Perspectives on DNP-enhanced NMR spectroscopy in solutions

Journal of Magnetic Resonance 264 (2016) 59–67

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Perspectives on DNP-enhanced NMR spectroscopy in solutions Jan van Bentum ⇑, Bas van Meerten, Manvendra Sharma, Arno Kentgens Radboud University, Nijmegen, The Netherlands

a r t i c l e

i n f o

Article history: Received 23 November 2015 Revised 12 January 2016

Keywords: Liquid state DNP Overhauser Supercritical solvent Rapid melt DNP

a b s t r a c t More than 60 years after the seminal work of Albert Overhauser on dynamic nuclear polarization by dynamic cross relaxation of coupled electron–nuclear spin systems, the quest for sensitivity enhancement in NMR spectroscopy is as pressing as ever. In this contribution we will review the status and perspectives for dynamic nuclear polarization in the liquid state. An appealing approach seems to be the use of supercritical solvents that may allow an extension of the Overhauser mechanism towards common high magnetic fields. A complementary approach is the use of solid state DNP on frozen solutions, followed by a rapid dissolution or in-situ melting step and NMR detection with substantially enhanced polarization levels in the liquid state. We will review recent developments in the field and discuss perspectives for the near future. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction The state-of-the-art sensitivity of NMR leads to detection limits in the liquid state in the low micromolar regime [1]. For multidimensional experiments, these concentrations might be either prohibitive or require long acquisition times, leading to low rates in sample processing and compromised information content. The tendency towards higher magnetic fields (20 T and above) combined with low noise cryoprobe detection can partly compensate for this drawback. In some specific cases it is possible with sample conditioning methods to extract and concentrate the sample of interest. For example, in the case of forensic applications, the sample size is intrinsically limited and absolute NMR detection limits (about one nanomole of molecules) can hardly compete with other methods such as mass-spectrometry. In many material science applications and in particular catalysis, the surface layer is essential, but the low dimensionality limits the number of NMR nuclei and analysis is not trivial. In the case of biological or medical research it would be desirable to monitor for example the blood or cerebral spine fluid of small animals during the development of a disease and to have direct feedback on the effects of treatment. NMR could in principle be the method of choice, allowing high throughput and fast response, if only the problem of the low sensitivity could be solved. For this reason, the quest for hyperpolarization methods in NMR is almost as old as NMR itself [2–4]. The first approach towards dynamic nuclear polarization was proposed by ⇑ Corresponding author. E-mail address: [email protected] (J. van Bentum). http://dx.doi.org/10.1016/j.jmr.2016.01.010 1090-7807/Ó 2016 Elsevier Inc. All rights reserved.

Overhauser [2] based on cross-relaxation of an interacting electron–nuclear spin system. The first observation by Carver and Slichter [4] in metals with mobile electron spins indeed confirmed the basic concept and soon it was realized by Abragam and Goldman [5] that the same method can also be applied with stable radicals in liquid solution. This has been a very active field of research in the 1970-s [6–10] but with the advent of high field NMR instrumentation, the momentum shifted to efficient pulsed NMR methods at magnetic fields above 9 T (400–900 MHz). One of the few exceptions is probably the work done in the group of Dorn [11– 13], which pioneered a flow setup with DNP at 0.35 T (X-band EPR) with NMR detection at high magnetic field. Currently, the most prominent method for boosting the nuclear polarization in the liquid state is based on the dissolution DNP route [14]. This method can indeed yield absolute polarization levels up to 60% for 13C nuclei, leading to an enhancement of the signals in the liquid state of more than 10.000 as compared to normal Boltzmann polarization levels. In a single scan it is possible to obtain signal to noise levels that would be nearly impossible by infinite signal averaging. For example, direct 13C NMR ligand binding studies at natural isotopic abundance of 13C become feasible in this way [15]. However, it is also fair to mention the drawbacks of the method, where the sample preparation and polarization step can take several hours. Most of the dissolution DNP effort is now concentrated on in vivo MRI with the promise of metabolic contrast for cancer research [16]. The application for screening low concentration drugs in the bloodstream has shown potential down to the low micro-molar concentration level [17], but the necessity for 13C isotope labelling does restrict the general applicability.

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Finally, it should be noted that the present generation of dissolution DNP instrumentation works with liquid state volumes of several mL, which is not optimized for the analysis of mass-limited samples. The typical time window for NMR experiments in dissolution DNP is only about 100 s for 13C nuclei and in general the polarization of fast relaxing nuclei such as protons is largely lost during the dissolution and transport step. Although proof of concept experiments have demonstrated the feasibility of single-scan 2D methods [18,19], more intricate 2D or 3D structural NMR with quantitative response is generally not possible. The Kockenberger group in Nottingham has spent a considerable effort in the development of a dual centre magnet aimed at solid phase DNP at a convenient field/frequency of 3.4/95 GHz, followed by a low temperature solid transport and in-situ dissolution and NMR detection at high field (400 MHz) [20]. Para-hydrogen hyperpolarization methods for liquid state samples, has seen a substantial progress to polarize low concentration substrates in solution. Initially [21] it was necessary to invoke an irreversible hydrogenation reaction with para-hydrogen molecules, limiting the method to typical probe molecules with double or triple carbon–carbon bonds. The recent invention of the SABRE method [22] has shown that the interaction with a metal centre catalyst can also repeatedly transfer the coherence of the singlet para-hydrogen to observable magnetization of substrate molecules. For example, it was demonstrated that in microtesla fields an efficient transfer to long-lived nuclei, such as 15N is feasible [23,24]. The application for the detection of low concentration substrates has been hampered by the fact that competition in binding limits the SABRE efficiency to millimolar concentrations. Recent advancements [25–28] have shown that the addition of a co-substrate can ameliorate the problem and low micro-molar concentrations can be measured in a quantitative way. It was also shown that a coherent transfer to a bound substrate is possible and the detection of the bound complex is possible with high sensitivity, allowing detection in the sub-micromolar regime. At present it is not clear how generic this method can become. Nevertheless, a subclass of biomolecules can indeed be hyperpolarized and a 2D selective filter can be applied to separate the different compounds in a complex mixture. More recently there has been a renaissance of Overhauser DNP. This is partly due to the fact that the DNP process is very dependent on local dynamics (solvent viscosity) and thus can be used to measure molecular motions in the hydration shell of biomolecules [29–34]. Even at low field, this effect can provide unique information that is very hard to obtain with conventional NMR methods. The development of high power microwave sources at 95 GHz (Extended Interaction Klystrons) and above (mostly Gyrotrons) triggered a series of high field Overhauser experiments on DNP polarization in water and other small molecule solvents [35–41]. Rather unexpected enhancement levels of
165 for water at a field of 3.4 T [42,43] and
80 at 9.4 T [44–49] were reported. The high enhancement is possible because of the very fast water dynamics at temperatures near the boiling point and above. While the results up to 3.4 T can be quantitatively understood within the original Overhauser model, the jury is still out if translational dynamics is sufficient to explain the high field results [50–52]. The main obstacle remains that high field Overhauser DNP has been mainly successful for very small molecules such as H2O, but the enhancement level rapidly decreases for larger molecules. A second methodological innovation came with the advent of rapid-melt DNP. In fact this is a kind of hybrid modification of Dissolution DNP, where the sample is polarized in the low temperature solid phase with NMR detection in the liquid after a fast melting step. This has the advantage that the irreversible dissolution step is avoided and repetitive polarization–detection cycles can be used for signal averaging or multidimensional NMR. The

initial step towards this technique was developed in the MIT group of Griffin [53,54], where they used a CO2 laser to melt the frozen sample. More recently, a full integration of miniaturized capillary NMR with stripline NMR chips and multi stage low temperature solid DNP and melting all within the homogeneous field ranges of a single centre wide bore magnet was realized [55]. As the melting of small capillary samples can be performed in times as short as 30 ms, the relaxation losses during melting can be reduced and fast cycle repetition becomes possible, including direct proton NMR detection. In this contribution we will summarize the state of the art of Overhauser and rapid-melt DNP in view of their perspective for high-field NMR spectroscopy. An outlook on their potential and the challenges faced in order to live up to that potential is discussed in the last section.

2. Liquid state Overhauser DNP In 1953 Albert Overhauser [2] considered the possibility that cross relaxation between two different spins (for example electron and proton) can lead to polarization transfer and thus induce a non-Boltzmann occupation of the spin levels. He showed that this indeed can lead to polarization enhancement for the spin with the lowest gyromagnetic ratio. As shown in Fig. 1, the four-level diagram of the interacting spin pair has various relaxation channels, where the electron and nuclear T1 is determined by the relaxation rates We and Wn. In general, the electronic relaxation rate We is fast. Wn includes the single quantum nuclear relaxation induced by the fluctuating dipolar field of the electron spin. The difference in the zero- and double-quantum cross relaxation terms W0 and W2 gives rise to a net transfer of polarization from electron to nucleus. As shown by averaging the dipolar interaction terms over all possible electron–nucleus interaction orientations, the double quantum rate is much larger than the zero quantum term (W2 = 6 W0) and dominates the liquid state polarization transfer if the dipolar matrix elements are much stronger than the scalar ones. If the microwave B1 field is sufficiently strong to saturate the allowed EPR transition, the vertical population levels will become equal. On the other hand, the presence of the cross relaxation channel will try to establish thermodynamic equilibrium between the energy levels connected by the cross relaxation, in this example dominated by W2. This will lead to a Boltzmann population difference of the nuclear states determined by the electronic Zeeman energy and the temperature of the lattice. The consequence is that the nuclear polarization (difference between left and right levels in the diagram) will now scale with the electronic Zeeman energy, which is 660 times larger than the nuclear energy scale for protons. As the net transfer between left and right is a balance between double quantum, zero and single quantum relaxation, the maximum enhancement is half the ratio of the gyromagnetic factors (330), and can be obtained in the low field limit. At higher magnetic fields, the energy difference of the double quantum transition cannot be dissipated by spin–lattice interactions. In the limit of very short correlation times, the Heisenberg uncertainty principle lifts this energy conservation bottleneck even at high magnetic field. The relative strength of the cross relaxation channels can be calculated using an appropriate spectral density function for the truncated dipolar interaction during rotational or translational collisions between radical and substrate molecule. The common spectral density function for Overhauser dynamics is based on the force-free hard sphere model [56]. In essence this model is based on an averaged dipolar interaction with a minimal isotropic spin–spin distance determined roughly by the van der Waals radii of the molecules. The correlation time is defined by

sc ¼ d2 =ðDs þ Di Þ, where d is the effective minimum distance

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Fig. 1. Level diagram for a coupled electron–nuclear spin system. The various transition rates are explained in the text.

parameter, Ds is the diffusion constant of the radical and Di is the diffusion constant of the molecule. As most molecules are not spherical, and the electron spin is generally not localized at the centre of the radical, the parameter d should be treated as a phenomenological parameter. Molecular dynamics simulations might give more insight in the physical basis of this approximation [51,52], but in most cases the simplified hard sphere model works surprisingly well. The most commonly used analytical expansion of the hard sphere spectral density function is given by Hwang and Freed [56]. At high magnetic fieldsðxsc > 1Þ, where x is the energy difference of the respective cross relaxation process, the probability decreases rapidly because of energy conservation restrictions. This model also explains why the enhancement observed for large molecules, with a low value of the diffusion constant, is rather low. In Fig. 2 we have reproduced the predictions from the hard sphere model for three distinct correlation times. The spectral density for the double quantum cross relaxation is shown in the upper panel and the corresponding maximum enhancement levels for ideal cases, assuming full EPR saturation and negligible intrinsic relaxation (or sufficiently high radical concentration). The green1 curves represent the typical case for water at room temperature. A selfdiffusion constant of 2  10
9 m2/s, combined with a typical minimum distance parameter d = 0.24 nm, leads to a correlation time of about 24 ps. At low magnetic fields it is still possible to achieve useful enhancement levels but at high fields this rapidly decreases as summarized in Table 1, first row. For water close to the boiling point (100 °C) the useful enhancement is extended to the more common NMR field ranges (second row). Finally, if a hypothetical correlation time of 1 ps can be realized, the Overhauser mechanism remains highly efficient up to the highest magnetic fields. Note that the actual experimental observations are within a factor two from these theoretical limits. For temperatures close to 100 °C, a maximum enhancement level of
165 was reported [43] for water–TEMPOL at 3.4 T (144 MHz) using an EIK amplifier source at 95 GHz. Prisner et al. [48,49,57] reported a maximum enhancement of about
80 for superheated water using a Gyrotron source and NMR detection at 400 MHz. Although the water polarization can be substantial, even at high magnetic fields, it is not trivial to use this for routine NMR experiments. First, the typical volumes used are limited by the size of the microwave resonators (hundreds of nanoliters), limiting the in-situ approach to microcoil experiments on small samples. Using chemical exchange via labile protons could provide a way to polarize

1 For interpretation of color in Fig. 2, the reader is referred to the web version of this article.

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biomolecules in combination with INEPT sequences or by spin diffusion to the relevant nuclei of interest [58–60]. Most biomolecules will not appreciate the elevated temperatures where the water dynamics is fast, so a compromise must be accepted. On the other hand, even at relatively low enhancement levels, the Overhauser effect itself can be used to create contrast based on local dynamics in bio-membranes or other heterogeneous structures. It is also possible to generate a flow of hyperpolarized water, for example combined with immobilized radicals [13]. This can be a very instructive way to create local contrast with enhanced sensitivity in microMRI experiments [61]. Most of the Overhauser liquid state DNP work has focused on the elucidation of mechanisms and relatively little attention was paid to applications. In particular, there have been no reports on Overhauser-DNP enhanced 2D correlation studies. Some work is done on nuclei other than protons (13C [62], 19F [63], 31P, 15 N [64]) but full optimization is missing and there is no detailed insight in the relevant interactions.

3. Overhauser DNP in supercritical solvents A more generic road towards Overhauser DNP could be the use of supercritical solvents. These solvents are commonly used in industrial extraction and for example in routine chromatography. The main characteristic of the supercritical phase diagram is that both density and viscosity are strongly dependent on pressure. For a proper combination of temperature and pressure it is possible to tune the conditions for a density close to that of usual liquids but with a viscosity that is in-between gas-phase and liquid phase. The increased molecular mobility allows for much faster diffusion in chromatography columns and the faster chemical kinetics in for example hydrogenation can be used in large scale chemical synthesis [65]. NMR in supercritical solvents represents a small but rather active community. Notable advantages are that fast rotational tumbling allows a higher resolution in particular for larger biomolecules [66]. The spectral resolution for quadrupolar nuclei (2H, 17 O, metal complexes [67]) in solution can also benefit from the fast averaging of quadrupolar couplings and nearly-isotropic resonances can be observed. For Overhauser DNP, the fast translational mobility is essential and solvents like for example supercritical CO2 provide a proton-free background. An interesting aspect of CO2 is that high concentrations of co-solvents are possible without severe changes in the supercritical properties. Also, the critical point is at a rather convenient temperature (31 °C) and pressure (73 bar). This allows a continuous variation of the polarity of the mixture and solubility can be tuned for both non-polar and polar solutes. In particular, this can be very useful to accommodate different radicals with polar and non-polar end-groups. The real exciting ingredient in supercritical DNP is that radical mobility of for example small nitroxide moieties can be quite high (approaching 10
8 m2/s). In the analysis above, it is the combined diffusion of radical and solute molecule that counts, and in principle also very large (immobile) molecules could be polarized by modulated interactions of the translational and rotational motion of the radical molecule. Apparently, this last point has been overlooked in literature. Typical diffusion constants in various supercritical solvents are in the range between 1 and 3  10
8 m2/s [68,69]. For nitroxide radicals, like TEMPO in CO2, this translates to an expected translational correlation time of 2–5 ps, which allows for enhancements in the region in between the red and blue lines in Fig. 2. This indicates that small molecule solutes could theoretically be enhanced to a level close to the theoretical limit. The main advantage then is that the sample temperature can remain close to room temperature. The intrinsic dielectric microwave heating of CO2 is much smaller than that of water, which may help in simplifying overmoded large volume cavity designs.

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Fig. 2. Top: Spectral density function for the double-quantum cross relaxation as function of the magnetic field, indicated by the corresponding NMR frequency for water doped with TEMPOL radicals at room temperature, 100 °C and for a solvent with a hypothetical correlation time of 1 ps. Bottom: The theoretical upper limit of the achievable Overhauser DNP enhancement for these three cases.

Table 1 Predictions for maximum Overhauser DNP in liquids. All calculations assume a negligible intrinsic proton relaxation rate (or sufficiently high radical concentration) and full microwave saturation of the EPR line. The upper row corresponds with the situation for TEMPOL radicals in water at room temperature. The second row is a typical situation for water close to the boiling point, and the bottom row corresponds with a hypothetical solvent that gives a correlation time of 1 ps. Di [m2/s]

sc [ps]

enh@144 MHz

enh@400 MHz

enh@600 MHz

2  10
9 8.6  10
9 5  10
8

24 5.6 1


50
180
280


10
80
230

5
50
200

The first attempts towards supercritical DNP were probably performed in the group of Dorn [11–13]. At the low magnetic field of 0.3 T, enhancement factors are realized above a factor 200. As the NMR detection is done in a separate high field magnet, the supercritical low viscosity helps in providing fast liquid transport to minimize relaxation losses during transport. A disadvantage in such flow experiments is that, although the enhancement at low magnetic field is impressive, the nuclear polarization is generated at the lower field, and the signals measured at high field should be compared with the Boltzmann polarization at the higher field. The net improvement in signal to noise, normalized to the thermal equilibrium at the higher field is reduced by a factor 9.4/0.33  30 and the effective signal enhancement is much more modest [12,13,70]. The same observation is also true for the more recent shuttling experiments with low field Overhauser DNP in normal solvents [36,38,71–73]. With the advent of high power THz microwave sources, it becomes possible to perform in situ DNP with substantially reduced relaxation losses. With the advent of the second generation supercritical chromatography instruments it has become possible to link chromatography with in-line flow NMR detection. The most convenient way to achieve the high pressure conditions without sacrificing the sensitivity is to use capillary flow probes. A specific version of these is the stripline NMR probe, where a lithographically defined structure

is used as the RF coil [74–79]. A modification of the instrument with additional valves and pumps allows the systematic introduction of co-solvent and radicals, as indicated in Fig. 3. As the high pressure setup also allows for measurements under superheated conditions, we revisited the water–TEMPOL system for temperatures up to 200 °C at a pressure of 150 bar. The results are shown in Fig. 4 [80], where the experimental points are normalized for the temperature-dependent Boltzmann factor and density. The solid line is a theoretical calculation, using the known diffusion constant from literature [43] and assuming a minimum contact distance of 0.245 nm. The temperature is determined from the proton chemical shift. Note that the observed maximum enhancement is the highest value reported for Overhauser DNP at a field of 3.4 T. Pulsed field gradient echo measurements provide a convenient way to determine the diffusion constants in an NMR setup. However, it is also possible to use a static RF B1 gradient to perform equivalent measurements without the added complexity of the switched gradient coils. As the stripline technology provides an elegant method to create arbitrary shape B1 profiles, this method can be used to measure on-chip capillary sample diffusion constants as function of the co-solvent fraction [80]. It is relevant to measure the diffusion dynamics in situ, as for example cosolvent modifiers will change the phase diagram and the diffusion constant becomes dependent on the molar fraction of the modifier. At a sample temperature of 18 °C, so actually in the sub-critical regime and at low co-solvent concentrations, the diffusion constant for toluene in CO2 exceeds 10
8 m2/s. For increasing (toluene) co-solvent concentrations the diffusion constant decreases. From the hard sphere Overhauser analysis it is clear that once the diffusion dynamics is known, the only parameter in the cross relaxation is the effective distance parameter d. A detailed study of the longitudinal relaxation at 600 MHz, as function of temperature, radical concentration and toluene co-solvent fraction in CO2 showed that the relaxation can be modelled within the hard sphere model with a constant contact distance (d = 0.234 and 0.252 nm for ring protons, respectively methyl protons) in toluene–TEMPO

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temperature and pressure make it feasible to approach the hypothetical 1 ps correlation time limit. A preliminary measurement of the maximum enhancement of a 50 mol% toluene in CO2 at 3.4 T showed a signal enhancement relative to the room temperature Boltzmann signal of about 160 [80]. As microwave heating, leading to a reduction in density, cannot be completely avoided, the actual enhancement is higher. More quantitative measurements are needed to verify the various details. Also in this case, the step towards real applications is not yet established. There is a clear need for additional work towards multi-nuclear, multidimensional NMR under the given conditions with DNP enhanced sensitivity.

4. Rapid-melt DNP

Fig. 3. Setup for microfluidic NMR with automated supercritical flow control.

Fig. 4. Observed saturation DNP enhancement for water at 95 GHz (3.4 T), and a pressure of 150 bar, as function of the temperature (open symbols). The solid line is a prediction for Overhauser DNP in a hard sphere model, using experimental diffusion constants and a distance parameter d = 0.245 nm.

interactions. This value is in good agreement with previous measurements on the water–TEMPOL interaction (0.25 nm) [43], and the more recent high pressure results shown in Fig. 4 (d = 0.245 nm) [80]. The correlation times for a rather high toluene/CO2 fraction of 8 mol% vary from 3.1 ps at 18 °C to 2.2 ps at 32 °C for the ring protons in toluene. For the methyl protons we find a slightly shorter correlation time of 2.5 ps resp. 1.7 ps at these two temperatures. The expected correlation times in the low co-solvent concentration limit are shorter, because of the increased diffusion characteristics. Further optimization of

An alternative approach to in-situ liquid state Overhauser DNP is to use a hybrid method where the polarization is done in the low temperature solid phase. The most common procedure is based on the dissolution method where superheated solvents are injected onto the frozen pellets in the DNP magnet. The dissolved sample is then transferred to the NMR magnet for quasi-single scan detection. An equivalent procedure is possible by in-situ melting of the sample. This was for example shown by the group of Griffin [53,54], where they used a high power infrared laser to melt the sample inside the cold region of the DNP probe. Using a CrossPolarization (CP) step in the solid phase to transfer proton polarization to 13C or other X (15N, etc.) nuclei extends the time window for melting as the low-gamma T1 is generally much longer, even in the presence of the radicals. Other groups are working towards a similar approach with either in-situ dissolution or melting within a dual centre magnet. The main advantage of melting, rather than dissolution is that the process becomes reversible and the polarization/detection cycle can be repeated for signal averaging or multidimensional analysis. Also, the in-situ dissolution or melting avoids the relaxation during the low field transport between the two magnets. A disadvantage is the fact that the radicals remain at a rather high concentration, leading to faster relaxation in the liquid state. A fairly recent development is the approach using microfluidic capillaries that are shuttled mechanically between the different zones of the probe [55]. In the low temperature zone, the sample is cooled to liquid nitrogen temperature. A non-resonant microwave concentrator is used to allow an effective broad-band microwave excitation for solid state DNP. As the power-density of the microwaves in the small volume of the concentrator can be much higher than for example in the usual case of non-resonant Dissolution-DNP or MAS-DNP, the local microwave B1e field can be quite high. With the availability of high power CW solid state sources (up to 100 W for a 95 GHz Extended Interaction Klystron), the phase space for different DNP mechanisms can be explored effectively. Since the sample is static, the dominant mechanism is in most cases not the Cross-Effect as the level crossings during mechanical rotation of a bi-radical do not occur. Commercially available mono-radicals such as TEMPOL and BDPA can be used and the dominant polarization mechanism at high power is the Solid Effect. In Fig. 5 we have reproduced the EPR spectrum and the normalized DNP enhancement spectrum for BDPA in a frozen toluene solution [81], in good agreement with theory. Although the probability for nominally forbidden Solid-Effect transitions is generally rather small, the transition rate for the well resolved case is basically linear with microwave power and for sufficiently high microwave B1e fields full polarization enhancement up to a factor 660 might be possible. The combination of small sample volume with the potential of high Q single mode resonators opens a sofar unexplored regime. Cooling with flowing liquid nitrogen of

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Fig. 5. (a) Simulated EPR spectrum for BDPA radicals in frozen solution, assuming a g-factor of 2.0026, a hyperfine tensor A = (1, 1.5, 5) MHz and a homogeneous linebroadening of 25 MHz. (b) Prediction for Solid Effect DNP (blue line) compared with normalized experimental results (open symbols). The red line represents an additional Gaussian on-resonance DNP contribution. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

small capillary samples can be quite effective and microwave heating can be partly compensated. A critical step in the rapid melt DNP approach is the transition from solid to liquid state. Near the melting point, the molecular mobility is low and paramagnetic relaxation rates can be quite high. On the other hand, the heat capacity of small capillary samples is low and heat conductivity of typical thin wall fused quartz capillaries is rather high. In practice, melting times of about 30–50 ms can be realized for solvents such as toluene that have a low latent heat for the solid/liquid phase transition [81]. Water on the other hand has a much higher specific- and latent heat, leading to typical melting times of 300– 500 ms. In Fig. 6 we have reproduced a set of 1H DNP buildup curves for frozen aqueous samples doped with TEMPOL radicals [81]. The buildup time is about 5 s at a temperature near 77 K, and saturation is achieved at power levels of about 10 W in the non-resonant microwave setup.

The actual relaxation in the liquid state depends strongly on the details of the radical–solute interaction. For nitroxide radicals in the liquid state the induced cross relaxation is very strong and forms the basis for Overhauser DNP. For radicals such as BDPA, the electron spin is more shielded on the internal bonds of the molecule and the liquid state relaxation is an order of magnitude lower. In the detection zone of the rapid-melt DNP approach, a microcoil NMR detector is used to achieve an optimal filling factor for these small sample volumes. Again, the stripline is the preferred structure as the open axial design allows for easy sample capillary shuttling along the axis of the magnet. In theory, the maximum enhancement, normalized to the room temperature Boltzmann signal, that can be realized by the rapid melt method is a combination of the low temperature DNP efficiency multiplied by the difference between the respective Boltzmann factors at the liquid and solid temperatures. For

Fig. 6. (a) Solid DNP buildup curves for a mixture of 25% D2O, 25% H2O and 50% d8-glycerol at various microwave power levels. (b) Maximum enhancement as function of the microwave power (open symbols). The solid line is a fit to a modified Solid-Effect DNP model, including the temperature jump.

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DNP at nitrogen temperatures with full theoretical efficiency, the proton enhancement could be as large as 660 ⁄ 293/77 = 2500. For 13 C, assuming a fully effective cross polarization in the solid phase, the enhancement can be 2–4 times larger. Also the more sensitive microcoil detection can add up to a factor 10 in improved sensitivity, when compared with standard large volume liquid state NMR. As this approach is still in its infancy, the DNP enhancement needs careful optimization in terms of sample formulation and radical design. Nevertheless, preliminary measurements have shown that an amount of 10–100 pmol pyruvate (corresponding with the total amount of molecules at a concentration of 0.05 lM in a traditional NMR tube volume of 200 lL) can be readily detected in about half an hour even at the low field of 3.4 T and with a rather compromised resolution in the prototype setup. A preliminary result obtained with this method is reproduced in Fig. 7 [55]. The pyruvate signals are indicated by the red arrows. The main peaks are DNP enhanced toluene signals from the 99% deuterated solvent. If a similar or improved performance can be realized at high magnetic field, then a detection limit in the sub-pmol regime should be possible. The rapid cycle option, with a total cycle delay of about 5 s, including the freezing/melting steps, allows for most common 1D and 2D NMR sequences. A critical aspect for multidimensional experiments is the enhancement stability. An interesting aspect of the capillary setup is that a variety of sample conditions is possible, including the high pressure (supercritical) liquid state discussed above. Also, the microwave power density for the miniaturized sample volumes can be very high, allowing for full saturation of the relevant DNP transitions. A detailed analysis of the power dependent enhancement and buildup time constant as function of the proton density of the sample mixture showed that the upper limit in the enhancement is largely due to a spin-diffusion bottleneck [81]. This insight might help to design future radical molecules with improved performance. For aqueous samples a typical melting time of 500 ms was found, and a direct proton detection in the liquid state is compromised because of the fast relaxation during and after melting. A solid-state cross polarization step to low gamma nuclei, as was demonstrated by the Griffin group [53,54], can alleviate this problem.

Fig. 7. DNP enhanced proton spectrum of a 50 nL sample volume, containing 1.5 nmol pyruvate in a d8-toluene solvent (99% deuteration). The signal is averaged over 300 scans with a cycle delay, (including freezing, DNP and melting) of 6 s.

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5. Outlook and conclusion In terms of physical concepts, there is no fundamental obstacle that prevents the application of high field liquid state Overhauser DNP. The use of supercritical solvents seems to provide a suitable pathway for this application, and correlation times approaching 1 ps have been achieved. This opens essentially the full NMR magnetic field range for future liquid state DNP. Nevertheless, there remain a number of technological issues that need to be addressed. First, the use of high pressures above 100 bar is not trivial in NMR. The usual autoclave option is not the best for routine measurements of many samples. Also, the thick wall sample cells lead to a reduced detection sensitivity as the filling factor for the RF coils is reduced. The use of capillary flow systems seems a more promising method, where high sensitivity microcoil detection can be combined with high throughput sample exchange. Also, the safety issues involved with high pressure is much less critical in low volume capillary systems. A down side of the capillary approach is that sample volumes remain rather low. A second important issue that needs to be solved is the construction of suitable high frequency resonators. The requirement of a high microwave B1 field, combined with minimal dielectric heating in a sufficiently large volume is a bit contradictory. Also, the field distortion of the resonator structure should be minimal if one aims for in-situ high resolution NMR detection. A way out can be the use of a short distance shuttling system within the homogeneous field of a single centre magnet [55] or in a dual centre magnet [20]. In that case one can optimize the problem of microwave excitation and RF detection in spatially separated volumes. If successful, the implementation of a high-conversion-factor resonator can help to reduce complexity and costs, in particular if lower power solid state sources can be used. A third issue is that the search for optimized radicals for high frequency Overhauser DNP has not been addressed in detail. Although small stable nitroxide radicals seem to be the obvious choice, the hyperfine coupling with the nitrogen nucleus does complicate the EPR spectrum and full saturation is less trivial. This is less of an issue at high radical concentrations, where the Heisenberg exchange effectively mixes the saturation over all hyperfine lines. The promise of supercritical solvents is that low concentration radicals are sufficient to dominate the cross-relaxation as the intrinsic T1 can be rather long. For low concentration radicals it is an option to use frequency swept or modulated excitation schemes to achieve broad-band saturation. The final proof of the pudding for Overhauser DNP will be the option to polarize larger molecules in the field of Bio NMR and of low gamma quadrupolar nuclei in both bio and (surface)materials science. The high mobility of the nitroxide radicals in supercritical solvents might provide a unique approach towards this goal, but experimental verification has not yet been demonstrated. The capability to invoke solid–liquid phase changes within the nuclear coherence times of protons and low gamma nuclei may allow novel NMR experiments, combining the potential of solid and liquid NMR in a single experiment. It is desirable to design water soluble radical molecules that combine well resolved Solid Effect DNP with low paramagnetic relaxation in the liquid state. Melting of non-polar solvents such as toluene, with a low specific heat and low latent heat for melting, can be achieved in less than 30 ms for a typical sample volume of 50 nL. As the paramagnetic relaxation of BDPA in this low viscosity solvent is slow, relaxation losses during the melting transition are minimized. The typical DNP enhancement of about 100–400 allows a reduction of measurement times by a factor above 10.000, reducing demanding experiments to single or few scan experiments. In combination with chromatography and intermediate concentration, an in-line

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detection is possible with fairly generic hyperpolarization. From a practical point of view we should note that the operational costs in terms of chemicals (including radicals) and cryogenic liquids are negligible, compared to the present commercial options. In the present study we used only the widely available and cheap TEMPO(L) and BDPA radicals. Also the investment costs are quite modest, as the use of a second superconducting magnet for either polariser (Dissolution) or Gyrotron oscillator is avoided. The miniaturized microwave excitation volume allows a future implementation of a high Q resonating structure and could facilitate the use of low cost solid-state microwave sources. It is relatively straightforward to scale the detection volume from the present 50 nL to a few microliters, possibly extending applicability for practical problems. The fast shuttling technique within the homogeneous bore of the magnet avoids relaxation losses and as the microwave DNP resonator is physically separated from the NMR detection both can be optimized separately. Finally, the sensitivity enhancement is a multiplication of factors given by the Boltzmann temperature jump (3 or more, depending on operating temperature) DNP enhancement (100–200, depending on radical optimization) and NMR detection efficiency (SNR improvement 10 for small volume microcoil or stripline), leading to a total potential enhancement by a factor exceeding 4000 for protons (or >10.000 for low gamma nuclei such as 13C with CP) as compared to routine measurements in a standard NMR system. Scaling to higher magnetic field levels will depend mostly on the microwave technology at these high frequencies. The successful demonstration of rapid melt DNP indicates that SNR enhancements are possible that can rival those of dissolution DNP, but in a fraction of the time and in-situ without irreversible changes to the sample. In principle this enhancement can be achieved without compromising resolution, and standard NMR operations are possible, including proton detection, multi scan averaging and generic 2D correlation spectroscopy. Possibly the most surprising aspect is that for the present low volume capillary samples the total cycle delay, including freezing, DNP polarization, melting and NMR acquisition can be as short as a few seconds. An extension to liquid Helium temperatures will increase the overall enhancement, at the cost of complexity and speed. More than 60 years after the conception of liquid state DNP, the general perception is still that this path is only viable at low magnetic fields and a combination with high field NMR is cumbersome at least. The outcome of various developments in the last few years is that this perception is too pessimistic and mobility in for example supercritical solvents is sufficiently fast to provide an effective partway for Overhauser DNP. As an alternative, rapid in-situ melting of a hyperpolarized solid matrix allows highly sensitive liquid state NMR with surprisingly fast recycle delays. The combination of microfluidic sample handling and on-chip microcoil detection with rapid-melt DNP polarization enhancement may provide a very effective way to measure NMR signals of mass limited samples. The basic physics of both approaches seems to be verified although substantial technological optimizations are needed to become a routine method for everyday use. Acknowledgments Part of this ‘perspective’ is based on ongoing and partly unpublished research at the Radboud University. This work would not be possible without the efforts of numerous people in our group. Hans Janssen designed and built many of the stripline probes. Jacob Bart and Koen Tijssen developed and tested many of the microfluidic NMR applications. Overhauser DNP in normal solvents was developed with the help of Jorge Villanueva-Garibay, Giuseppe Annino and Gijs van der Heijden. Michael Tayler was instrumental in setting up the coupling between supercritical chromatography and NMR, together with Bas van Meerten. Gerrit Janssen designed

and built the rapid-melt probes. Jim Leggett debugged many of the early flaws in the rapid-melt setup and Manvendra Sharma did most of the measurements related with rapid-melt DNP. We are grateful for the financial support from the European Union and the provinces of Gelderland and Overijssel for support through the EFRO Ultrasense NMR project.

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