Environ. Sci. Technol. 2001, 35, 4289-4294
Two-Dimensional NMR Studies of Size Fractionated Suwannee River Fulvic and Humic Acid Reference S. HAIBER, H. HERZOG, P. BURBA, B. GOSCINIAK, AND J. LAMBERT* Institut fu ¨ r Spektrochemie und Angewandte Spektroskopie (ISAS), P.O. Box 10 13 52, D-44 013 Dortmund, Germany
Two-dimensional phase sensitive 13C,1H correlation spectra were applied to the investigation of substructures in size fractions obtained by tangential flow multistage ultrafiltration (MST-UF) of humic substances (HS) Suwannee River Fulvic Acid Reference (HS SR FA) and Suwannee River Humic Acid Reference (HS SR HA), purchased from the International Humic Substances Society (IHSS). After size fractionation with MST-UF the HS samples give well resolved two-dimensional 13C,1H-correlated NMR spectra which offer a great potential for substructure elucidation and even quantification. It is shown that low molecular size lignin moieties undergo demethylation of the methoxy groups, accompanied by removal of the phenylpropane side chains and subsequent reaggregation of the aromatic rings. These findings provide insight into the processes of lignin degradation. Only the fraction >100 kDa contains macromolecules that have spin-spin relaxation times too short for investigations employing NMR multipulse sequences.
Introduction Humic substances (HS) (1) are very complex mixtures of primarily organic low and high molecular weight components comprising a broad spectrum of substructures and functionalities. HS are involved in the transport, deposition, and availability of organic and inorganic pollutants in soils and natural waters. A fundamental understanding of these environmental processes requires investigation of HS structures. NMR has proven the most powerful tool for structural investigations of humic substances that are very difficult to perform as complete HS samples show only broad and featureless NMR spectra. While for peptides or nucleic acids three-dimensional structural investigations are nowadays feasible (2), for HS only substructure information, i.e., data on certain well-defined structural moieties can be obtained. Especially two-dimensional NMR has been shown (3-9) to be a valuable tool for substructure elucidation in humic substances. Cross-peaks in two-dimensional NMR spectra of polydisperse natural HS, however, still show considerable overlap. A reduction of structural complexity is therefore mandatory even for qualitative investigations. As has been suggested in the literature (3) and has been shown recently (8), an additional fractionation step is helpful to reduce the strong polydispersity of HS. Tangential-flow multistage ultrafiltration (MST-UF) under standardized conditions, which has just recently been optimized and successfully * Corresponding author phone: +49 231 1392 147; fax +49 231 1392 120; e-mail:
[email protected]. 10.1021/es010033u CCC: $20.00 Published on Web 10/05/2001
2001 American Chemical Society
applied (10-12) to the molecular size fractionation of aquatic HS, can be supposed to be promising for size fractionation of dissolved HS. Recently (8), we could show that the size fractions obtained from MST-UF give 2D NMR spectra which are more easily interpretable than the 2D NMR spectrum of the complete HS sample before separation. In this paper, we demonstrate the potential of the combination of MST-UF with twodimensional 13C,1H correlated NMR spectroscopy. This combinated technique delivers new information on substructures present in aquatic HS. It is shown that lignin components representing different stages of decomposition can be readily identified in the spectra of the fractions.
Experimental Section Humic Substances. The river water HS Suwannee River Fulvic Acid Reference (SR FA) and Suwannee River Humic Acid Reference (SR HA) were purchased from the International Humic Substances Society (IHSS). MST-UF. Five hundred milligrams of the HS samples as delivered by the IHSS was dissolved in 5 L of H2O and adjusted to pH 8.5. The carbon contents (see: DOC measurements) of the HS solutions were 207 mg and 201 mg for HS SR FA and HS SR HA, respectively. Differences between carbon contents obtained from DOC measurements and the originally weighed in carbon quantity are mainly due to moisture (FA 8.9%; HA 9.8%) and ash (FA: 0.98%; HA 3.46%) contents of the supplied HS. The size fractionation was performed with a preparative tangential flow multistage ultrafiltration cascade similar to that described in refs 10-12 and equipped with appropriate UF membranes (Pall Filtron OMEGA, diameter: 47 mm, nominal molecular weight-cutoff: 100, 50, 10, 5, and 3 kDa). In this setup several tangential-flow UF cells are online coupled together, which enables an easy handling and working in a closed flow system. The tangential flow, that is necessary to reduce polarization effects on the membranes, was adjusted to 25 mL/min. The penetrate flow of 0.5 mL/min as well as the tangential flow were obtained with a multichannel peristaltic pump working with an initial pressure of 2.5 bar. After fractionation, the HS fractions obtained were washed by introducing 50 mL high-purity water (Millipore Q) into the MST-UF system. The molecular weight ranges of the fractions were F1 (>100 kDa), F2 (10050 kDa), F3 (50-10 kDa), F4 (10-5 kDa), F5 (5-3 kDa), and F6 (
