Environ. Sci. Technol. 2003, 37, 2929-2935
Solid-State and Multidimensional Solution-State NMR of Solid Phase Extracted and Ultrafiltered Riverine Dissolved Organic Matter E D I T H K A I S E R , * ,† A N D R E J . S I M P S O N , ‡ KARL J. DRIA,‡ BARBARA SULZBERGER,† AND PATRICK G. HATCHER‡ Swiss Federal Institute of Environmental Science and Technology (EAWAG), Ueberlandstrasse 133, CH-8600 Duebendorf, Switzerland, and Department of Chemistry, The Ohio State University, 120 West 18th Avenue, Columbus, Ohio 43210
In this study we used multidimensional solution-state NMR to elucidate the differences in the chemical composition of solid phase extracted and ultrafiltered DOM isolates. DOM was isolated from water sampled from an oligotrophic river, the River Tagliamento (Italy). The recovery of total DOM was up to 42% with both isolation techniques. In addition to 1- and 2-D solution-state NMR, we also applied 1-D solidstate 13C NMR spectroscopy for DOM characterization. 13C NMR spectroscopy only produced broad overlapping resonances, thus allowing a bulk characterization of DOM composition. However, it demonstrated that the bulk chemical composition of the two DOM fractions exhibited minor spatial-temporal changes. The 2-D experiments (TOCSY, HMQC) showed that the solid phase extracted hydrophobic DOM contained predominantly aliphatic esters, ethers, and hydroxyl groups, whereas the ultrafiltered DOM was comprised partially of peptides/protein, with further evidence for a small amount of aliphatic/fatty acid material. Sugars were present in both DOM fractions. The results show the two isolation techniques selected for different suites of compounds within the bulk DOM pool.
Introduction Multiple analysis techniques, ranging from NMR spectroscopy to GC-MS, have been applied to examine the specific chemical features of DOM (1-4). Most of these techniques require that DOM, present in low concentrations in natural water samples, be concentrated using isolation methods such as XAD or C18 solid phase extraction and ultrafiltration (5). These methods also allow recovering DOM as a solid powder that can be analyzed by solid-state NMR (6, 7). Hitherto, mostly one-dimensional (1-D) NMR spectroscopy, solid-state 13 C NMR in particular, was applied to elucidate the DOM chemical composition. However, many published solid-state 13C NMR spectra exhibit only broad resonance peaks and often suffer from poor spectral quality due to sample complexity, and some even contain “spinning sidebands”. Advances in solid-state NMR have drastically reduced the * Corresponding author phone: +41-1-823 5096; fax: +41-1-823 5028; e-mail:
[email protected]. † EAWAG. ‡ The Ohio State University. 10.1021/es020174b CCC: $25.00 Published on Web 05/30/2003
2003 American Chemical Society
effect of spinning sidebands and allow the analysis of smaller sample quantities with increased sensitivity (8). Nevertheless, these NMR spectra still often exhibit broad overlapping peaks, due to the presence of paramagnetic materials and the dipolar interactions present in solid materials. In contrast, multidimensional solution-state NMR can give highly resolved information, even for complex mixtures (9). Multidimensional experiments (10) can be used to correlate the chemical shifts of different carbon and proton atoms and identify shortand long-range connectivities. In using isolation techniques to concentrate DOM, one must be aware that different isolation methods, e.g., C18 solid phase extraction and ultrafiltration, select for chemically different compounds. This is the first study in which homoand multinuclear solution-state NMR methods were used to compare the chemical composition of DOM isolated by C18 solid phase extraction and ultrafiltration. The goals of our study were (i) to use state-of-the-art NMR methods to elucidate the differences in the chemical composition of DOM isolates derived by chemical and physical fractionation techniques and (ii) to demonstrate that solution-state NMR spectroscopy is a tool of high potential for investigating the molecular nature of heterogeneous DOM mixtures. We show that NMR methods improve functional group assignments and aid in the understanding of DOM chemical structure.
Materials and Methods Study Area and Sample Collection. C18 solid phase extracted hydrophobic and ultrafiltered DOM samples come from water of the Tagliamento River, northeastern Italy. The river is the last unregulated river in Europe, flowing unrestrained from the alpine headwaters to its mouth in the northern Adriatic Sea (11). Water samples for C18 solid phase extraction and ultrafiltration were collected seasonally from the main channel in the headwater, major, and transition flood plains from spring 1999 to 2000. The samples are part of a larger study in which material was collected seasonally from three longitudinally aligned stations (Kaiser et al., in preparation). Water was collected during low-, medium-, and high-flow conditions (Table 1). Throughout the year, the main channel river water displayed temperatures of 10.9 ( 2.5 °C, pH values of 8.13 ( 0.14, and alkalinities of 2.8 ( 0.5 mM. Water samples for DOM fractionation were collected with clean 50 L highdensity polyethylene (HDPE) carboys and transported back to the field station. Immediately following collection, water samples were filtered through muffled glass fiber filters (GF/F) and prerinsed 0.2 µm Durapore filters (142 mm diameter, Millipore) and stored in clean 50 L carboys. DOM Fractionation and Isolation. For chemically fractionating DOM into hydrophobic and hydrophilic compounds, a Mega Bond Elute C18 column (C18 loaded silica, 60CC, Varian) was used after acidification with 32% hydrochloric acid (Suprapur) to pH 2.8. Flow rates ranged between 3 and 5 L h-1. Of the total dissolved organic carbon (DOC
