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Nuclear Magnetic Resonance Spectroscopy and Its Key Role in Environmental Research Andre J. Simpson,* Myrna J. Simpson, and Ronald Soong Environmental NMR Centre, Department of Chemistry, University of Toronto, Toronto, Canada frequency is referred to as the “chemical shift” and identifies the different types of chemical groups within a molecule and their bonding environment. Also the peak area, in a properly acquired spectrum, is fully quantitative. Thus in its most basic form one-dimensional (1D) NMR identifies the types of nuclei present (via chemical shift) and how many of each type is present (via peak intensity). Multidimensional NMR on the other hand can be used to identify correlations that depict how nuclei are connected to form a complete structure. A series of multidimensional spectra are analogous to a “molecular jigsaw puzzle”, and assuming no spectral overlap, can be pieced together in one way to produce a single unambiguous chemical structure. NMR however, is not restricted to simple chemical structures but can be used to solve the primary structure of proteins as well as their folding, dynamics, 3D solvated shape, and inter/intramolecular interactions. In proteins, many similar nuclear environments are present and can be resolved through Nuclear magnetic resonance (NMR) is arguably the most additional spectral dispersion afforded by multidimensional powerful and versatile tool in modern science. It has the NMR experiments. The theoretical resolving power of NMR is capability to solve complex structures and interactions in situ considerable,1 with 1D 1H and 13C NMR having a peak even in complex heterogeneous multiphase samples such as capacity on the order of 5000 and 30 000, respectively, 2D soil, plants, and tissues. NMR has vast potential in environ1 H−13C >1 000 000, 3D NMR 1H−13C−15N 100 000 000, and mental research and can provide insight into a diverse range of 4−8D >1011−1026. The key message is that NMR, if employed environmental processes at the molecular level be it identifying to its full potential, has considerable capability to resolve the binding site in human blood for a specific contaminant or overlapping signals be it in a complex structure such a protein the compositional dynamics of soil with climate change. or complex environmental matrices such as soil, sediment, Modern NMR-based metabonomics is elucidating contaminant water, or air particles. toxicity and toxic mode of action rapidly and at sub lethal
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concentrations. Combined modern NMR approaches provide a powerful framework to better understand carbon cycling and sustainable agriculture, as well as contaminant fate, bioavailability, toxicity, sequestration, and remediation.
WHY IS NMR IMPORTANT FOR ENVIRONMENTAL RESEARCH? NMR is unique in that it can be used to study liquids, gels, solids, gases, and any combination of the aforementioned. This is beneficial with respect to environmental samples. For example, in a soil, pore water and dissolved organic matter (DOM) will be present as a liquid, swollen organic matter can be “gel-like” and microbial cell walls, dry organic matter, and minerals will behave more like pure solids. Thus if we are to truly understand what a soil is we must not only study the various components in isolation but require the eventual capacity to study structure and organization in samples in their natural unaltered state. The same is true if we then want to determine how and why a contaminant strongly binds to soil. Ultimately a technology to monitor the process with molecular resolution in its natural state is required to describe both how (molecular orientation), and with what (for example lignin, protein, clay, etc.), in the soil the contaminant binds to, which in turn determine the best remediation practices.
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WHAT IS NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROSCOPY? Nuclear Magnetic Resonance (NMR) is arguably the most powerful analytical tool in modern science. Seven Nobel prizes have been awarded in the field of NMR and the technology has revolutionized medicine (largely in the form of magnetic resonance imaging) and biochemistry (complete protein structure determination), and is central for routine structure determination as well as a wide range of research in the chemical and physical sciences. Nearly all elements in the periodic table are NMR observable to some extent, meaning nearly all matrices, both inorganic and organic, can be studied by some form of NMR spectroscopy. NMR is especially useful for organic structures as all the commonly occurring nuclei (hydrogen, carbon, nitrogen, phosphorus) produce relatively narrow NMR signals. When a molecule is placed in a magnetic field, the nuclei resonate at specific frequencies that are characteristic of their chemical environment. The specific © 2012 American Chemical Society
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TYPES OF NMR SPECTROSCOPY AND THEIR APPLICATION TO ENVIRONMENTAL RESEARCH NMR is highly adaptable and can be applied to study most environmental matrices. Different techniques are applied at different scales (angstrom to millimeter) to unravel the molecular complexities found in various environmental samples. However, before this can be demonstrated it is important to briefly introduce the different types of NMR available. A more in-depth treatise of all the techniques, history, applications, and experimental protocols in environmental NMR are covered in the comprehensive reviews by Simpson et al.2 and Cardoza et al.3 Solution-State NMR Spectroscopy. Solution-state NMR spectroscopy provides the highest resolution data and comprehensive molecular information but only for soluble components. Solution-state NMR probes range in diameter from 1 mm (∼5 μL sample) to 10 mm (∼5 mL sample) with the most common sample size being 5 mm (∼600 μL sample). If a sample is naturally soluble, such as DOM, solution-state studies are recommended. Alternatively, it is often advantageous to extract/isolate various components from the matrices such as with the extraction of humic and fulvic acid from soils or sediments. The isolation can decrease the heterogeneity of the sample as well as remove paramagnetics, improving both sensitivity and resolution. The additional information from the isolated fractions is often very complementary and important for source apportionment of broader resonances observed with unfractionated samples. More recently it has been shown that solution-state NMR can be performed on practically all natural water samples without preconcentration.4 The advantage is that samples are completely unperturbed, but the disadvantages are the analysis takes at least 24 h of instrument time due to the low concentration of organic molecules, the water suppression involved is challenging, and only 1D NMR can be collected routinely. In specific cases, 2D NMR is possible at natural abundance.5 In a recent study of glacial ice constituents at natural abundance, over 90% of the components were observed and identified as simple biomolecules which were assigned using NMR databases.6 Multidimensional solution-state NMR spectroscopy is very useful for environmental research and has considerable impact on the elucidation of organic matter in soils, sediments, and more recently air particulates.7 Solid-State NMR Spectroscopy. Solid-state NMR is traditionally performed on dried samples packed into a rotor. The most common rotor sizes are 7 mm (∼500 mg sample) and 4 mm (∼100 mg sample). The sample is spun between 5 and 13 kHz at the magic angle to reduce any unwanted linebroadening and nuclear spin interactions. In the solid state 1 H−1H dipole interactions are very strong generally resulting in 1 H NMR with a very wide spectral profile. In specific situations 1 H line shape is sufficiently narrow to permit chemical shift information to be obtained directly, such as the structural water in clays.8 In other cases techniques such as combined rotation and multiple-pulse spectroscopy (CRAMPS) can be used to narrow the 1H line width.9 As such, 13C is the most commonly detected nucleus in the solid state for environmental samples. Due to the low sensitivity of 13C detection, cross-polarization (CP) is often used to enhance the 13C signal. During CP some of the magnetization is passed from proton to carbon enhancing the signal up to 4 fold. CP-MAS is not fully quantitative as carbons far removed from protons (for example COO− carbon) are less enhanced than other moieties. If fully
quantitative solid-state NMR is essential, direct polarization (DP-MAS) must be performed.10 In general though acquiring a DP-MAS spectrum takes a lot longer than acquiring a CP-MAS spectrum. In complex matrices such as soil with low carbon content DP-MAS may take as long as 1 week to acquire, thus CP-MAS (which usually takes less than 24 h) tends to be performed on a more routine basis.11 Solid-state 13C NMR of environmental samples gives an excellent overview of the type and distribution of carbon within a sample with little pretreatment. Samples such as plant biomass that are organic rich may benefit from drying. Whereas, soils sediments or air particles that have high inorganic contents can benefit from hydrofluoric acid treatment12 which dissolves paramagnetic minerals and concentrates organic matter resulting in high-quality NMR data. Studies employing other NMR nuclei such as phosphorus13 and nitrogen14 have great potential to understand nutrient cycling whereas fluorine NMR is useful to study contaminant interactions.15 Multidimensional NMR in the solid state is possible and has proved extremely useful for the analysis of environmental samples including understanding humification processes,16 discovery of crystalline domains,17 unravelling mechanisms of contaminant sorption,18 and aggregate soil structure.19 Gel-Phase NMR Spectroscopy. Gel-phase NMR (commonly referred to as HR-MAS NMR) was first introduced in 1996.20 HR-MAS NMR probes use low power circuitry and are optimized for 1H detection. Swellable or gel-like samples can be analyzed with line-shape similar to that observed in solutionstate NMR. With 1H detection, gel-phase NMR can observe signals from structures that are not in the true solid state (i.e., exhibit some local mobility). The 1H signals from true solids may be too broad for conventional 1H acquisition and linenarrowing approaches such as CRAMPS cannot be applied as HR-MAS probes cannot handle the RF power required for these experiments. The advantage of gel-phase NMR is that information from the liquid or gel-like components can be obtained from intact samples. In the case of tissues, organisms, and plant materials, the natural water component acts as the “solvent” permitting information from the flexible biopolymers and metabolites to be obtained in situ. In other cases the solvent system can be manipulated to obtain new information. For example if a dry whole soil is wetted only the components in contact with the water will appear in gel-phase NMR which in turn describes the structural moieties at the key soil−water interface.21 Other environmental studies have used gel-phase NMR to monitor sorption kinetics of contaminants across the soil−water interface,22 as well as provide a wealth of structural information on a range of environmental matrices such as plants,23 microbes,24 clay−organo complexes,25 vegetables,26 and in vivo organisms.27 Comprehensive Multiphase (CMP) NMR Spectroscopy. Comprehensive multiphase (CMP) NMR spectroscopy is a recently introduced approach for performing NMR on natural intact samples. The technology incorporates all of the aforementioned methods into one single approach.28 CMP includes magic angle spinning, a gradient coil, a lock, is fully susceptibility matched for excellent line shape, and includes full high power solids circuitry that permits the most demanding solids experiments. The result is a universal approach that can observe all bonds, in all phases in natural, unaltered samples. Samples are deposited directly in the sample rotor and a lock solvent is added. This can be added directly to the sample or in an external capillary if required to keep the lock solvent 11489
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Figure 1. (A) Relating NMR and environmental scale and (B) relating NMR and different environmental matrices demonstrating that modern NMR is very versatile and can be applied to study liquids, gels, solids, intact samples, interfaces, structures, and interactions at varying scales.
the bond distance between two aromatic protons in a molecule). Scale (ii) represents nanometers and would be appropriate when using, for example, diffusion-based solutionstate NMR to study how individual molecules associate and aggregate to form colloids. Scale (iii) deals with micrometers which may be the case when using gel-phase or CMP-NMR to study interactions of either individual molecules or colloids with mineral surfaces. Scale (iv) considers micrometer to millimeter interaction as would be the case when studying the association, layering, and conformation of components in whole soils. In this case solid-state NMR experiments (using either a conventional solids probe or a CMP probe) would be most useful as the large dipoles in solid materials permit interactions to be measured over large distance. Finally, scale (v) connects to the visual scale in terms of magnetic resonance imaging (MRI) spectroscopy. In MRI localized spectroscopy is possible permitting the NMR spectra of soluble components to be extracted from a single voxel helping to understand how molecular components and interactions vary spatially as well as providing a neat “bridge” between molecular and visual observations. The important message is that NMR is very versatile in terms of the techniques and experiments that can be employed and studies can be varied to probe different phenomena in different sample matrices and different scales. This is depicted diagrammatically in Figure 1B which aims to relate different NMR approaches to different environmental matrices. Position 1 shows an MRI of soil. MRI studies are excellent for understanding water or contaminant mobility and flow in situ. However, it could be argued that little direct information is provided as to the molecular mechanisms producing the larger scale dynamics. Position 2 represents the other extreme, showing a very high resolution 3D NMR experiment for organic matter isolated from soil. Multidimensional solution-state NMR studies provide unrivalled molecular information even for mixtures of novel molecules but only for soluble components. Position 3 depicts that targeted studies can be employed to obtain information at key environmental interfaces, for example the solid−water interface. Here gelphase approaches can be used to assess the interactions and preservation of organic matter on minerals surface in the presence of water. Position 4 presents direct NMR of ice (4a) and river water (4b). The DOM in natural water can be
separate from the sample itself. The technology was developed specifically with environmental and biological samples in mind such that they could be studied in their intact and natural state.28 Not only can the individual phases (solid, gel, solution) be studied and fully differentiated in situ, but interactions and kinetics at interfaces can also be monitored. Consider, for example, the fate of a hydrophobic pesticide after a spill. Immediately after introduction, the hydrophobic pesticide will be solvated but it will quickly interact with the soil interface and take on properties of a “semi-solid”, eventually migrating into the hydrophobic interior of the soil structure where it becomes entrapped or sequestered in a solid domain. CMP-NMR permits for the first time such processes to be followed in situ with full molecular resolution and in real time. CMP-NMR provides a detailed insight into the processes that facilitate the entrapment of contaminants in a soil matrix and as such it represents a powerful tool to assess the efficiency and effectiveness of existing and novel remediation approaches. In many ways CMP-NMR may be thought of as “changing NMR technology to match the sample” rather than changing the sample to suit a specific type of NMR analysis. Because of its unique abilities to study samples in their native state CMPNMR will have a considerable impact on the field of environmental research. Low Field and Portable NMR. While most NMR measurements are obtained at high magnetic field, interesting measurements can be made at lower fields. Fast field cycling (FFC) NMR is becoming a powerful technique to study dynamics in liquid systems as well as liquid−solid suspensions. Example studies have looked to dynamics in decaying leaf litter,29 soil pore size,30 and soil contamination.31 Mobile NMR studies are also becoming more common with measurements predicting the porosity of rocks,32 analysis of pipes,33 determination wood moisture content,34 and even water diffusion in the Antarctic.35 Applying Different NMR Technologies to Environmental Samples. Different NMR techniques can be utilized to probe molecular bonds, interactions, and distances over varying scales. This is illustrated in Figure 1A using soil as an example. The smallest scale (i) represents the Angstrom scale and would be appropriate for solution-state studies looking at the molecular structure of individual components (for example 11490
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Figure 2. (Left) Two dimensional 1H−13C HSQC NMR spectra of ultrafiltered dissolved organic matter isolated from Pacific Ocean water. The main assignments can be summarized as 1 = methyl bound to carbon and sulfur (dotted circle), and in the lower left corner, branched purely aliphatic CH pairs and polymethylene, 2 = methylene and methine cross-peaks without direct bonds to heteroatoms, 3 = low intensity cross-peaks from methoxyl, 4 = cross-peaks representative mainly of α-CH in proteins and vicinal dicarboxylic acids, 5 = carbohydrate methylene cross-peaks, 6 = carbohydrate methine cross-peaks, and 7 = anomeric units in carbohydrates. A1C = (poly)alanine-CH3; A2C = methylated carbohydrates; A3C = Nacetyl carbohydrate. Reproduced with permission from Hertkorn et al.43 2006 Elsevier Science. (Right) (B) Representation of the molecular organization of components in the Saguenay Fjord, Canada, and (C) model of a Gram-negative cell wall. Reprinted with permission from Mao et al.46 2007 Elsevier Science.
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QUESTIONS IN ENVIRONMENTAL RESEARCH NMR spectroscopy plays a key role in most areas of environmental research. In general however, the questions can be categorized into two main areas, namely, molecular structure and molecular interactions.
analyzed completely and without preconcentration using a solution-state NMR probe in combination with advanced water suppression. Unfortunately this is not the case for most environmental matrices which often contain a combination of phases (liquid, gel, solid). This is exemplified at position 5 which demonstrates how a combined NMR approach can be used to assess all the components in air particles.36 In this case soluble components were extracted and studied with solution NMR (a), swellable components by gel-phase NMR (b), and the insoluble reside by solid-state NMR (c). The result is that all the organic components in the sample could be assessed and excellent structural information gained, with the drawbacks being that sample preparation was extremely laborious, required harsh extracting solvents, and the study could only performed by a NMR lab that had access to solution, gel, and solid-state NMR technology, which is a rarity rather than the norm. With the advent of comprehensive multiphase NMR such studies are now possible on whole untreated samples and every molecular bond can be studied and differential in its native unaltered state. Position 6 highlights an example on a whole plant. 6a shows a high resolution 2D 1H−13C correlation which depicts the chemical environment of the C−H bonds in the soluble and gel-like components of the plant, including all the metabolites and structural biopolymers that demonstrate mobility. Conversely, 6b shows the components in the same sample that cannot be observed by the former approach and is dominated by cellulose and lignin which form the rigid “backbone” of the plant. The result is that if combined, MRI, solution-, gel-, and solid-state NMR can provide a comprehensive and nondestructive description of intact natural samples, and probe specific interactions or structural components as required.
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NMR AND MOLECULAR STRUCTURE Soil Organic Matter. Thus far NMR has played a pivotal role in unravelling the structure of soil organic matter (SOM). For many decades SOM was thought to consist mainly of humic substances and that these substances were cross-linked to form a unique chemical category. However, solution-state NMR has demonstrated that while SOM is extremely complex, the alkaline extractable component of soil is dominated by plant and microbial biopolymers at various states of decay.37 In a separate study, novel solvent systems were used to solubilize 70% of the humin (traditionally insoluble fraction) and solution NMR demonstrated predominance of microbial and plant biopolymers and their degradation products.38 Furthermore, NMR has shown that microbial cells themselves contribute much more to soil biomass than previously thought (can account for up to 50% of the total NMR signal) explaining the high amounts of protein often found in soil extracts.24 This work is supported by solid-state NMR of whole soils which demonstrates that known biopolymers make up much of the signal in whole soil.19 Understanding SOM as a complex mixture of microbial and plant residues at various stages of degradation better permits advanced NMR studies to understand key aspects such as soil aggregate structure, humification processes, fertility, and stability, and in turn better predict how this vast carbon pool responds to climate change, intensive agriculture, and land-use change.39,40 11491
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Figure 3. Probing molecular interactions in soil using saturation transfer difference (STD) NMR. (A) Epitope map for Trifluralin and its interaction at the whole soil−water interface. Adapted with permission from Shirzadi et al.22. 2008 American Chemical Society. (B) How RHSTD NMR can used to identify components in extracted SOM with which contaminants interact. Fluoro-naphthol shows a preference for the lignin component of soil, whereas the PFOA shows a preference for protein. Reproduced with permission from Simpson et al.2 2011 Elsevier Science. (C) The same concept in a whole fully swollen soil using a CMP-NMR probe, the major difference being that only the very rigid sequestered fraction is selected. This is achieved by using 19F−1H cross-polarization. The result identifies the components in soil that bind to sequestered molecules. The aromatic contaminant shows bias toward lignin whereas the PFOA appears to show preference for protein. Adapted with permission from Courtier-Murias et al.28 2012 American Chemical Society.
Atmospheric Organic Matter. Compared with the study of DOM and SOM the study of atmospheric organic matter is still very young. While a handful of studies using 1D and 2D NMR have been performed (see references in Simpson et al.2) there is huge potential to understand key processes such as secondary aerosol formation. In addition, techniques such as gel-phase NMR can be used to understand molecular interactions at aqueous−solid interfaces that are important for rain formation and surface reactions and CMP-NMR to understand the interfaces, layering, and composition of air particles in situ. Traditionally, collecting enough atmospheric organic matter for NMR is challenging but new small diameter probes are facilitating the acquisition of 2D NMR on as little as ng quantities of homogeneous material and μg quantities of heterogeneous material.
Dissolved Organic Matter. Dissolved organic matter has been described as one of the most complex mixtures known. Understanding DOM structure and function is essential for understanding global carbon cycling, contaminant transport, and ocean chemistry in general.41 This is summarized best by John Hedges who wrote:42 “The 1012 diverse organic molecules dissolved in every milliliter of seawater are the only constituents whose stored information approaches the richness needed to understand where the water has been and what has happened within it over time. The f uture of oceanographic research belongs in part to those who can learn to read these molecular messages.” While DOM clearly contains some carbohydrates and proteins, these biomolecules do not dominate the NMR spectral profile and in general DOM appears much to be more heterogeneous than SOM. Figure 2A shows the complexity and spectral overlap in the solution-state 1H−13C correlation for Pacific Ocean DOM.43 In combination with a range of other NMR techniques and high resolution mass spectrometry Hertkorn et al.,43 in pioneering work, identified one of the more abundant subfractions to be carboxylic rich alicyclic material (CRAM). Other work defined a second aliphatic fraction consistent with material derived from linear terpenoids (MDLT).44 More recently it has been shown by 2D HPLC combined with 3D NMR detection that the CRAM component is likely to be an incredibly complex overlap of oxidized sterols and hopanoids.45 Complementary work using solid-state NMR has shown the organization of structural component in sinking aquatic particles to be similar to those found in bacterial cell walls,46 as shown in Figure 2B and C.
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MOLECULAR INTERACTIONS NMR-based interaction studies can measure a range of phenomena including spatial interactions, diffusion, relaxation, line-shape, and chemical shift perturbations, many of which have been applied in the solid state, gel state, and solution state to environmental samples.2,3 Here only a couple of examples based on saturation transfer are considered to demonstrate the potential of this more recently applied approach. Often the first question in an interaction study is “how does the ligand interact with the receptor”? In environmental research the ligand is often a contaminant and the “receptor” can be anything from soil to a protein in human blood. This question can be answered by using epitope mapping based on saturation transfer.47,48 In this case the receptor (soil, protein, 11492
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studies have collectively shown that NMR-based methods are more sensitive than conventionally used ecotoxicity indicators. For example, a recent study showed that earthworm exposure to nanosized TiO2 in soil did not result in any detectable toxic responses; however, NMR-based metabolomics was able to detect signs of oxidative stress.64 NMR-based metabolomics has also been used to determine the toxic mode of action of contaminants at sub-lethal concentrations.65 As such, NMRbased environmental metabolomics has great potential to reshape the fundamental understanding of ecosystem functions and shifts with both acute and chronic external stressors.
air particulate, etc.) is selectively saturated which in turn perturbs the properties of the ligand nuclei that are in close proximity to the receptor. The results can converted into an epitope map, an example of which is shown in Figure 3A (top) for the herbicide Trifluralin interacting with a soil surface. The protons that are closest to the soil surface are perturbed the most during the experiment and are assigned 100% with protons further from the surface having increasingly lower values. The result is a “molecular map” that describes the binding orientation of the contaminant with the receptor. To date, studies have identified how a range of contaminants interact with extracted SOM,49 at the soil−water interface,22 in plants,50,51 and biological tissues.52 A second key question in interaction studies is “what does the contaminant bind to?” This can be answered by reversing the process and selectively saturating the ligand. To avoid spectral overlap, selective saturation is achieved using heteronuclei in the ligand such as 19F, 2H that are not present in the receptor. As such, the overall NMR approach is termed reverse heteronuclear saturation transfer difference (RHSTD) NMR spectroscopy.53 In this case the saturation of the ligand perturbs nuclei in the receptor. As the receptor is generally a macromolecule (for example, protein, lignin, cellulose, etc. in soil) the saturation is distributed throughout the rigid dipolar network in the macromolecule. The result is a difference spectrum which shows only the components in the mixture interacting with the contaminant and provides a powerful approach to identify molecules which bind in complex environmental matrices. Thus far RHSTD has been applied to directly identify that human serum albumin (HSA) binds perfluorinated carboxylic acids (PFCAs) in whole blood54 as well as identifying the exact binding sites on the protein. In soil, RHSTD has been used to demonstrate that PFCAs bind to soil protein, whereas perfluorinated aromatic compounds preferentially bind to lignin.53,55 Figure 3B illustrates this for extracted SOM, whereas Figure 3C shows the result for fully swollen soil using CMP-NMR. In the latter case, only the most rigid components, (i.e., the fully sequestered contaminant molecules) were targeted using 19F−1H cross-polarization for magnetization transfer. The ability for NMR to describe, how, and where contaminants bind in complex matrices in soil will have a profound implication on improving selective, targeted, and cost-effective remediation strategies. This is summarized well by Bertsch and Seaman in PNAS56 who stated “a complete understanding of the chemistry of complex soil assemblages is prerequisite to accurately assessing environmental and human health risks of contaminants or in designing environmentally sound, cost-ef fective chemical and biological remediation strategies”. CMPNMR with its ability to probe all bonds and interactions in unaltered natural samples will provide the key information required to truly understand soil sequestration and subsequently permit the design of optimal remediation strategies. Metabolomics. NMR is a widely used analytical platform for ecological and environmental metabolomics, a rapidly growing area of study that examines rapid and quantitative changes to the metabolome in the presence of an external stressor, such as a contaminant, or nutrient stress.57−63 NMR is an excellent discovery tool because it is nonselective, nondestructive, and provides a wealth of metabolite information. To date, NMR-based metabolomic studies have examined a variety of environmental settings (terrestrial versus aquatic) and focused on a number of model organisms (fish, earthworms, water fleas, mussels, and plants).57,58,61−63 These
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CONCLUSIONS AND FUTURE DIRECTIONS NMR spectroscopy is still very much in its infancy in environmental research. However, considering molecular interactions, associations, and dynamics fundamentally define environmental reactivity, and that NMR provides unrivalled molecular information in whole samples in their native state, it is clear it has a profound role to play in the field. Future considerations include the following. Sensitivity Improvements. NMR is traditionally considered a relatively insensitive technique. However, modern microcoil probes can detect and even run multidimensional correlations on solutes in the ng range.66 1H NMR of seawater at natural abundance has been demonstrated where only 600 ng of organic matter (itself containing thousands of components) were present in the detection coil. However, due to the extremely heterogeneous nature of environmental matrices additional sensitivity is always beneficial. Recent improvements such as cryogenically cooled NMR probes (× 4 signal enhancement),67 magic angle spinning microcoils (× 10 signal enhancement or more),68 dynamic nuclear polarization (× 10 000 signal enhancement),69 signal amplification by reversible exchange (∼800 signal enhancement),70 parahydrogen (∼31 000 signal enhancement),71 all show promise to improve detection limits and/or reduce experimental run time and sample size. Multiplatform Analysis and Hyphenation. Both NMR and mass spectrometry (MS) are powerful discovery tools for molecular characterization of environmental matrices. It has been shown that NMR and MS based metabolomics can be directly correlated to important genes and enzymes by integrating information from many different platforms in a “Trans-omics” approach in turn leading to a better understanding of ecosystem function.72 In other cases combining NMR with a range of analytical approaches has demonstrate that litter decomposition rates are determined by the lignin chemistry of the parent vegetation.73 When applied independently to the same sample NMR and MS can greatly increase our understanding of the even the most complex mixtures such as marine DOM.74 However, identifying a range of novel chemical structures in such complex environmental mixtures will likely require direct hyphenation of NMR, MS, and chromatography. If chromatographic separation can be achieved then accurate MS provides the molecular formulas and NMR determines how these nuclei are linked to form the chemical structure. Combining in-line solid-phase extraction (SPE) has improved detection limits and permits NMR to be performed with deuterated solvents and MS with protonated solvents to optimize both techniques. Such approaches have already proved useful for the nontarget analysis of wastewater75 and identifying novel transformation products of mononitrotoluenes.76 The limitations for solving the structural components 11493
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in the most complex environmental mixtures such as dissolved organic matter arise in large part from the lack of chromatographic separation rather than NMR or MS limitations.45 More development is required into advanced separations such as 2D GC, 2D HPLC, 2D CE, and combinations thereof. If excellent chromatography can be achieved even novel structures up to 1000 Da can be solved by combining the power of modern MS, NMR, and structure elucidation software.
Environmental Science in 2010). M. Simpson currently serves as an Associate Editor for the Journal of Environmental Quality, Canadian Journal of Soil Science, Organic Geochemistry, and serves on the editorial board for Scientific Reports (Nature Publishing Group). Dr. Ronald Soong completed his PhD (Toronto) and Postdoc (Michigan) in biological NMR and joined the Environmental NMR Center as a Senior Scientist & Manager in 2010.
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ACKNOWLEDGMENTS A.J.S. and M.J.S. thank the Natural Sciences and Engineering Research Council of Canada (NSERC, Strategic and Discovery Grants Programs), the Canada Foundation for Innovation (CFI), and the Ontario Ministry of Research and Innovation (MRI) for providing funding. A.J.S. thanks the Government of Ontario for an Early Researcher Award. We would like to thank everyone at Bruker BioSpin (Germany, U.S., and Canada) for providing continued support and collaboration, especially Dr. Manfred Spraul, Dr. Werner Maas, and Dr. Henry Stronks and Dr. Michael Fey.
FUTURE DIRECTIONS Structure. Assignments of the major organic components of soil have been performed. This now permits more targeted studies to follow the effect of natural and anthropogenic processes such as climate change and sustainable agriculture. In atmospheric research, detailed NMR analysis is required to understand the composition and structure of atmospheric particles as well as atmospheric reactions in general. In aquatic research more work is required to better understand DOM and its role in contaminant transport and carbon cycling. Considerable additional NMR work is required in industry to better understand biofuel conversion, anaerobic digestion, and waste treatment. CMP-NMR with its ability to understand conformation, structure, layering, and interfacial interaction and reaction in situ should have a major role to play in all these fields. Interactions. Modern NMR is important in understanding where and how contaminants bind in soils, sediments, and living species.77 Understanding the interactions with soil and sediment will improve our understanding of bioavailability and how to best proceed with remediation. Understanding where and how contaminants bind in living species can help explain uptake, distribution, excretion, and bioaccumulation possibly explaining more subtle long-term effects of exposure which at present are very challenging to predict or explain. In addition, metabolomics can be used to measure environmental stress and determine contaminant modes of action in situ. Combining these modern NMR approaches should allow one to monitor a contaminant from source, through soil/sediment, and into a living organism at the molecular level. While challenging, such studies are likely required to fully understand and predict true environmental fate, toxicity, and long-term risk.
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REFERENCES
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest. Biography André Simpson is Professor of Chemistry and Director of the Environmental NMR Center at the University of Toronto. He has developed technologies and applications in Environmental NMR for the past 15 years, publishing over 100 journal articles and 14 book chapters. He has received numerous awards including RSC/SETAC environmental science award (2008), CSC Fred Beamish Award (2010), CSC McBryde Medal (2011), RSC Joseph Black Award/ medal, and was elected as a fellow of the RSC (2010). Prof. Myrna Simpson is Professor of Environmental Chemistry at the University of Toronto and the Associate Director of the Environmental NMR Centre. She is a pioneer in the development of environmental metabolomics. She has published over 100 articles and is the recipient of a number of prestigious awards (NSERC UFA 2002-07, NSERC Discovery Accelerator Supplement in 2010, RSC/SETAC Award for 11494
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(14) Berns, A. E.; Conte, P.; Philipp, H.; Witte, E. G.; Lewandowski, H. Interactions between 2-aminobenzothiazole and natural organic matter as evidenced by CPMAS nitrogen-15 NMR spectroscopy. Vadose Zone J. 2009, 8 (3), 670−676. (15) Kohl, S. D.; Toscano, P. J.; Hou, W. H.; Rice, J. A. Solid-state 19 F NMR investigation of hexafluorobenzene sorption to soil organic matter. Environ. Sci. Technol. 2000, 34 (1), 204−210. (16) Fan, T. W. M.; Bird, J. A.; Brodie, E. L.; Lane, A. N. 13C Isotopomer-based metabolomics of microbial groups isolated from two forest soils. Metabolomics 2009, 5 (1), 108−122. (17) Hu, W. G.; Mao, J. D.; Xing, B. S.; Schmidt-Rohr, K. Poly(methylene) crystallites in humic substances detected by nuclear magnetic resonance. Environ. Sci. Technol. 2000, 34 (3), 530−534. (18) Sachleben, J. R.; Chefetz, B.; Deshmukh, A.; Hatcher, P. G. Solid-state NMR characterization of pyrene - Cuticular matter interactions. Environ. Sci. Technol. 2004, 38 (16), 4369−4376. (19) Lattao, C.; Birdwell, J.; Wang, J. J.; Cook, R. L. Studying organic matter molecular assemblage within a whole organic soil by nuclear magnetic resonance. J. Environ. Qual. 2008, 37 (4), 1501−1509. (20) Maas, W. E.; Laukien, F. H.; Cory, D. G. Gradient, high resolution, magic angle sample spinning NMR. J. Am. Chem. Soc. 1996, 118 (51), 13085−13086. (21) Simpson, A. J.; Kingery, W. L.; Shaw, D. R.; Spraul, M.; Humpfer, E.; Dvortsak, P. The application of 1H HR-MAS NMR spectroscopy for the study of structures and associations of organic components at the solid−aqueous interface of a whole soil. Environ. Sci. Technol. 2001, 35 (16), 3321−3325. (22) Shirzadi, A.; Simpson, M. J.; Kumar, R.; Baer, A.; Xu, Y.; Simpson, A. J. Molecular interactions of pesticides at the soil−water interface. Environ. Sci. Technol. 2008, No. 42, 5514−5520. (23) Kelleher, B. P.; Simpson, M. J.; Simpson, A. J. Assessing the fate and transformation of plant residues in the terrestrial environment using HR-MAS NMR spectroscopy. Geochim. Cosmochim. Acta 2006, 70 (16), 4080−4094. (24) Simpson, A. J.; Simpson, M. J.; Smith, E.; Kelleher, B. P. Microbially derived inputs to soil organic matter: Are current estimates too low? Environ. Sci. Technol. 2007, 41 (23), 8070−8076. (25) Simpson, A. J.; Simpson, M. J.; Kingery, W. L.; Lefebvre, B. A.; Moser, A.; Williams, A. L.; Kvasha, M.; Kelleher, B. P. The application of 1H HR-MAS NMR for the study of clay-organic associations in natural and synthetic complexes. Langmuir 2006, 22, 4498−4503. (26) Miglietta, M. L.; Lamanna, R. 1H HR-MAS NMR of carotenoids in aqueous samples and raw vegetables. Magn. Reson. Chem. 2006, 44 (7), 675−685. (27) Bunescu, A.; Garric, J.; Vollat, B.; Canet-Soulas, E.; GraveronDemilly, D.; Fauvelle, F. In vivo proton HR-MAS NMR metabolic profile of the freshwater cladoceran Daphnia magna. Mol. Biosyst. 2010, 6 (1), 121−125. (28) Courtier-Murias, D.; Farooq, H.; Masoom, H.; Botana, A.; Soong, R.; Longstaffe, J. G.; Simpson, M. J.; Maas, W. E.; Fey, M.; Andrew, B.; Struppe, J.; Hutchins, H.; Krishnamurthy, S.; Kumar, R.; Monette, M.; Stronks, H. J.; Hume, A.; Simpson, A. J. Comprehensive multiphase NMR spectroscopy: Basic experimental approaches to differentiate phases in heterogeneous samples. J. Magn. Reson. 2012, 217, 61−76. (29) Berns, A. E.; Bubici, S.; De Pasquale, C.; Alonzo, G.; Conte, P. Applicability of solid state fast field cycling NMR relaxometry in understanding relaxation properties of leaves and leaf-litters. Org. Geochem. 2011, 42 (8), 978−984. (30) Jaeger, F.; Bowe, S.; Van As, H.; Schaumann, G. E. Evaluation of 1 H NMR relaxometry for the assessment of pore-size distribution in soil samples. Eur. J. Soil Sci. 2009, 60 (6), 1052−1064. (31) Nakashima, Y.; Mitsuhata, Y.; Nishiwaki, J.; Kawabe, Y.; Utsuzawa, S.; Jinguuji, M. Non-destructive analysis of oil-contaminated soil core samples by X-ray computed tomography and low-field nuclear magnetic resonance relaxometry: A case study. Water, Air, Soil Pollut. 2011, 214 (1−4), 681−698. (32) Pape, H.; Arnold, J.; Pechnig, R.; Clauser, C.; Talnishnikh, E.; Anferova, S.; Blumich, B. Permeability prediction for low porosity 11495
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hydrogen enhance NMR sensitivity by polarization transfer. Science 2009, 323 (5922), 1708−1711. (71) Duckett, S. B.; Wood, N. J. Parahydrogen-based NMR methods as a mechanistic probe in inorganic chemistry. Coord. Chem. Rev. 2008, 252 (21−22), 2278−2291. (72) Ogata, Y.; Chikayama, E.; Morioka, Y.; Everroad, R. C.; Shino, A.; Matsushima, A.; Haruna, H.; Moriya, S.; Toyoda, T.; Kikuchi, J. ECOMICS: A Web-based toolkit for investigating the biomolecular web in ecosystems using a trans-omics approach. PLOS One 2012, 7, (2). (73) Talbot, J. M.; Yelle, D. J.; Nowick, J.; Treseder, K. K. Litter decay rates are determined by lignin chemistry. Biogeochemistry 2012, 108 (1−3), 279−295. (74) Hertkorn, N.; Harir, M.; Koch, B. P.; Michalke, B.; Grill, P.; Schmitt-Kopplin, P. High field NMR spectroscopy and FTICR mass spectrometry: Powerful discovery tools for the molecular level characterization of marine dissolved organic matter from the South Atlantic Ocean. Biogeosci. Discuss. 2012, 9 (1), 745−833. (75) Godejohann, M.; Berset, J. D.; Muff, D. Non-targeted analysis of wastewater treatment plant effluents by high performance liquid chromatography-time slice-solid phase extraction-nuclear magnetic resonance/time-of-flight-mass spectrometry. J. Chromatogr., A 2011, 1218 (51), 9202−9209. (76) Preiss, A.; Berger-Preiss, E.; Elend, M.; Reineke, A. K.; Hollender, J. Unusual polar metabolites in the groundwater of a contaminated waste site indicate a new pathway of mononitrotoluene transformation. Chemosphere 2011, 84 (11), 1650−1657. (77) Seger, M. R.; Maciel, G. E. NMR investigation of the behavior of an organothiophosphate pesticide, chlorpyrifos, sorbed on soil components. Environ. Sci. Technol. 2006, 40 (3), 791−796.
spectroscopy, solid-phase assays and cell binding for a plant toxin model. ChemMedChem 2010, 5 (3), 415−419. (51) Koistinen, K. M.; Soininen, P.; Venalainen, T. A.; Hayrinen, J.; Laatikainen, R.; Perakyla, M.; Tervahauta, A. I.; Karenlampi, S. O. Birch PR-10c interacts with several biologically important ligands. Phytochemistry 2005, 66 (21), 2524−2533. (52) Zakhour, M.; Ruvoen-Clouet, N.; Charpilienne, A.; Langpap, B.; Poncet, D.; Peters, T.; Bovin, N.; Le Pendu, J. The alpha gal epitope of the histo-blood group antigen family is a ligand for bovine norovirus Newbury2 Expected to prevent cross-species transmission. PLoS Pathog. 2009, 5, (7). (53) Longstaffe, J. G.; Simpson, M. J.; Maas, W.; Simpson, A. J. Identifying components in dissolved humic acid that bind organofluorine contaminants using 1H {19F} reverse heteronuclear saturation transfer difference NMR spectroscopy. Environ. Sci. Technol. 2010, 44 (14), 5476−5482. (54) D’eon, J. C.; Simpson, A. J.; Kumar, R.; Baer, A. J.; Mabury, S. A. Determining the molecular interactions of perfluorinated carboxylic acids with human sera and isolated human serum albumin using nuclear magnetic resonance spectroscopy. Environ. Toxicol. Chem. 2010, 29 (8), 1678−1688. (55) Longstaffe, J. G.; Simpson, A. J. Understanding solution-state noncovalent interactions between xenobiotics and natural organic matter using 19F/1H heteronuclear saturation transfer difference nuclear magnetic resonance spectroscopy. Environ. Toxicol. Chem. 2011, 30 (8), 1745−1753. (56) Bertsch, P. M.; Seaman, J. C. Characterization of complex mineral assemblages: Implications for contaminant transport and environmental remediation. Proc. Natl. Acad. Sci., U.S.A. 1999, 96 (7), 3350−3357. (57) Bundy, J. G.; Davey, M. P.; Viant, M. R. Environmental metabolomics: A critical review and future perspectives. Metabolomics 2009, 5 (1), 3−21. (58) Lin, C. Y.; Viant, M. R.; Tjeerdema, R. S. Metabolomics: Methodologies and applications in the environmental sciences. J. Pestic. Sci. 2006, 31, 245−251. (59) Peñuelas, P.; Sardans, J. Ecological metabolomics. Chem. Ecol. 2009, 25, 305−309. (60) Robertson, D. G. Metabonomics in toxicology: A review. Toxicol. Sci. 2005, 85, 809−822. (61) Simpson, M. J.; McKelvie, J. R. Environmental metabolomics: New insights into earthworm ecotoxicity and contaminant bioavailability in soil. Anal. Bioanal. Chem. 2009, 394 (1), 137−149. (62) Viant, M. R. Metabolomics of aquatic organisms: the new 'omics' on the block. Mar. Ecol.-Prog. Ser. 2007, 332, 301−306. (63) Viant, M. R. Recent developments in environmental metabolomics. Mol. BioSyst. 2008, 4, 980−986. (64) Whitfield Aslund, M.; McShane, H.; Simpson, M. J.; Simpson, A. J.; Whalen, J.; Hendershoot, W.; Sunahara, G. Earthworm sub-lethal responses to titanium dioxide nanomaterial in soil detected by 1H NMR metabolomics. Environ. Sci. Technol. 2012, 46, 1111−1118. (65) Lankadurai, B. P.; Wolfe, D. M.; Simpson, A. J.; Simpson, M. J. Concentration-dependence of earthworm responses to phenanthrene exposure. Environ. Chem. 2011, 8, 105−114. (66) Jones, C. J.; Larive, C. K. Could smaller really be better? Current and future trends in high-resolution microcoil NMR spectroscopy. Anal. Bioanal. Chem. 2012, 402 (1), 61−68. (67) Kim, Y. W.; Earl, W. L.; Norberg, R. E. Cryogenic probe with low-loss transmission-line for nuclear-magnetic-resonance. J. Magn. Reson., Ser. A 1995, 116 (2), 139−144. (68) Inukai, M.; Takeda, K. Double-resonance magic angle coil spinning. J. Magn. Reson. 2010, 202 (2), 274−278. (69) Ardenkjaer-Larsen, J. H.; Fridlund, B.; Gram, A.; Hansson, G.; Hansson, L.; Lerche, M. H.; Servin, R.; Thaning, M.; Golman, K. Increase in signal-to-noise ratio of >10,000 times in liquid-state NMR. Proc. Natl. Acad. Sci., U.S.A. 2003, 100 (18), 10158−10163. (70) Adams, R. W.; Aguilar, J. A.; Atkinson, K. D.; Cowley, M. J.; Elliott, P. I. P.; Duckett, S. B.; Green, G. G. R.; Khazal, I. G.; LopezSerrano, J.; Williamson, D. C. Reversible interactions with para11496
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