Quantitative reaction monitoring using parahydrogen-enhanced benchtop NMR spectroscopy.

The parahydrogen-induced polarisation (PHIP) NMR signal enhancement technique is used to study H2 addition to Vaska's complex (trans-[IrCl(CO)(PPh3)2]) with both standard high-field (9.4 T) NMR and benchtop (1 T) NMR detection. Accurate and repeatable rate constants of (0.84 ± 0.03) dm3 mol-1 s-1 and (0.89 ± 0.03) dm3 mol-1 s-1 were obtained for this model system using standard high-field and benchtop NMR, respectively. The high-field NMR approach is shown to be susceptible to systematic errors associated with interference from non-hyperpolarised signals, which can be overcome through a multiple-quantum filtered acquisition scheme. This challenge is avoided when using benchtop NMR detection because the non-hyperpolarised signals are much weaker due to the lower magnetic field, enabling the use of a simpler and more efficient single RF pulse detection scheme. Method validation against several experimental parameters (NMR relaxation, %pH2 enrichment and temperature) demonstrates the robustness of the benchtop NMR approach but also highlights the need for sample temperature control throughout reaction monitoring. A simple temperature equilibration protocol, coupled with use of an insulated sample holder while manipulating the sample outside the spectrometer, is found to provide sufficient temperature stabilisation to ensure that accurate and repeatable rate constants are obtained. Finally, the benchtop NMR reaction monitoring protocol is applied to the analysis of a complex mixture, where multiple reaction products form simultaneously. H2 addition to a mixture of three Vaska's complex derivatives was monitored, revealing the presence of competitive reaction pathways within the mixture.

Enhancing 19F Benchtop NMR Spectroscopy by Combining para-Hydrogen Hyperpolarization and Multiplet Refocusing.

Benchtop NMR spectrometers provide a promising alternative to high-field NMR for applications that are limited by instrument size and/or cost. 19F benchtop NMR is attractive due to the larger chemical shift range of 19F relative to 1H and the lack of background signal in most applications. However, practical applications of benchtop 19F NMR are limited by its low sensitivity due to the relatively weak field strengths of benchtop NMR spectrometers. Here we present a sensitivity-enhancement strategy that combines SABRE (Signal Amplification By Reversible Exchange) hyperpolarization with the multiplet refocusing method SHARPER (Sensitive, Homogeneous, And Resolved PEaks in Real time). When applied to a range of fluoropyridines, SABRE-SHARPER achieves overall signal enhancements of up to 5700-fold through the combined effects of hyperpolarization and line-narrowing. This approach can be generalized to the analysis of mixtures through the use of a selective variant of the SHARPER sequence, selSHARPER. The ability of SABRE-selSHARPER to simultaneously boost sensitivity and discriminate between two components of a mixture is demonstrated, where selectivity is achieved through a combination of selective excitation and the choice of polarization transfer field during the SABRE step.

SHARPER-enhanced benchtop NMR: improving SNR by removing couplings and approaching natural linewidths.

We present a signal enhancement strategy for benchtop NMR that produces SNR increases on the order of 10 to 30 fold by collapsing the target resonance into an extremely narrow singlet. Importantly, the resultant signal is amenable to quantitative interpretation and therefore can be applied to analytical applications such as reaction monitoring.

In Situ SABRE Hyperpolarization with Earth's Field NMR Detection.

Hyperpolarization methods, which increase the sensitivity of nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI), have the potential to expand the range of applications of these powerful analytical techniques and to enable the use of smaller and cheaper devices. The signal amplification by reversible exchange (SABRE) method is of particular interest because it is relatively low-cost, straight-forward to implement, produces high-levels of renewable signal enhancement, and can be interfaced with low-cost and portable NMR detectors. In this work, we demonstrate an in situ approach to SABRE hyperpolarization that can be achieved using a simple, commercially-available Earth's field NMR detector to provide 1H polarization levels of up to 3.3%. This corresponds to a signal enhancement over the Earth's magnetic field by a factor of ε > 2 × 108. The key benefit of our approach is that it can be used to directly probe the polarization transfer process at the heart of the SABRE technique. In particular, we demonstrate the use of in situ hyperpolarization to observe the activation of the SABRE catalyst, the build-up of signal in the polarization transfer field (PTF), the dependence of the hyperpolarization level on the strength of the PTF, and the rate of decay of the hyperpolarization in the ultra-low-field regime.

Towards measuring reactivity on micro-to-millisecond timescales with laser pump, NMR probe spectroscopy.

We present a quantitative analysis of the timescales of reactivity that are accessible to a laser pump, NMR probe spectroscopy method using para-hydrogen induced polarisation (PHIP) and identify three kinetic regimes: fast, intermediate and slow. These regimes are defined by the relative rate of reaction, k, compared to δω, the frequency of the NMR signal oscillations associated with the coherent evolution of the hyperpolarised 1H NMR signals created after para-hydrogen (p-H2) addition during the pump-probe delay. The kinetic regimes are quantitatively defined by a NMR dephasing parameter, ε = δω/k. For the fast regime, where k ≫ δω and ε tends to zero, the observed NMR signals are not affected by the chemical evolution of the system and so only an upper bound on k can be determined. In the slow regime, where k ≪ δω and ε tends to infinity, destructive interference leads to the complete dephasing of the coherent NMR signal intensity oscillations. As a result, the observed NMR signal evolution during the pump-probe delay reflects only the chemical change of the system and NMR relaxation. Finally, in the intermediate regime, where k ∼ δω, characteristic partial dephasing of the NMR signal oscillations is predicted. In the limit where the dephasing parameter is small but non-zero, chemical evolution manifests itself as a phase shift in the NMR signal oscillation that is equal to the dephasing parameter. As this phase shift is predicted to persist for pump-probe delays much longer than the timescale of the formation of the product molecules, it provides a route to measure reactivity on micro-to-millisecond timescales through NMR detection. We predict that the most significant fundamental limitations of the accessible reaction timescales are the duration of the NMR excitation pulse (∼1 µs) and the chemical shift difference (in Hz) between the p-H2-derived protons in the product molecule.

Reaction Monitoring Using SABRE-Hyperpolarized Benchtop (1 T) NMR Spectroscopy.

The conversion of [IrCl(COD)(IMes)] (COD = cis, cis-1,5-cyclooctadiene, IMes = 1,3-bis(2,4,6-trimethyl-phenyl)imidazole-2-ylidene) in the presence of an excess of para-hydrogen ( p-H2) and a substrate (4-aminopyridine (4-AP) or 4-methylpyridine (4-MP)) into [Ir(H)2(IMes)(substrate)3]Cl is monitored by 1H NMR spectroscopy using a benchtop (1 T) spectrometer in conjunction with the p-H2-based hyperpolarization technique signal amplification by reversible exchange (SABRE). A series of single-shot 1H NMR measurements are used to monitor the chemical changes that take place in solution through the lifetime of the hyperpolarized response. Non-hyperpolarized high-field 1H NMR control measurements were also undertaken to confirm that the observed time-dependent changes relate directly to the underlying chemical evolution. The formation of [Ir(H)2(IMes)(substrate)3]Cl is further linked to the hydrogen isotope exchange (HIE) reaction, which leads to the incorporation of deuterium into the ortho positions of 4-AP, where the source of deuterium is the solvent, methanol- d4. Comparable reaction monitoring results are achieved at both high-field (9.4 T) and low-field (1 T). It is notable that the low sensitivity of the benchtop (1 T) NMR enables the use of protio solvents, which when used here allows the effects of catalyst formation and substrate deuteration to be separated. Collectively, these methods illustrate how low-cost low-field NMR measurements provide unique insight into a complex catalytic process through a combination of hyperpolarization and relaxation data.

Rapid 13C NMR hyperpolarization delivered from para-hydrogen enables the low concentration detection and quantification of sugars.

Monosaccharides, such as glucose and fructose, are important to life. In this work we highlight how the rapid delivery of improved 13C detectability for sugars by nuclear magnetic resonance (NMR) can be achieved using the para-hydrogen based NMR hyperpolarization method SABRE-Relay (Signal Amplification by Reversible Exchange-Relay). The significant 13C signal enhancements of 250 at a high field of 9.4 T, and 3100 at a low field of 1 T, enable the detection of trace amounts of these materials as well as the quantification of their tautomeric makeup. Using studies on 13C and 2H isotopically labelled agents we demonstrate how hyperpolarization lifetime (T 1) values can be extended, and how singlet states with long lifetimes can be created. The precise quantification of d-glucose-13C6-d 7 at the millimolar concentration level is shown to be possible within minutes in conjunction with a linear hyperpolarized response as a function of concentration. In addition to the measurements using labelled materials, low concentration detection is also illustrated for millimolar samples with natural abundance 13C where isomeric form quantification can be achieved with a single transient.

Quantitative In Situ Monitoring of Parahydrogen Fraction Using Raman Spectroscopy.

Raman spectroscopy has been used to provide a rapid, noninvasive, and nondestructive quantification method for determining the parahydrogen fraction of hydrogen gas. The basis of the method is the measurement of the ratio of the first two rotational bands of hydrogen at 355 cm-1 and 586 cm-1 corresponding to parahydrogen and orthohydrogen, respectively. The method has been used to determine the parahydrogen content during a production process and a reaction. In the first example, the performance of an in-house liquid nitrogen cooled parahydrogen generator was monitored both at-line and on-line. The Raman measurements showed that it took several hours for the generator to reach steady state and, hence, for maximum parahydrogen production (50%) to be reached. The results obtained using Raman spectroscopy were compared to those obtained by at-line low-field nuclear magnetic resonance (NMR) spectroscopy. While the results were in good agreement, Raman analysis has several advantages over NMR for this application. The Raman method does not require a reference sample, as both spin isomers (ortho and para) of hydrogen can be directly detected, which simplifies the procedure and eliminates some sources of error. In the second example, the method was used to monitor the fast conversion of parahydrogen to orthohydrogen in situ. Here the ability to acquire Raman spectra every 30 s enabled a conversion process with a rate constant of 27.4×10-4 s-1 to be monitored. The Raman method described here represents an improvement on previously reported work, in that it can be easily applied on-line and is approximately 500 times faster. This offers the potential of an industrially compatible method for determining parahydrogen content in applications that require the storage and usage of hydrogen.

Quantification of hyperpolarisation efficiency in SABRE and SABRE-Relay enhanced NMR spectroscopy.

para-Hydrogen (p-H2) induced polarisation (PHIP) is an increasingly popular method for sensitivity enhancement in NMR spectroscopy. Its growing popularity is due in part to the introduction of the signal amplification by reversible exchange (SABRE) method that generates renewable hyperpolarisation in target analytes in seconds. A key benefit of PHIP and SABRE is that p-H2 can be relatively easily and cheaply produced, with costs increasing with the desired level of p-H2 purity. In this work, the efficiency of the SABRE polarisation transfer is explored by measuring the level of analyte hyperpolarisation as a function of the level of p-H2 enrichment. A linear relationship was found between p-H2 enrichment and analyte 1H hyperpolarisation for a range of molecules, polarisation transfer catalysts, NMR detection fields and for both the SABRE and SABRE-Relay transfer mechanisms over the range 29-99% p-H2 purity. The gradient of these linear relationships were related to a simple theoretical model to define an overall efficiency parameter, E, that quantifies the net fraction of the available p-H2 polarisation that is transferred to the target analyte. We find that the efficiency of SABRE is independent of the NMR detection field and exceeds E = 20% for methyl-4,6-d2-nicotinate when using a previously optimised catalyst system. For the SABRE-Relay transfer mechanism, efficiencies of up to E = 1% were found for 1H polarisation of 1-propanol, when ammonia was used as the polarisation carrier.

Refocused linewidths less than 10 Hz in 1H solid-state NMR.

Coherence lifetimes in homonuclear dipolar decoupled 1H solid-state NMR experiments are usually on the order of a few ms. We discover an oscillation that limits the lifetime of the coherences by recording spin-echo dephasing curves. We find that this oscillation can be removed by the application of a double spin-echo experiment, leading to coherence lifetimes of more than 45 ms in adamantane and more that 22 ms in ß-AspAla, corresponding to refocused linewidths of less than 7 and 14 Hz respectively.

SABRE hyperpolarization enables high-sensitivity 1H and 13C benchtop NMR spectroscopy.

Benchtop NMR spectrometers operating with low magnetic fields of 1-2 T at sub-ppm resolution show great promise as analytical platforms that can be used outside the traditional laboratory environment for industrial process monitoring. One current limitation that reduces the uptake of benchtop NMR is associated with the detection fields' reduced sensitivity. Here we demonstrate how para-hydrogen (p-H2) based signal amplification by reversible exchange (SABRE), a simple to achieve hyperpolarization technique, enhances agent detectability within the environment of a benchtop (1 T) NMR spectrometer so that informative 1H and 13C NMR spectra can be readily recorded for low-concentration analytes. SABRE-derived 1H NMR signal enhancements of up to 17 000-fold, corresponding to 1H polarization levels of P = 5.9%, were achieved for 26 mM pyridine in d4-methanol in a matter of seconds. Comparable enhancement levels can be achieved in both deuterated and protio solvents but now the SABRE-enhanced analyte signals dominate due to the comparatively weak thermally-polarized solvent response. The SABRE approach also enables the acquisition of 13C NMR spectra of analytes at natural isotopic abundance in a single scan as evidenced by hyperpolarized 13C NMR spectra of tens of millimolar concentrations of 4-methylpyridine. Now the associated signal enhancement factors are up to 45 500 fold (P = 4.0%) and achieved in just 15 s. Integration of an automated SABRE polarization system with the benchtop NMR spectrometer framework produces renewable and reproducible NMR signal enhancements that can be exploited for the collection of multi-dimensional NMR spectra, exemplified here by a SABRE-enhanced 2D COSY NMR spectrum.

A simple hand-held magnet array for efficient and reproducible SABRE hyperpolarisation using manual sample shaking.

Signal amplification by reversible exchange (SABRE) is a hyperpolarisation technique that catalytically transfers nuclear polarisation from parahydrogen, the singlet nuclear isomer of H2 , to a substrate in solution. The SABRE exchange reaction is carried out in a polarisation transfer field (PTF) of tens of gauss before transfer to a stronger magnetic field for nuclear magnetic resonance (NMR) detection. In the simplest implementation, polarisation transfer is achieved by shaking the sample in the stray field of a superconducting NMR magnet. Although convenient, this method suffers from limited reproducibility and cannot be used with NMR spectrometers that do not have appreciable stray fields, such as benchtop instruments. Here, we use a simple hand-held permanent magnet array to provide the necessary PTF during sample shaking. We find that the use of this array provides a 25% increase in SABRE enhancement over the stray field approach, while also providing improved reproducibility. Arrays with a range of PTFs were tested, and the PTF-dependent SABRE enhancements were found to be in excellent agreement with comparable experiments carried out using an automated flow system where an electromagnet is used to generate the PTF. We anticipate that this approach will improve the efficiency and reproducibility of SABRE experiments carried out using manual shaking and will be particularly useful for benchtop NMR, where a suitable stray field is not readily accessible. The ability to construct arrays with a range of PTFs will also enable the rapid optimisation of SABRE enhancement as function of PTF for new substrate and catalyst systems.

Coherent evolution of parahydrogen induced polarisation using laser pump, NMR probe spectroscopy: Theoretical framework and experimental observation.

We recently reported a pump-probe method that uses a single laser pulse to introduce parahydrogen (p-H2) into a metal dihydride complex and then follows the time-evolution of the p-H2-derived nuclear spin states by NMR. We present here a theoretical framework to describe the oscillatory behaviour of the resultant hyperpolarised NMR signals using a product operator formalism. We consider the cases where the p-H2-derived protons form part of an AX, AXY, AXYZ or AA'XX' spin system in the product molecule. We use this framework to predict the patterns for 2D pump-probe NMR spectra, where the indirect dimension represents the evolution during the pump-probe delay and the positions of the cross-peaks depend on the difference in chemical shift of the p-H2-derived protons and the difference in their couplings to other nuclei. The evolution of the NMR signals of the p-H2-derived protons, as well as the transfer of hyperpolarisation to other NMR-active nuclei in the product, is described. The theoretical framework is tested experimentally for a set of ruthenium dihydride complexes representing the different spin systems. Theoretical predictions and experimental results agree to within experimental error for all features of the hyperpolarised 1H and 31P pump-probe NMR spectra. Thus we establish the laser pump, NMR probe approach as a robust way to directly observe and quantitatively analyse the coherent evolution of p-H2-derived spin order over micro-to-millisecond timescales.

Photochemical pump and NMR probe to monitor the formation and kinetics of hyperpolarized metal dihydrides.

On reaction of IrI(CO)(PPh3)21 with para-hydrogen (p-H2), Ir(H)2I(CO)(PPh3)22 is formed which exhibits strongly enhanced 1H NMR signals for its hydride resonances. Complex 2 also shows similar enhancement of its NMR spectra when it is irradiated under p-H2. We report the use of this photochemical reactivity to measure the kinetics of H2 addition by laser-synchronized reactions in conjunction with NMR. The single laser pulse promotes the reductive elimination of H2 from Ir(H)2I(CO)(PPh3)22 in C6D6 solution to form the 16-electron precursor 1, back reaction with p-H2 then reforms 2 in a well-defined nuclear spin-state. The build up of this product can be followed by incrementing a precisely controlled delay (τ), in millisecond steps, between the laser and the NMR pulse. The resulting signal vs. time profile shows a dependence on p-H2 pressure. The plot of kobs against p-H2 pressure is linear and yields the second order rate constant, k2, for H2 addition to 1 of (3.26 ± 0.42) × 102 M-1 s-1. Validation was achieved by transient-UV-vis absorption spectroscopy which gives k2 of (3.06 ± 0.40) × 102 M-1 s-1. Furthermore, irradiation of a C6D6 solution of 2 with multiple laser shots, in conjunction with p-H2 derived hyperpolarization, allows the detection and characterisation of two minor reaction products, 2a and 3, which are produced in such low yields that they are not detected without hyperpolarization. Complex 2a is a configurational isomer of 2, while 3 is formed by substitution of CO by PPh3.

Protein dynamics. Direct observation of hierarchical protein dynamics.

One of the fundamental challenges of physical biology is to understand the relationship between protein dynamics and function. At physiological temperatures, functional motions arise from the complex interplay of thermal motions of proteins and their environments. Here, we determine the hierarchy in the protein conformational energy landscape that underlies these motions, based on a series of temperature-dependent magic-angle spinning multinuclear nuclear-magnetic-resonance relaxation measurements in a hydrated nanocrystalline protein. The results support strong coupling between protein and solvent dynamics above 160 kelvin, with fast solvent motions, slow protein side-chain motions, and fast protein backbone motions being activated consecutively. Low activation energy, small-amplitude local motions dominate at low temperatures, with larger-amplitude, anisotropic, and functionally relevant motions involving entire peptide units becoming dominant at temperatures above 220 kelvin.

Macroscopic nuclear spin diffusion constants of rotating polycrystalline solids from first-principles simulation.

A method for quantitatively calculating nuclear spin diffusion constants directly from crystal structures is introduced. This approach uses the first-principles low-order correlations in Liouville space (LCL) method to simulate spin diffusion in a box, starting from atomic geometry and including both magic-angle spinning (MAS) and powder averaging. The LCL simulations are fit to the 3D diffusion equation to extract quantitative nuclear spin diffusion constants. We demonstrate this method for the case of (1)H spin diffusion in ice and L-histidine, obtaining diffusion constants that are consistent with literature values for (1)H spin diffusion in polymers and that follow the expected trends with respect to magic-angle spinning rate and the density of nuclear spins. In addition, we show that this method can be used to model (13)C spin diffusion in diamond and therefore has the potential to provide insight into applications such as the transport of polarization in non-protonated systems.

Photochemical pump and NMR probe: chemically created NMR coherence on a microsecond time scale.

We report pump-probe experiments employing laser-synchronized reactions of para-hydrogen (para-H2) with transition metal dihydride complexes in conjunction with nuclear magnetic resonance (NMR) detection. The pump-probe experiment consists of a single nanosecond laser pump pulse followed, after a precisely defined delay, by a single radio frequency (rf) probe pulse. Laser irradiation eliminates H2 from either Ru(PPh3)3(CO)(H)2 1 or cis-Ru(dppe)2(H)2 2 in C6D6 solution. Reaction with para-H2 then regenerates 1 and 2 in a well-defined nuclear spin state. The rf probe pulse produces a high-resolution, single-scan (1)H NMR spectrum that can be recorded after a pump-probe delay of just 10 µs. The evolution of the spectra can be followed as the pump-probe delay is increased by micro- or millisecond increments. Due to the sensitivity of this para-H2 experiment, the resulting NMR spectra can have hydride signal-to-noise ratios exceeding 750:1. The spectra of 1 oscillate in amplitude with frequency 1101 ± 3 Hz, the chemical shift difference between the chemically inequivalent hydrides. The corresponding hydride signals of 2 oscillate with frequency 83 ± 5 Hz, which matches the difference between couplings of the hydrides to the equatorial (31)P nuclei. We use the product operator formalism to show that this oscillatory behavior arises from a magnetic coherence in the plane orthogonal to the magnetic field that is generated by use of the laser pulse without rf initialization. In addition, we demonstrate how chemical shift imaging can differentiate the region of laser irradiation thereby distinguishing between thermal and photochemical reactivity within the NMR tube.

Improved phase-modulated homonuclear dipolar decoupling for solid-state NMR spectroscopy from symmetry considerations.

We explore the effects of symmetry on the performance of phase-modulated homonuclear dipolar decoupling in (1)H solid-state NMR. We demonstrate that the symmetry of the DUMBO family of decoupling sequences is the result of two well-defined symmetry expansions. The first is an antipalindromic expansion that arises from the symmetrization step that was built into the original architecture of the DUMBO sequence. The second is a mirror-pair expansion that inverts the sign of the phase modulation in the second half of the pulse sequence relative to the first. The combination of these two symmetry expansions generates a sequence of four Lee-Goldburg-type rotations in the rotating frame. The axes of rotation, oriented at the magic angle, are separated in the transverse plane by 2α, where α is chosen to minimize the sensitivity of the sequence to instrument imperfections such as rf inhomogeneity. The efficiency of the DUMBO symmetry for decoupling is demonstrated experimentally, and the effect of the α-phase-shift parameter is investigated. A new decoupling sequence (LG4) that combines the DUMBO symmetry with α = 55° is introduced and is shown to produce very efficient decoupling as well as a nearly 2-fold increase in coherence lifetimes when compared to standard PMLG/FSLG decoupling.

Quasi-equilibria in reduced Liouville spaces.

The quasi-equilibrium behaviour of isolated nuclear spin systems in full and reduced Liouville spaces is discussed. We focus in particular on the reduced Liouville spaces used in the low-order correlations in Liouville space (LCL) simulation method, a restricted-spin-space approach to efficiently modelling the dynamics of large networks of strongly coupled spins. General numerical methods for the calculation of quasi-equilibrium expectation values of observables in Liouville space are presented. In particular, we treat the cases of a time-independent Hamiltonian, a time-periodic Hamiltonian (with and without stroboscopic sampling) and powder averaging. These quasi-equilibrium calculation methods are applied to the example case of spin diffusion in solid-state nuclear magnetic resonance. We show that there are marked differences between the quasi-equilibrium behaviour of spin systems in the full and reduced spaces. These differences are particularly interesting in the time-periodic-Hamiltonian case, where simulations carried out in the reduced space demonstrate ergodic behaviour even for small spins systems (as few as five homonuclei). The implications of this ergodic property on the success of the LCL method in modelling the dynamics of spin diffusion in magic-angle spinning experiments of powders is discussed.