Sandwich-ELISE NMR: reducing the sample volume of NMR samples

We present Sandwich-ELISE, a concatenated version of our previously proposed Experimental LIquid SEaling (ELISE) protocol, in which an aqueous sample is effectively sealed by the addition of a small layer of mineral oil, or, alternatively, a chloroform sample was sealed by a water layer. With Sandwich-ELISE, a triple layered geometry composed of deuterated chloroform/aqueous buffer/mineral oil can be used to limit the sample to the active coil volume, effectively replacing the popular Shigemi tubes. Importantly, this procedure is readily applicable to smaller diameter tubes, for which no Shigemi tubes are available. We further present spectra of a 1 microl protein sample sandwiched between the chloroform and Nujol phases in a 1mm tube, demonstrating thereby that the volume of the aqueous phase of interest can be reduced even further.     

Experimental method for low-temperature reaction studies in sealed samples by 13C CP/MAS NMR

A new, simple, and inexpensive technique is presented for monitoring high-resolution solid-state NMR of ¹³C at temperatures ranging between 85 and 450 K. In this procedure, the reaction conditions are controlled by preparing samples at 77 K in 5 mm NMR tubes, while attached to a vacuum system. The NMR tubes are prefitted with a rotor for spinning. After preparation, the samples are sealed, transferred to the double-resonance MAS NMR probe, and analyzed, all while the sample temperature is maintained as low as 85 K. The spinning rates vary from 3.0 kHz at 85 K to 5.2 kHz at 300 K using nitrogen drive gas. Probe design and performance, sample-preparation procedure, and details of the low-temperature experiment are described. In general, the technique may be applied in studies of low-temperature reaction mechanisms and kinetics. ¹³C CP/MAS spectra of ethylene adsorbed on silica-supported ruthenium catalyst are presented to illustrate its performance and possible application.     

ELISE NMR: experimental liquid sealing of NMR samples

We present a simple, generally applicable approach to prevent sample evaporation when working at elevated temperatures in high resolution NMR. It consists of experimentally sealing the NMR sample by a second liquid (Experimental Liquid Sealing, ELISE). For aqueous samples, we identified the mineral oil commonly used in PCR application as the best candidate, because it contains only a very limited amount of water-soluble contaminants, is stable over time and heat resistant. The procedure does not interfere with shim settings, and is compatible with a wide variety of samples, including oligosaccharides and proteins. For chloroform samples, a simple drop of water allows to efficiently seal the sample, avoiding solvent evaporation even over lengthy time periods.     

Optimal electric fields for different sample shapes in high resolution NMR spectroscopy

For many applications, reducing sample resistance, rather than increasing probe Q or filling factor, is the only way to further improve the signal-to-noise ratio of cryogenically cooled NMR probes. In this paper, bounds are calculated for the minimum sample resistance that can be achieved for various sample geometries. The sample resistance of 100 mM NaCl in H(2)O in 5 mm sample tubes was measured on a 600 MHz cold probe to be within 14% of the optimum value. The minimum sample resistance can however be lowered by altering the tube cross section. Rectangular tubes oriented with the long axis along the RF magnetic field are particularly favourable.     

垂直场中环形样品核磁共振信号的特征线型

观察到了垂直场中环形样品核磁共振的U形信号不但具有文献所描述的特征,还具有细微的结构。经理论分析指出了这种细微结构的成因,修正了文献给出的裂距公式。     

Development of a dual cell, flow-injection sample holder, and NMR probe for comparative ligand-binding studies

NMR based ligand screening is becoming increasingly important for the very early stages of drug discovery. We have proposed a method that makes highly efficient use of a single sample of a scarce target, or one with poor or limited solubility, to screen an entire compound library. This comparative method is based on immobilizing the target for the screening procedure. In order to support the method, a dual cell, flow injection probe with a single receiver coil has been constructed. The flow injection probe has been mated to a single high performance pump and sample handling system to enable the automated analysis of large numbers of compound mixes for binding to the target. The probe, having an 8 mm 1H/2H dual tuned coil and triple axis gradients, is easily shimmed and yields NMR spectra of comparable quality to a standard 5 mm high-resolution probe. The lineshape in the presence of a solid support is identical to that in glass NMR tubes in a 5 mm probe. Control spectra of each cell are identical and well separated, while ligand binding in a complex mixture can be readily detected in 20-30 min, thus paving the way for use of the probe for actual drug discovery efforts.     

A temperature-jump design for conventional NMR probes

The design and performance of a simple probe insert for temperature-jump experiments in conventional NMR probes is described. The insert uses the output from conventional NMR amplifiers for heating conductive aqueous samples with a rate of 30-80 K/s for 200 W radiofrequency power. The observed dependence of the heating rate on sample conductivity is explained by the dominance of dielectric heating. Factors governing the temperature gradient within the sample are discussed.     

Performance of cryogenic probes as a function of ionic strength and sample tube geometry

The pursuit for more sensitive NMR probes culminated with development of the cryogenic cooled NMR probe. A key factor for the sensitivity is the overall resistance of RF circuitry and sample. Lowering the coil temperature to approximately 25 K and the use of superconducting coil material has greatly reduced the resistance contribution of the hardware. However, the resistance of a salty sample remains the same and evolves as the major factor determining the signal-to-noise ratio. Several approaches have been proposed to reduce the resistance contribution of the sample. These range from encapsulating proteins in a water cavity formed by reverse micelles in low viscosity fluids to the optimal selection of low mobility, low conductivity buffer ions. Here we demonstrate that changing the sample diameter has a pronounced effect on the sample resistance and this results in dramatic improvements of the signal-to-noise ratio and shorter pi/2 pulses. We determined these parameters for common 5 mm NMR tubes under different experimental conditions and compared them to the 2, 3 and 4 mm tubes, in addition, 5mm Shigemi tubes were included since these are widely used. We demonstrate benefits and applicability of studying NMR samples with up to 4M salt concentrations in cryogenic probes. Under high salt conditions, best results in terms of short pi/2 pulses and high signal-to-noise ratios are obtained using 2 or 3mm NMR tubes, especially when limited sample is available. The 4 mm tube is preferred when sample amounts are abundant at intermediate salt conditions.     

一种手持便携式核磁共振样品管高压清洗器的设计与实现

在液体核磁共振（NMR）实验使用的耗材中,NMR样品管用量非常大,通常需经清洗后回收利用.NMR样品管的清洗效率及清洗后的洁净程度直接影响到NMR实验的进程,清洗过程中产生的废液也将对环境产生影响.本文首先介绍了一款自主设计的手持便携式NMR样品管高压清洗器,并对该装置的结构和功能做了详细的说明.然后,对曾装存过不同溶剂和溶液的NMR样品管,分别利用该装置和商用NMR样品管清洗装置进行清洗,并比较了清洗效率和效果.最后,对利用此高压清洗器清洗NMR样品管时,NMR样品管的损伤程度进行了评估.结果证实,对于没有样品严重附着管壁的NMR样品管,本文设计的装置清洗效果良好、效率高;并且清洗过程中不使用有毒的有机溶剂,非常环保;而且对NMR样品管损伤较小.     

Construction and performance of an NMR tube with a sample cavity formed within magnetic susceptibility-matched glass

We describe the construction and performance of an NMR tube with a magnetic susceptibility matched sample cavity that confines the solution within the detection zone in the axial direction and in a quasi-rectangular region in the radial direction. The slot-like sample cavity provides both good sample volume efficiency and tolerance to sensitivity loss in the sample space. The signal-to-noise ratio per unit volume of the constructed tube was 2.2 times higher than that of a cylindrical tube of 5mm outer diameter with a sample containing 300 mM NaCl at a static magnetic field of 14.1T. Even the overall signal-to-noise ratio of the slot tube was 35% higher than that of the conventional 5mm tube for a sample containing 300 mM NaCl. Similar improvements over existing sample tube geometries were obtained at 950 MHz. Moreover the temperature rise resulting from RF heating was found to be significantly lower for the slot tube even when compared to 3 and 4mm outer diameter cylindrical tubes as measured in a 5mm cryoprobe. A further advantage of this type of tube is that a sample cavity of any desired size and shape can be formed within a cylindrical tube for use in a single cryogenic probe.     

On-line Surface Area Measurement of Concentrated Slurries using low field spin-lattice relaxation NMR

A method for the rapid on-line determination of surface area and solids content in flowing concentrated slurries using low field NMR spin-lattice relaxation measurements has been developed and demonstrated. The relationship between flow and spin-lattice relaxation time (T₁) of protons in water at 20 MHz was examined using aqueous copper sulfate solutions. The ability to measure surface area and solids concentration in both stagnant (stopped flow) and flowing systems via NMR was demonstrated using several different concentrated aqueous titania and glass slurries (20 to 80 weight percent) for which the dried powder surface area was previously determined via nitrogen adsorption/BET analysis and the solids content determined gravimetrically. Surface areas were also calculated from particle size analysis and found to vary by up to an order of magnitude from the adsorption and NMR results.     

Shift of the proton NMR field in aqueous solutions of paramagnetic ions

In the paper there is discussed the proton NMR field shift in aqueous solutions of transition metal ions, which has to be taken into account in exact measurements of the static magnetic field. Using the results of Luz's and Shulman's measurements (1965) the empirical expression for the field shift is evaluated, which is a function of the sample shape, of the concentration and the susceptibility of paramagnetic ions in the solution. It is shown that in exact field measurements the diamagnetic part of the susceptibility of the solution cannot be neglected and that one may eliminate the field shift by a suitable choice of the paramagnetic-ions concentration in transversely magnetized cylindrical samples.     

An apparatus for NMR of laser polarized 129Xe on single crystal surfaces

Due to a lack of at least 2 orders of magnitude in the amount of sample nuclei, single crystal surfaces are out of reach for conventional NMR measurements. Our aim is to prove that highly polarized ¹²⁹Xe provides a technique to overcome this restriction. Therefore an apparatus for polarizing ¹²⁹Xe up to 0.7 by spin transfer from optically pumped Rb has been designed as well as an NMR spectrometer in combination with a UHV chamber with sample cleaning, cooling and characterization abilities and a special manifold of glass stopcocks with a liquid nitrogen cooled trap for dosing nitrogen free polarized Xe into the chamber onto the surface. This revised version was published online in August 2006 with corrections to the Cover Date.     

High resolution 1H NMR of a lipid cubic phase using a solution NMR probe

The cubic mesophase formed by monoacylglycerols and water is an important medium for the in meso crystallogenesis of membrane proteins. To investigate molecular level lipid and additive interactions within the cubic phase, a method was developed for improving the resolution of (1)H NMR spectra when using a conventional solution state NMR probe. Using this approach we obtained well-resolved J-coupling multiplets in the one-dimensional NMR spectrum of the cubic-Ia3d phase prepared with hydrated monoolein. A high resolution t-ROESY two-dimensional (1)H NMR spectrum of the cubic-Ia3d phase is also reported. Using this new methodology, we have investigated the interaction of two additive molecules, L-tryptophan and ruthenium-tris(2,2-bipyridyl) dichloride (rubipy), with the cubic mesophase. Based on the measured chemical shift differences when changing from an aqueous solution to the cubic phase, we conclude that L-tryptophan experiences specific interactions with the bilayer interface, whereas rubipy remains in the aqueous channels and does not associate with the lipid bilayer.     

High-pressure,high-resolution NMR probe working at 400 MHZ

Abstract

A probe for obtaining high-resolution multinuclear NMR spectra at elevated pressures with a Bruker AM-400 spectrometer is described. The probe is designed for pressures up to 200 MPa and has been used between -40 to 150°C. We obtain routinely a resolution of about 1 Hz for proton (400 MHz) spectra using deuterium as an internal field lock. This probe is easily interchangeable with a commercial probe. We also describe a simple sample tube made of a 5 mm commercial NMR tube and a machinable glass cap with a total volume of about 1 cm³.     

Capillary scale NMR flow mapping

Stopped-flow NMR at capillary scale has many advantages over traditional methods of introducing the sample into the probe, particularly when large numbers of samples must be examined. This work describes application of a simple method for direct visualization of a sample inside the flow cell of flow NMR systems to capillary scale analysis. We describe the details of the method and show how it can be used to measure the optimum flow rate for a capillary NMR system and how to determine the optimum sampling efficiency for small samples.     

NMR observations of fluorine ion-implanted into polyacetylene and graphite

¹⁹FNMR spectroscopy has been used to characterize fluorine implanted into polyacetylene and highly orientated pyrolytic graphite. The intensities of the ¹⁹F NMR spectra at 4.5 K show that essentially all the fluorine is retained in these materials after the implantation process is over. The Une width of the spectrum of the graphite sample indicates that the fluorine remains where it was deposited.For the polyacetylene sample the ratio of the NMR solid echo height to the spin echo height, as calibrated herein, shows that the predominant nuclear diploar interaction of the implanted fluorine is heteronuclear.     

Spectral unraveling by space-selective Hadamard spectroscopy

Spectral unraveling by space-selective Hadamard spectroscopy (SUSHY) enables recording of NMR spectra of multiple samples loaded in multiple sample tubes in a modified spinner turbine and a regular 5mm liquids NMR probe equipped with a tri-axis pulsed field gradient coil. The individual spectrum from each sample is extracted by adding and subtracting data that are simultaneously obtained from all the tubes based on the principles of spatially resolved Hadamard spectroscopy. The well-known Hadamard spectroscopy has been applied for spatial selection and the method utilizes standard configuration of NMR instrument hardware. The SUSHY method can be easily incorporated in multi-dimensional multi-tube NMR experiments. This method combines the excitation multiplexing, natural advantage of FTNMR, and sample multiplexing and offers high-throughput by reducing the total experimental time by up to a factor of four in a 4-tube mode.     

Measurement of diffusion coefficients in polystyrene using xenon-129 NMR

A sample of polystyrene beads, 18 μm in diameter, has been sealed in an NMR tube under 10 atm of xenon gas. Two dimensional,¹²⁹Xe NMR spectra show cross peaks between the resonances corresponding to xenon in the free gas and the sorbed state, indicating that appreciable exchange occurs during the mixing time of the NMR experiment. Selective saturation of the free gas resonance attenuates the integrated intensity of the sorbed xenon resonance as a function of saturation time, thus allowing the accurate measurement of the exchange rates between the gas and the sorbed states. A model has been developed using a slightly modified form of Crank’s treatment of diffusion in a sphere which allows for the accurate determination of the diffusion coefficient for xenon in the sorbed state. The diffusion coefficient for xenon in polystyrene at 25°C is determined to be 2.9·10^?9 cm²/s.     

Sensitive and robust electrophoretic NMR: instrumentation and experiments

Although simple as a concept, electrophoretic NMR (eNMR) has so far failed to find wider application. Problems encountered are mainly due to disturbing and partly irreproducible convection-like bulk flow effects from both electro-osmosis and thermal convection. Additionally, bubble formation at the electrodes and rf noise pickup has constrained the typical sample geometry to U-tube-like arrangements with a small filling factor and a low resulting NMR sensitivity. Furthermore, the sign of the electrophoretic mobility cancels out in U-tube geometries. We present here a new electrophoretic sample cell based on a vertically placed conventional NMR sample tube with bubble-suppressing palladium metal as electrode material. A suitable radiofrequency filter design prevents noise pickup by the NMR sample coil from the high-voltage leads which extend into the sensitive sample volume. Hence, the obtained signal-to-noise ratio of this cell is one order of magnitude higher than that of our previous U-tube cells. Permitted by the retention of the sign of the displacement-related signal phase in the new cell design, an experimental approach is described where bulk flow effects by electro-osmosis and/or thermal convection are compensated through parallel monitoring of a reference signal from a non-charged species in the sample. This approach, together with a CPMG-like pulse train scheme provides a superior first-order cancellation of non-electrophoretic bulk flow effects.