Skip to main content

Liquid State NMR for Organic Matter/Complex Mixtures

Hood
Hood
NMR
Liquids NMR

Liquid state nuclear magnetic resonance (NMR) can study mixtures or pure compounds, returning spectra rich in chemical information. Experiments using NMR can be targeted or untargeted, and a high degree of automation lends the technique to high-throughput studies, where established statistical methods can pull out key features to discriminate between sample groups. 

Complex mixtures, such as organic matter, are challenging to analyze with any technique. NMR can provide quantitative information on chemical functionality present in a sample, but limits on sensitivity and resolution make in-depth characterization challenging. 

EMSL hosts five solution-state NMR instruments that are a combination of high-field superconducting magnets, various electronics, and “probes.” These probes are tuned to specific atomic nuclei and allow for the direct (and indirect) characterization of various environmentally relevant nuclei including 1H, 13C, 15N, 19F, and 31P, as well as broadband tunable probes for other nuclei. Fully automated acquisition on two of our systems allows for 24/7 operation and is suited to high-throughput studies. 

Research application 

Supporting the Biogeochemical Transformations Integrated Research Platform and the Terrestrial-Atmospheric Processes Integrated Research Platform, liquid state NMR can be used to characterize natural organic matter, including dissolved organic matter from waterways (fresh or salt) or pore water, and liquid state soil organic matter extracts.

In addition: 

  • 1H and 13C NMR can provide quantitative or semi-quantitative analysis of the chemical functionality present in a sample. 

  • 31P NMR can directly characterize types of phosphorous present in a sample, for example quantifying and speciating inorganic and organic phosphorous in soil or sediment extracts. 

  • The intrinsically nuclei-specific nature of NMR supports stable isotope probing experiments with 13C or 15N enriched tracers. 

  • Effects of metal binding to organic matter may be probed directly through X-nuclei or indirectly through induced NMR property changes. 

  • Other complex mixtures, such as biofuels or lignin, are well suited to NMR characterization. For example, key structural motifs in lignin (such as β-O-4 linkages) can be identified and semi-quantified using two-dimensional 1H, 13C Heteronuclear Single Quantum Coherence experiments. Diffusion ordered spectroscopy NMR can inform on approximate molecular size in solution (hydrodynamic radii). 

Available instruments 

EMSL has a number of high-field, liquid state NMR instruments. The following summarizes key resources, but reach out to our subject matter experts to discuss technical needs and system suitability. 

  • 800 MHz Bruker Avance Neo (Tava), 24-position SampleCase sample changer, supporting 1H, 13C, 15N, 19F, and 31P nuclei across two CryoProbes. 

  • 750 MHz Bruker Avance III (Bokan), with 510-position cooled SampleJet sample changer, with a 1H, 13C, 15N CryoProbe and a broadband X-nuclei probe. 

  • 750 MHz Varian VNMRS (Rainier), with a 1H, 13C, 15N probe and a broadband X-nuclei probe.  

  • 600 MHz Agilent VNMRS (Hood): 5 mm HCN salt tolerant Z-gradient cold probe with VT control of 0 to 80 °C 

  • 600 MHz Agilent DD2 (Baker): 5 mm HCN Z-gradient cold probe with VT control of 0 to 50 °C 

Tips for success 

The following is a non-exhaustive list of common NMR tips. Contact the staff person listed on this page at your earliest opportunity to discuss details about sample preparation and experimental design considerations. 

  • Solution-state NMR is non-destructive and only requires small volumes of sample (200 μl for 3 mm tubes, 600 μl for 5 mm tubes). It is possible, and routine, to use a 3 mm sample tube in a 5 mm probe when samples are limited. 

  • NMR is not as sensitive as mass spectrometry, so providing more samples will be advantageous. 

  • NMR is sensitive to salt—higher salts reduce sensitivity, so limiting salts below 100 mM is ideal. Desalting methods (i.e., solid-phase extraction) are an option but will bias the later-detected compounds. 

  • CryoProbe or Cold Probe equipped instruments have higher sensitivity but are more affected by salt levels. 

  • 1H NMR is the most sensitive nuclei, with approximate limits-of-detection (in practical instrument time on our best instruments) for small molecules around 1–10 μM. Our most sensitive instrument for 1H experiments is the 800 MHz (Tava). 

  • NMR is quantitative and non-selective (in its normal operation), so highly abundant features may obscure less-abundant compounds. In biological applications, this means we have to suppress the water signal. Similar approaches can be used for other solvents, but this can affect quantitation. 

  • Solution-state NMR will only “see” what is in a solution. Particulates will not only go undetected but will seriously impede analysis. Centrifugation or filtration is key for samples that are not fully dissolved. Careful selection and filter washing is necessary to avoid leaching of common contaminants. 

  • Many solvents are NMR compatible—water, methanol, and chloroform are among the most common. We routinely add approximately 10% of a deuterated solvent and an internal standard to the sample for referencing. 

  • For most organic matter studies, data analysis is based on integrating signal regions. The throughput limiting factor in these studies is typically experimental time for low-concentration or high-salt samples. Where cleaned up (desalted) samples can be provided with high concentration, high-throughput studies are more feasible. 

Contacts