Cryogenic signal amplification combined with helium-temperature MAS DNP toward ultimate NMR sensitivity at high field conditions.

The low sensitivity of NMR spectroscopy is of historical concern in the field, and various approaches have been developed to mitigate this limitation. On the shoulder of giants, today one can routinely implement, for example, the pulse/Fourier transform NMR with the cross polarization together with the ultra-low temperature MAS DNP under high-field conditions. We show in this work this current opportunity should further be augmented by combining them with the cryogenic signal amplification. Our presented MAS DNP probe operates with the closed-cycle helium MAS system, and cools the internal preamplifier-duplexer module with the "return" helium gas on its way back to the compressor in the loop. The signal-to-noise (S/N) gain relative to the room-temperature measurements of a factor of 4.6 and 2.4 was found for the measurement using the cold- and room-temperature preamplifier, respectively, at the sample temperature of T = 20 K at B0 = 16.4 T. The ratio of these factors reveals âˆ¼ two-fold sensitivity improvement that results purely from the introduction of the cold signal amplification, i.e., noise reduction. Together with the increase of the thermal Boltzmann polarization at low temperatures, the combined S/N gain of max. ∼70-fold is possible without DNP. The DNP enhancement factor of ∼40 as we found in this work for a microcrystalline MLF sample may be multiplied to this gain. We also demonstrated the sensitivity improvement with a 13C-detected 2D NCaCx spectrum, illustrating the generality of the S/N gain from combining DNP with the cold signal amplification.

A mixing microfluidic chip for real-time NMR monitoring of macromolecular reactions.

NMR spectroscopy permits real-time monitoring of reactions that involve changes in the spectra of reactants. MICCS (MIcro Channelled Cell for Synthesis monitoring) is a microfluidic chip for such purposes, which is used to rapidly activate reactions by mixing the reactant solutions in the chip inserted into the typical NMR tube. Although it allows monitoring of chemical reactions of small compounds, its simple mixing system dependent on diffusion in the microchannel was not suitable for macromolecules such as proteins with low diffusion rates. Here, we developed a new microfluidic chip based on MICCS by incorporating a mixer of split-and-recombination type within the microchannel. We applied it to monitoring of the protein-folding reaction in a stopped-flow mode. A solution of denaturant-unfolded RNase A was injected from a syringe pump into the microchip set inside the NMR magnet and mixed with a buffer for dilution to reach the folding condition. Immediately after dilution, the reaction was initiated and detected by a series of NMR measurements that were synchronized with activation and inactivation of the pump. The process was repeated for accumulation of the data. By analysing the change of the spectra by factor analysis, a kinetic constant of 0.57 min-1 was obtained.

24 T High-Resolution and -Sensitivity Solid-State NMR Measurements of Low-Gamma Half-Integer Quadrupolar Nuclei 35Cl and 37Cl.

Solid-state NMR observations of low-gamma half-integer quadrupolar nuclei, 35Cl and 37Cl, were demonstrated using a 24 T hybrid magnet (1H resonance frequency of 1.02 GHz) comprised of the high-temperature (HTS) and low-temperature (LTS) superconductors, and compared with results using a 14.1 T standard NMR magnet. While at 24 T the linewidth is 1.7 times narrower than that at 14.1 T, the gain in the sensitivity is 7.0 times because of enhanced polarization, reduced linewidth, and the use of larger rotor. A simple theoretical model was used to rationalize the sensitivity enhancements. The ratio of 35Cl and 37Cl quadrupolar couplings agrees well with the ratio of quadrupolar moments, and no isotope-dependent chemical shift has been observed. In addition, the 3QMAS spectrum of 35Cl is shown to demonstrate the high sensitivity rendered by the 24 T spectrometer.

1020MHz single-channel proton fast magic angle spinning solid-state NMR spectroscopy.

This study reports a first successful demonstration of a single channel proton 3D and 2D high-throughput ultrafast magic angle spinning (MAS) solid-state NMR techniques in an ultra-high magnetic field (1020MHz) NMR spectrometer comprised of HTS/LTS magnet. High spectral resolution is well demonstrated.

Closed-cycle cold helium magic-angle spinning for sensitivity-enhanced multi-dimensional solid-state NMR.

Magic-angle spinning (MAS) NMR is a powerful tool for studying molecular structure and dynamics, but suffers from its low sensitivity. Here, we developed a novel helium-cooling MAS NMR probe system adopting a closed-loop gas recirculation mechanism. In addition to the sensitivity gain due to low temperature, the present system has enabled highly stable MAS (vR=4-12 kHz) at cryogenic temperatures (T=35-120 K) for over a week without consuming helium at a cost for electricity of 16 kW/h. High-resolution 1D and 2D data were recorded for a crystalline tri-peptide sample at T=40 K and B0=16.4 T, where an order of magnitude of sensitivity gain was demonstrated versus room temperature measurement. The low-cost and long-term stable MAS strongly promotes broader application of the brute-force sensitivity-enhanced multi-dimensional MAS NMR, as well as dynamic nuclear polarization (DNP)-enhanced NMR in a temperature range lower than 100 K.

Achievement of 1020MHz NMR.

We have successfully developed a 1020MHz (24.0T) NMR magnet, establishing the world's highest magnetic field in high resolution NMR superconducting magnets. The magnet is a series connection of LTS (low-Tc superconductors NbTi and Nb3Sn) outer coils and an HTS (high-Tc superconductor, Bi-2223) innermost coil, being operated at superfluid liquid helium temperature such as around 1.8K and in a driven-mode by an external DC power supply. The drift of the magnetic field was initially ±0.8ppm/10h without the (2)H lock operation; it was then stabilized to be less than 1ppb/10h by using an NMR internal lock operation. The full-width at half maximum of a (1)H spectrum taken for 1% CHCl3 in acetone-d6 was as low as 0.7Hz (0.7ppb), which was sufficient for solution NMR. On the contrary, the temporal field stability under the external lock operation for solid-state NMR was 170ppb/10h, sufficient for NMR measurements for quadrupolar nuclei such as (17)O; a (17)O NMR measurement for labeled tri-peptide clearly demonstrated the effect of high magnetic field on solid-state NMR spectra.

Development of an NMR interface microchip "MICCS" for direct detection of reaction products and intermediates of micro-syntheses using a "MICCS-NMR".

The development of an NMR interface microchip and its applications to the real-time monitoring of chemical reactions are described. The microchip device was named "MICCS" (MIcro Channeled Cell for Synthesis monitoring), and the method using it was named "MICCS-NMR". MICCS was inserted into a 5 mm Phi NMR sample tube. Thus standard solution NMR probes without any modifications can be used in MICCS-NMR measurements. A gap between MICCS and the sample tube was filled with a deuterated solvent for an NMR lock. The reaction temperature and reaction time in MICCS can be easily changed by adjusting the temperature of the NMR probe and changing the flow rates, respectively. The effectiveness of the MICCS-NMR was verified in the real-time monitoring of the Wittig reaction. Preliminary data on the direct detection of intermediates of the Grignard reaction is also reported. Besides real-time monitoring of chemical reactions, MICCS-NMR would be useful as a qualitative detection method for microchip-based synthesis.