 resin seems to have more precise column characteristics compared with the XAD-8 resin. According to the tetramethylammonium hydroxide (TMAH) procedure followed up with pyrolysis gas chromatography–mass spectrometry (Py-GC–MS) the content and quality of aliphatic carbons were the most systematic and powerful discriminating factors between the humic extracts obtained by these two resins. Generally speaking, the DAX-8 and XAD-8 resins seem to isolate humic solute bulks almost equally, although the content of aliphatics is slightly greater for the former, producing mixtures with similar structural compositions for general purposes. The environmental impact was also visible on the quality of the structural fine chemistry of the different humic isolates obtained both by the DAX-8 and XAD-8 resins.

Critical comments on accuracy of quantitative determination of natural humic matter by solid state 13C NMR spectroscopy

Structural information of natural organic matter (NOM) at the molecular level is very essential in understanding their nature and reactivity. Nuclear magnetic resonance (NMR) is an excellent tool for estimating the gross chemical composition of the very complex humic matter (HM). A well-known fact is that the solid state (13)C NMR spectral analysis is very parameter-sensitive especially in the study of the heterogenous HM (e.g. baseline corrections, different pulse techniques and spinning rates of the rotor vs. different disruptive sidebands in the spectra). This being the case, it has been emphasized the importance of qualitative and quantitative analyses for generating as real spectra as possible by means of different pulse and polarization techniques, sampling spinning rates as well as certain correction factors. In the present study a practical accuracy for quantitative determination of NOM type material by solid state (13)C NMR spectroscopy was assessed using a known HM sample. Different magnetic-field strengths, sampling spinning rates, single and ramped amplitude cross polarization techniques and TOSS pulse sequence were applied for obtaining a more reliable insight into the disruptive effect of the chemical shift anisotropy (CSA), especially the most disturbing first order spinning side bands (SSB). The results demonstrated that the SSB problem is not so significant as sometimes stated, at least in the context of HM samples and in the light of the overall reproducibility and uncertainty connected with the sample itself.

Isolation and characterization of natural organic matter from lake water : Comparison of isolation with solid adsorption and tangential membrane filtration

Abstract It is well known that the different steps in applying non-ionic macroporous sorbing solids (such as XAD resins or their analogous) to isolation of aquatic humic substances at preadjusted acidities contain some risks for uncontrolled fractionation or reactions. According to this work, it is likely that structural alterations take place in the block of dissolved organic matter, in addition to the removal of certain ‘impurities’, during isolation of aquatic humic substances with the XAD technique. Evidently macromolecular organic solutes with different molecular sizes in an aquatic environment, separated from the dissolved organic matter and defined as humic substances based on a special isolation technique, cannot be merely accidental products of the isolation procedure. However, the partition of aquatic humic substances into distinct humic- and fulvic-acid fractions at strongly acidic conditions does not lead to a chemically unique result. For that reason, it is questionable whether these humic- and fulvic-acid entities are originally present in the dissolved organic matter or created by chemical alterations at strongly acidic conditions.

Multimethod characterization of lake aquatic humic matter isolated with sorbing solid and tangential membrane filtration

According to this work, it is evident that structural alterations take place in the organic solutes defined as humic substances, in addition to the removal of certain “impurities”, during specific isolation of aquatic humic solutes at preadjusted acidity by non-ionic sorbing solids (e.g. XAD-resins). The acidic partitioning of the humic solutes at strongly acidic conditions into humic- (HA) and fulvic-acid (FA) fractions does not lead to a chemically unambiguous result, especially in the case of humic solutes with smaller molecular sizes (most significant fraction of humic solutes). The combined molecular formula (C,H,O) recalculated from distinct HA- and FA-entities, after acid precipitation of the smaller size humic solutes, was ca. 50% greater than that obtained for their corresponding unseparated HA↔FA mixtures. Also, the combined relative unsaturation (mainly due to aromatics and in particular cycloalkyl groups) recalculated from distinct HA- and FA-entities after acid precipitation of the smaller size humic solutes was ca. 10% greater than that obtained for their corresponding unseparated mixture. The combined degree of substitution of aromatic rings recalculated from distinct HA- and FA-entities after acid precipitation was, on an average, 40% greater than that estimated for their total unseparated mixture, and even ca. 90% greater in the case of smaller size humic solutes. The combination of the distinct HA- and FA-subfractions with different molecular sizes generates a molecular formula close to that obtained for the corresponding individual HA- and FA-solutes, respectively (relative differences of C, H and O: 5±2%). Also, the composition, based on the NMR spectra of different types of carbons and hydrogens, was particularly similar (r=0.98 and 0.82, respectively) for the computationally combined distinct HA- and FA-subfractions with different molecular sizes and for the corresponding individual HA- and FA-solutes, respectively. Isolation/fractionation of aquatic humic solutes with the XAD technique appears to occur with nearly the same mechanism independent of the molecular size or of the degree of polydispersity of the solutes. Therefore the isolated humic solutes (especially the unseparated mixture of HA and FA) must play a role as certain definite entities in the dissolved organic matter (DOM) and they cannot be merely accidental products of the isolation processes, though they do not seem to be fully in accord with the original DOM. With a computational method, the most probable carbon distribution was generated for a humus sample.