Category Archives: Sample Preparation

DNP NMR spectroscopy of cross-linked organic polymers: rational guidelines towards optimal sample preparation #DNPNMR

Tanaka, Shinji, Wei-Chih Liao, Atsuko Ogawa, Kazuhiko Sato, and Christophe Copéret. “DNP NMR Spectroscopy of Cross-Linked Organic Polymers: Rational Guidelines towards Optimal Sample Preparation.” Physical Chemistry Chemical Physics 22, no. 6 (2020): 3184–90.

Cross-linked polystyrenes (PS) are an important class of polymers, whose properties are strongly dependent on incorporated functionalities, for which detailed understanding of their structure remains a challenge. Here, we develop a rational guideline for dynamic nuclear polarization (DNP) sample formulation for cross-linked PS to interrogate their structure. We show that the DNP enhancement on a series of cross-linked PS bearing alkylammonium groups as prototypical organic polymers correlates with the polymer swelling properties in both apolar and polar formulations (TEKPol/1,1,2,2-tetrachloroethane and AMUPol/dimethyl sulfoxide). This work provides guidelines to easily optimize DNP formulation using a simple swelling test and enables natural abundance 15N NMR to be recorded on a series of PS-supported quaternary alkylammonium salts, allowing a detailed structural analysis.

Solvent suppression in solid-state DNP NMR using Electronic Mixing-Mediated Annihilation (EMMA) #DNPNMR

Ziarelli, Fabio, Pierre Thureau, Stéphane Viel, and Giulia Mollica. “Solvent Suppression in Solid-State DNP NMR Using Electronic Mixing-Mediated Annihilation (EMMA).” Magnetic Resonance in Chemistry, January 23, 2020.

We show here that the Electronic Mixing-Mediated Annihilation (EMMA) method, previously reported for the suppression of background signals in solid-state NMR spectra, can be successfully applied to remove the solvent signals observed in the case of NMR spectra obtained with dynamic nuclear polarization (DNP). The methodology presented here is applied to two standard sample-preparation methods for DNP, namely glass forming and incipient wetness impregnation. It is demonstrated that the EMMA method is complementary to the different methods for solvent suppression based on relaxation filters, and that it can be used to preserve the quantitative information that might be present in the pristine spectra.

Sensitivity analysis of magic angle spinning dynamic nuclear polarization below 6 K #DNPNMR

Judge, Patrick T., Erika L. Sesti, Edward P. Saliba, Nicholas Alaniva, Thomas Halbritter, Snorri Th. Sigurdsson, and Alexander B. Barnes. “Sensitivity Analysis of Magic Angle Spinning Dynamic Nuclear Polarization below 6 K.” Journal of Magnetic Resonance 305 (August 2019): 51–57.

Dynamic nuclear polarization (DNP) improves signal-to-noise in nuclear magnetic resonance (NMR) spectroscopy. Signal-to-noise in NMR can be further improved with cryogenic sample cooling. Whereas MAS DNP is commonly performed between 25 and 110 K, sample temperatures below 6 K lead to further improvements in sensitivity. Here, we demonstrate that solid effect MAS DNP experiments at 6 K, using trityl, yield 3.2Â more sensitivity compared to 90 K. Trityl with solid effect DNP at 6 K yields substantially more signal to noise than biradicals and cross effect DNP. We also characterize cross effect DNP with AMUPol and TEMTriPol-1 biradicals for DNP magic angle spinning at temperatures below 6 K and 7 Tesla. DNP enhancements determined from microwave on/off intensities are 253 from AMUPol and 49 from TEMTriPol-1. The higher thermal Boltzmann polarization at 6 K compared to 298 K, combined with these enhancements, should result in 10,000Â signal gain for AMUPol and 2000Â gain for TEMTriPol-1. However, we show that AMUPol reduces signal in the absence of microwaves by 90% compared to 41% by TEMTriPol-1 at 7 Tesla as the result of depolarization and other detrimental paramagnetic effects. AMUPol still yields the highest signal-to-noise improvement per unit time between the cross effect radicals due to faster polarization buildup (T1DNP = 4.3 s and 36 s for AMUPol and TEMTriPol-1, respectively). Overall, AMUPol results in 2.5Â better sensitivity compared to TEMTriPol-1 in MAS DNP experiments performed below 6 K at 7 T. Trityl provides 6.0Â more sensitivity than TEMTriPol-1 and 1.9Â more than AMUPol at 6 K, thus yielding the greatest signal-to-noise per unit time among all three radicals. A DNP enhancement profile of TEMTriPol-1 recorded with a frequency-tunable custom-built gyrotron oscillator operating at 198 GHz is also included. It is determined that at 7 T below 6 K a microwave power level of 0.6 W incident on the sample is sufficient to saturate the cross effect mechanism using TEMTriPol-1, yet increasing the power level up to 5 W results in higher improvements in DNP sensitivity with AMUPol. These results indicate MAS DNP below 6 K will play a prominent role in ultra-sensitive NMR spectroscopy in the future.

The effects of sample conductivity on the efficacy of dynamic nuclear polarization for sensitivity enhancement in solid state NMR spectroscopy #DNPNMR

Svirinovsky-Arbeli, Asya, Dina Rosenberg, Daniel Krotkov, Ran Damari, Krishnendu Kundu, Akiva Feintuch, Lothar Houben, Sharly Fleischer, and Michal Leskes. “The Effects of Sample Conductivity on the Efficacy of Dynamic Nuclear Polarization for Sensitivity Enhancement in Solid State NMR Spectroscopy.” Solid State Nuclear Magnetic Resonance 99 (July 2019): 7–14.

In recent years dynamic nuclear polarization (DNP) has greatly expanded the range of materials systems that can be studied by solid state NMR spectroscopy. To date, the majority of systems studied by DNP were insulating materials including organic and inorganic solids. However, many technologically-relevant materials used in energy conversion and storage systems are electrically conductive to some extent or are employed as composites containing conductive additives. Such materials introduce challenges in their study by DNP-NMR which include microwave absorption and sample heating that were not thoroughly investigated so far.

Dynamic Nuclear Polarization / solid-state NMR of membranes. Thermal effects and sample geometry #DNPNMR

Salnikov, Evgeniy Sergeevich, Fabien Aussenac, Sebastian Abel, Armin Purea, Paul Tordo, Olivier Ouari, and Burkhard Bechinger. “Dynamic Nuclear Polarization / Solid-State NMR of Membranes. Thermal Effects and Sample Geometry.” Solid State Nuclear Magnetic Resonance 100 (August 2019): 70–76.

Whereas specially designed dinitroxide biradicals, reconstitution protocols, oriented sample geometries and NMR probes have helped to much increase the DNP enhancement factors of membrane samples they still lag considerably behind those obtained from glasses made of protein solutions. Here we show that not only the MAS rotor material but also the distribution of the membrane samples within the NMR rotor have a pronounced effect on the DNP efficiency. These observations are rationalized with the cooling efficiency and the internal properties of the sample, monitored by their T1 relaxation, microwave on versus off signal intensities and DNP enhancement. The data are suggestive that for membranes the speed of cooling has a pronounced effect on membrane phase transitions and concomitantly the distribution of biradicals within the sample.

A biradical-tagged phospholipid as a polarizing agent for solid-state MAS Dynamic Nuclear Polarization NMR of membrane proteins #DNPNMR

Good, Daryl B., Maxim A. Voinov, David Bolton, Meaghan E. Ward, Ivan V. Sergeyev, Marc Caporini, Peter Scheffer, et al. “A Biradical-Tagged Phospholipid as a Polarizing Agent for Solid-State MAS Dynamic Nuclear Polarization NMR of Membrane Proteins.” Solid State Nuclear Magnetic Resonance, April 2019, S0926204018301140.

A novel Dynamic Nuclear Polarization (DNP) NMR polarizing agent ToSMTSL-PTE representing a phospholipid with a biradical TOTAPOL tethered to the polar head group has been synthesized, characterized, and employed to enhance solid-state Nuclear Magnetic Resonance (SSNMR) signal of a lipid-reconstituted integral membrane protein proteorhodopsin (PR). A matrix-free PR formulation for DNP improved the absolute sensitivity of NMR signal by a factor of ca. 4 compared to a conventional preparation with TOTAPOL dispersed in a glassy glycerol/water matrix. DNP enhancements measured at 400 MHz/263 GHz and 600 MHz/395 GHz showed a strong field dependence but remained moderate at both fields, and comparable to those obtained for PR covalently modified with ToSMTSL. Additional continuous wave (CW) X-band electron paramagnetic resonance (EPR) experiments with ToSMTSL-PTE in solutions and in lipid bilayers revealed that an unfavorable conformational change of the linker connecting mononitroxides could be one of the reasons for moderate DNP enhancements. Further, differential scanning calorimetry (DSC) and CW EPR experiments indicated an inhomogeneous distribution and/or a possibility of a partial aggregation of ToSMTSLPTE in DMPC:DMPA bilayers when the concentration of the polarizing agent was increased to 20 mol% to maximize the DNP enhancement. Thus, conformational changes and inhomogeneous distribution of the lipid-based biradicals in lipid bilayers emerged as important factors to consider for further development of this matrix-free approach for DNP of membrane proteins.

Contrasting effects of glycerol and DMSO on lipid membrane surface hydration dynamics and forces #DNPNMR #ODNP

Schrader, Alex M., Chi-Yuan Cheng, Jacob N. Israelachvili, and Songi Han. “Communication: Contrasting Effects of Glycerol and DMSO on Lipid Membrane Surface Hydration Dynamics and Forces.” The Journal of Chemical Physics 145, no. 4 (July 28, 2016): 041101.

Glycerol and dimethyl sulfoxide (DMSO) are commonly used cryoprotectants in cellular systems, but due to the challenges of measuring the properties of surface-bound solvent, fundamental questions remain regarding the concentration, interactions, and conformation of these solutes at lipid membrane surfaces. We measured the surface water diffusivity at gel-phase dipalmitoylphosphatidylcholine (DPPC) bilayer surfaces in aqueous solutions containing ≤7.5 mol. % of DMSO or glycerol using Overhauser dynamic nuclear polarization. We found that glycerol similarly affects the diffusivity of water near the bilayer surface and that in the bulk solution (within 20%), while DMSO substantially increases the diffusivity of surface water relative to bulk water. We compare these measurements of water dynamics with those of equilibrium forces between DPPC bilayers in the same solvent mixtures. DMSO greatly decreases the range and magnitude of the repulsive forces between the bilayers, whereas glycerol increases it. We propose that the differences in hydrogen bonding capability of the two solutes leads DMSO to dehydrate the lipid head groups, while glycerol affects surface hydration only as much as it affects the bulk water properties. The results suggest that the mechanism of the two most common cryoprotectants must be fundamentally different: in the case of DMSO by decoupling the solvent from the lipid surface, and in the case of glycerol by altering the hydrogen bond structure and intermolecular cohesion of the global solvent, as manifested by increased solvent viscosity.

Efficiency of Water-Soluble Nitroxide Biradicals for Dynamic Nuclear Polarization in Rotating Solids at 9.4 T: bcTol-M and cyolyl-TOTAPOL as New Polarizing Agents #DNPNMR

Geiger, Michel-Andreas, Anil P. Jagtap, Monu Kaushik, Han Sun, Daniel Stöppler, Snorri T. Sigurdsson, Björn Corzilius, and Hartmut Oschkinat. “Efficiency of Water-Soluble Nitroxide Biradicals for Dynamic Nuclear Polarization in Rotating Solids at 9.4 T: BcTol-M and Cyolyl-TOTAPOL as New Polarizing Agents.” Chemistry – A European Journal 24, no. 51 (September 12, 2018): 13485–94.

Nitroxide biradicals are very efficient polarizing agents in magic angle spinning (MAS) cross effect (CE) dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR). Many recently synthesized, new radicals show superior DNP-efficiency in organic solvents but suffer from insufficient solubility in water or glycerol/water for biological applications. We report DNP efficiencies for two new radicals, the water-soluble bcTol-M and cyolyl-TOTAPOL, and include a comparison with three known biradicals, TOTAPOL, bcTol, and AMUPol. They differ by linker groups, featuring either a 3-aminopropane-1,2-diol or a urea tether, or by the structure of the alkyl substituents that flank the nitroxide groups. For evaluating their performances, we measured both signal enhancements e and DNP-enhanced sensitivity k, and compared the results to electron spin relaxation data recorded at the same magnetic field strength (9.4 T). In our study, differences in DNP efficiency correlate with changes in the nuclear polarization dynamics rather than electron relaxation.

The ratios of their individual e and k differ by up to 20%, which is explained by starkly different nuclear polarization build-up rates. For the radicals compared here empirically, using proline standard solutions, the new radical bcTol-M performs best while being most soluble in water/glycerol mixtures.

Effect of water/glycerol polymorphism on dynamic nuclear polarization #DNPNMR

Leavesley, Alisa, Christopher B. Wilson, Mark Sherwin, and Songi Han. “Effect of Water/Glycerol Polymorphism on Dynamic Nuclear Polarization.” Physical Chemistry Chemical Physics 20, no. 15 (April 18, 2018): 9897–9903.

A paramount feature of robust experimental methods is acquiring consistent data. However, in dynamic nuclear polarization (DNP), it has been observed that the DNP-induced NMR signal enhancement of nominally the same sample can vary between different experimental sessions. We investigated the impact of various freezing conditions on the DNP results for a standard sample, a 50/40/10 by volume d8-glycerol/D2O/H2O solution of 40 mM 4-amino TEMPO, and found that annealing the samples 10 K above the glass transition temperature (Tg) causes significant changes to the DNP profiles and enhancements compared to that in rapidly frozen samples. When varying the glycerol composition to yield a solution of 60/30/10 d8-glycerol/D2O/H2O, the DNP performance became markedly more consistent, even for samples prepared under vastly different sample freezing methods, in stark contrast with that of the 50/40/10 solution. The EPR lineshapes, Tm, and glass transition temperature, Tg, were measured under the same sample and experimental conditions as used for the DNP experiments to support the conclusion that different freezing methods change the distribution of 4-amino TEMPO radials in the 50/40/10 solution due to the formation of different polymorphs of the glass, which is mitigated in the 60/30/10 solution and is consistent with the water/glycerol vitrification literature.

Unusual Local Molecular Motions in the Solid State Detected by Dynamic Nuclear Polarization Enhanced NMR Spectroscopy #DNPNMR

Hoffmann et al., “Unusual Local Molecular Motions in the Solid State Detected by Dynamic Nuclear Polarization Enhanced NMR Spectroscopy.”

Polyethylene glycol (PEG) and three related surfactants were studied by dynamic nuclear polarization (DNP) enhanced solid state NMR spectroscopy and differential scanning calorimetry (DSC). DNP enhanced solid state NMR surprisingly reveals the presence of local molecular motions that are normally understood to be inactive at temperatures ∼100 K. This surprising phenomenon could be explained by the experimentally necessary rapid freezing of the studied samples. Specifically, DSC shows that PEG 200 forms a glass upon freezing and that the three PEG-related surfactants are at least partially in a glass state or some other thermodynamic nonequilibrium state when rapidly frozen to the temperatures of the DNP enhanced solid state NMR experiments. This effect of preserving local molar motions by rapid freezing also holds true for solutions of organic solutes in the PEG 200 solvent matrix.

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