Name: high-temperature and high-pressure in situ magic angle spinning nuclear magnetic resonance spectroscopy
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The review of catalyst applications was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Biosciences, and Geosciences Catalysis Program under contract DE-AC05-RL01830 and FWP-47319. The review of biomedical applications was supported by the National Institute of Health, National Institute of Environmental Health Sciences under grant R21ES029778. Experiments were conducted at EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory (PNNL). PNNL is a multi-program national laboratory operated by Battelle for the U.S. Department of Energy under contract DE-AC05-RL01830 and FWP-47319.

The protocol is divided into four sections which specify 1) the preparation of any solid materials being used in the system or activation or clearing of undesired adsorbed species, 2) addition of the solid and liquid materials to the NMR rotor, 3) addition of gases to the rotor, and 4) conducting the NMR experiments in the spectrometer. The procedure is representative of a typical sequence but may be modified to fit the specific needs of the experiment.
1. Pretreating solid samples
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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-pressure+in+situ+129Xe+NMR+spectroscopy+and+computer+simulations+of+breathing+transitions+in+the+metal-organic+framework+Ni2(2,6-ndc)2(dabco)+(DUT-8(Ni)).">High-pressure in situ 129Xe NMR spectroscopy and computer simulations of breathing transitions in the metal-organic framework Ni2(2,6-ndc)2(dabco) (DUT-8(Ni)).</a> Journal of the American Chemical Society. 133 (22), 8681-8690 (2011).</li><li>Turcu, R. V. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotor+design+for+high+pressure+magic+angle+spinning+nuclear+magnetic+resonance.">Rotor design for high pressure magic angle spinning nuclear magnetic resonance.</a> Journal of Magnetic Resonance. 226, 64-69 (2013).</li><li>Jaegers, N. R., Hu, M. Y., Hoyt, D. W., Wang, Y., Hu, J. 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R., Linehan, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+supercritical+fluids+as+solvents+for+NMR+spectroscopy.">The use of supercritical fluids as solvents for NMR spectroscopy.</a> Progress in Nuclear Magnetic Resonance Spectroscopy. 47 (1), 95-109 (2005).</li><li>Deuchande, T., Breton, O., Haedelt, J., Hughes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+performance+of+a+high+pressure+insert+for+use+in+a+standard+magic+angle+spinning+NMR+probe.">Design and performance of a high pressure insert for use in a standard magic angle spinning NMR probe.</a> Journal of Magnetic Resonance. 183 (2), 178-182 (2006).</li><li>Hoyt, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-pressure+magic+angle+spinning+nuclear+magnetic+resonance.">High-pressure magic angle spinning nuclear magnetic resonance.</a> Journal of Magnetic Resonance. 212 (2), 378-385 (2011).</li><li>Vjunov, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Following+Solid-Acid-Catalyzed+Reactions+by+MAS+NMR+Spectroscopy+in+Liquid+Phase-Zeolite-Catalyzed+Conversion+of+Cyclohexanol+in+Water.">Following Solid-Acid-Catalyzed Reactions by MAS NMR Spectroscopy in Liquid Phase-Zeolite-Catalyzed Conversion of Cyclohexanol in Water.</a> Angewandte Chemie International Edition. 53 (2), 479-482 (2014).</li><li>Chamas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+temperature/pressure+MAS-NMR+for+the+study+of+dynamic+processes+in+mixed+phase+systems.">High temperature/pressure MAS-NMR for the study of dynamic processes in mixed phase systems.</a> Magnetic Resonance Imaging. 56, 37-44 (2019).</li><li>Hu, J. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sealed+rotors+for+in+situ+high+temperature+high+pressure+MAS+NMR.">Sealed rotors for in situ high temperature high pressure MAS NMR.</a> ChemComm. 51 (70), 13458-13461 (2015).</li><li>Hu, J. Z., Hu, M. Y., Townsend, M. R., Lercher, J. A., Peden, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-pressure,+high-temperature+magic+angle+spinning+nuclear+magnetic+resonance+devices+and+processes+for+making+and+using+same.">High-pressure, high-temperature magic angle spinning nuclear magnetic resonance devices and processes for making and using same.</a> US patent. , (2015).</li><li>Jaegers, N. R., Mueller, K. T., Wang, Y., Hu, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+Temperature+and+Pressure+Operando+MAS+NMR+for+Catalysis+Science+and+Related+Materials.">Variable Temperature and Pressure Operando MAS NMR for Catalysis Science and Related Materials.</a> Accounts of Chemical Research. 53 (3), 611-619 (2020).</li><li>Dagle, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-step+Conversion+of+Ethanol+to+n-butenes+over+Ag-ZrO2/SiO2+catalysts.">Single-step Conversion of Ethanol to n-butenes over Ag-ZrO2/SiO2 catalysts.</a> ACS Catalysis. 10 (18), 10602-10613 (2020).</li><li>Jaegers, N. R., Wang, Y., Hu, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+perturbation+of+NMR+properties+in+small+polar+and+non-polar+molecules.">Thermal perturbation of NMR properties in small polar and non-polar molecules.</a> Scientific Reports UK. 10 (1), 6097 (2020).</li><li>Jaegers, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Applications of In situ Magnetic Resonance Spectroscopy for Structural Analysis of Oxide-supported Catalysts. , (2019).</li><li>Mehta, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+high-temperature+MAS+probe+with+optimized+temperature+gradient+across+sample+rotor+for+in-situ+monitoring+of+high-temperature+high-pressure+chemical+reactions.">A novel high-temperature MAS probe with optimized temperature gradient across sample rotor for in-situ monitoring of high-temperature high-pressure chemical reactions.</a> Solid State Nuclear Magnetic Resonance. 102, 31-35 (2019).</li><li>Hu, J. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large+sample+volume+magic+angle+spinning+nuclear+magnetic+resonance+probe+for+in+situ+investigations+with+constant+flow+of+reactants.">A large sample volume magic angle spinning nuclear magnetic resonance probe for in situ investigations with constant flow of reactants.</a> Physical Chemistry Chemical Physics. 14 (7), 2137-2143 (2012).</li><li>Jiang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+MAS+NMR-UV/Vis+investigation+of+H-SAPO-34+catalysts+partially+coked+in+the+methanol-to-olefin+conversion+under+continuous-flow+conditions+and+of+their+regeneration.">In situ MAS NMR-UV/Vis investigation of H-SAPO-34 catalysts partially coked in the methanol-to-olefin conversion under continuous-flow conditions and of their regeneration.</a> Microporous and Mesoporous Materials. 105 (1-2), 132-139 (2007).</li><li>Xu, S., Zhang, W., Liu, X., Han, X., Bao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+In+situ+Continuous-Flow+MAS+NMR+for+Reaction+Kinetics+in+the+Nanocages.">Enhanced In situ Continuous-Flow MAS NMR for Reaction Kinetics in the Nanocages.</a> Journal of the American Chemical Society. 131 (38), 13722-13727 (2009).</li><li>Graham, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+Al-27+NMR+Spectroscopy+of+Aluminate+in+Sodium+Hydroxide+Solutions+above+and+below+Saturation+with+Respect+to+Gibbsite.">In situ Al-27 NMR Spectroscopy of Aluminate in Sodium Hydroxide Solutions above and below Saturation with Respect to Gibbsite.</a> Inorganic Chemistry. 57 (19), 11864-11873 (2018).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boehmite+and+Gibbsite+Nanoplates+for+the+Synthesis+of+Advanced+Alumina+Products.">Boehmite and Gibbsite Nanoplates for the Synthesis of Advanced Alumina Products.</a> ACS Applied Nano Materials. 1 (12), 7115-7128 (2018).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+Gibbsite+to+Boehmite+in+Caustic+Aqueous+Solution+at+Hydrothermal+Conditions.">Transformation of Gibbsite to Boehmite in Caustic Aqueous Solution at Hydrothermal Conditions.</a> Crystal Growth &amp; Design. 19 (10), 5557-5567 (2019).</li><li>Hu, J. 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The method of conducting MAS NMR spectroscopic measurements outlined herein represents the state of the art for conducting high-temperature, high-pressure MAS NMR. Such methods enable the observation of interactions occurring in vacuum atmospheres up to several hundred bar and from low temperatures (well below 0 &#176;C to 250 &#176;C) in a reliable, reproducible fashion. The ability to probe systems containing mixtures of solids, liquids, and gases under flexible chemical environments enables a wide range of experiments...

Spectroscopic analysis of samples is an analytical tool used to gain valuable information about materials of interest such as their chemical state, structure, or reactivity. In a simplistic view, nuclear magnetic resonance (NMR) is one such technique that utilizes a strong magnetic field to manipulate the spin state of atomic nuclei to better understand the chemical environment of the species of interest. The nuclear spin state refers to the relative direction of the magnetic moment induced by the motion of the spinning nucleus, a positively charged particle. In the absence of a magnetic field, the nuclear spins are randomly oriented but in the presence of a magnetic field, nuclear spins preferentially align with the external field of the magnet in a low energy spin state. This splitting of spin states to discrete energy values is known as the Zeeman effect. The difference between these energy levels (&#916;E) is modeled by Equation 1: 
<img alt="Equation 1" src="/files/ftp_upload/61794/61794equ01.jpg" /> 
where h is Plank&#8217;s constant, B0 is the strength of the external magnetic field and &#947; is the gyromagnetic ratio of the nucleus. The chemical environment of these spins also applies slight perturbations to these energy levels. Radio waves of corresponding frequencies can be used to excite the nuclei, which generates a transverse magnetization due to spins gaining phase coherence as longitudinal magnetization (based on the population of spins in parallel and anti-parallel states) is decreased. As the nuclei continue precessing about the axis of the magnetic field, the rotating magnetic movement creates a magnetic field that is also rotating and generating an electric field. This field modulates the electrons in the NMR detection coil, generating the NMR signal. Slight differences in the chemical environment of the nuclei in the sample affect the frequencies detected in the coil.
NMR analysis of solid samples introduces complexities not found in fluids. In fluids, the molecules tumble at fast rates, averaging the chemical environment spatially around the nuclei. In solid samples, no such averaging effect occurs, introducing an orientation-dependent chemical environment and broad spectral lines in the NMR signal. To mitigate these challenges, a technique known as magic angle spinning (MAS) is employed1,2. In MAS NMR, the samples are quickly rotated (several kilohertz) at an angle of 54.7356&#176; with respect to the external magnetic field using an external spinning mechanism to address the orientation-dependent (anisotropic) interactions of NMR. This substantially narrows the NMR features and enhances the spectral resolution by averaging the orientation-dependent terms of the chemical shift anisotropy, dipolar interactions, and quadrupolar interactions. Two notable exceptions do hinder the line narrowing abilities of MAS NMR. The first is strong homonuclear coupling sometimes present in 1H NMR that requires high spinning speeds (~70 kHz) to remove. However, the significantly elevated temperatures of the high temperature applications will greatly suppress the 1H homonuclear interaction by imparting enhanced thermal motion such that a much reduced sample spinning rate can be utilized for significantly enhanced spectral resolution. Furthermore, with the technology continuously evolving, rotors with smaller diameters can now be fabricated to achieve spinning rates far exceeding 5 kHz, which helps to further suppress the 1H homonuclear dipolar interactions. The second exception is residual second-order quadrupolar interactions for nuclei with spin that exceeds one-half since only the first order term is eliminated at the magic angle, leaving more complex lineshapes that can only be improved by stronger external magnetic fields. It should be emphasized that 2D MQMAS techniques can be readily incorporated into the current technology so that a true isotropic chemical shift spectrum can be obtained in a similar way as to the standard MQMAS experiments3.
MAS NMR has enabled detailed characterization of solid materials, strengthening the quality of observations. However, the necessity of spinning the samples in NMR rotors (the sample holder) at high rates also imposes challenges in conducting experiments at elevated temperatures and pressures which may be more relevant to the conditions of interest. It may, at times, be desirable to examine materials under conditions that are relatively harsh for NMR rotors. A number of efforts have successfully adapted liquid-state NMR technologies to conduct high-temperature, high-pressure NMR4,5,6,7; however, commercial rotor caps used for solid-state MAS NMR may be expelled from the rotor at high pressures, causing significant damage to the equipment. Such effects may be compounded by examining a decomposition reaction that greatly increases the pressure in the sample holder. As such, new designs are required to effectively and safely conduct in situ NMR experiments. For example, the rotor must adhere to several qualities for effective use in MAS NMR, namely non-magnetic, lightweight, durable, temperature resistant, low NMR background material, sealable, high-strength, and chemical resistant. The pressures the rotor must withstand are quite large. Not only must the rotor withstand the pressure of the sample contained within (e.g., high-pressure gas), the rotation of the device imparts centrifugal force which has its own contribution to the total system pressure8, PT, by equation 2: 
<img alt="Equation 2" src="/files/ftp_upload/61794/61794equ02.jpg" /> 
RI and RO are the inner and outer rotor radii, respectively, &#969; is the rotational frequency in radians per second, and Ps is the sample pressure.
A number of strategies have been developed to address these concerns9. Early examples resembled flame-sealed tubes10,11,12 or polymer inserts13,14, which were insufficient for extended, fine-controlled operation at elevated temperatures and pressures. Iterations to rotor designs have suffered from limitations in the maximum operating temperature imparted by the use of epoxy or sample volume reductions from ceramic inserts8,15,16. A recent technology reduces unit production costs by employing simple snap-in features in a commercial rotor sleeve, but offers relatively less control over the conditions with which it can operate17. The design employed herein is an all-zirconia, cavern-style rotor sleeve milled with a threaded top18. A cap is also threaded to allow for a secure seal. Reverse threading prevents sample rotation from loosening the zirconia cap and an O-ring constitutes the sealing surfaces. This rotor design is visible in Figure 1 and similar rotors and instructions to make them have been patented19. Such a strategy enables high mechanical strength, chemical resistance, and temperature tolerance.
These designs are suitable for temperatures and pressures of at least 250 &#176;C and 100 bar, limited in temperature by readily-available NMR probe technology. When combined with specialized sample preparation equipment, it represents a truly powerful technique that has been employed for far-reaching applications as carbon sequestration, catalysis, energy storage, and biomedicine20. Such equipment includes a way to pretreat the solid materials to remove unwanted surface species such as water. A furnace is often employed for this step. A dry box is typically used to load the solid samples into the NMR rotor. From there, the rotor is transferred into an exposure device which enables the rotor to be opened under a tightly controlled atmosphere to load a desired gas or mixture into the rotor. Such a device is depicted in Figure 2.

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			<td>1) Preparation of Solids Samples</td>
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			<td>Gas maniforld</td>
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			<td>Gas Mass Flow Controllers</td>
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			<td>Vacuum Pump</td>
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			<td>Tube Furnace</td>
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			<td>Temperature Controller</td>
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			<td>Thermocouple</td>
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			<td>Quartz Tube</td>
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			<td>Isolation Valves</td>
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			<td>Quartz Wool</td>
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			<td>2) Loading solid samples into the rotor</td>
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			<td>Dry glove box</td>
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			<td>High-temperature, high-pressure NMR rotor</td>
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			<td>Sample funnel</td>
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			<td>Sample packing rod</td>
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			<td>Rotor holder</td>
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			<td>Analytical Balance</td>
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			<td>Microsyringe</td>
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			<td>Rotor cap bit</td>
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			<td>3) Addition of gases to the rotor</td>
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			<td>NMR loading chamber</td>
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			<td>Rotor stage and appropriately sized inserts</td>
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			<td>Vacuum Pump</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gas maniforld</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gas Mass Flow Controllers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Pump</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature Controller</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocouple</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Allen wrench</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Threaded rod</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wrenchs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Gauge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High-pressure syringe pump</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid syringe pump</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4) Conducting the NMR experiments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MAS NMR probe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NMR spectrometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer to control the spectrometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

high-temperature and high-pressure in situ magic angle spinning nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy represents an important technique to understand the structure and bonding environments of molecules. There exists a drive to characterize materials under conditions relevant to the chemical process of interest. To address this, in situ high-temperature, high-pressure MAS NMR methods have been developed to enable the observation of chemical interactions over a range of pressures (vacuum to several hundred bar) and temperatures (well below 0 &#176;C to 250 &#176;C). Further, the chemical identity of the samples can be comprised of solids, liquids, and gases or mixtures of the three. The method incorporates all-zirconia NMR rotors (sample holder for MAS NMR) which can be sealed using a threaded cap to compress an O-ring. This rotor exhibits great chemical resistance, temperature compatibility, low NMR background, and can withstand high pressures. These combined factors enable it to be utilized in a wide range of system combinations, which in turn permit its use in diverse fields as carbon sequestration, catalysis, material science, geochemistry, and biology. The flexibility of this technique makes it an attractive option for scientists from numerous disciplines.

The output from the NMR spectrometer takes the form of a free induction decay (FID) which is the time-domain signal from the excited spins as they relax back to thermodynamic equilibrium. Such an FID resembles Figure 3. When Fourier transformed from the time domain to the frequency domain (frequency to PPM by Equation 3, whereby the difference absolute frequency and a reference is divided by the carrier frequency of the NMR spectrometer), it represents the NMR spectrum for which each peak in...

Watch this Scientific Journal Video about High-Temperature and High-Pressure In situ Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy at JoVE.com

High-Temperature and High-Pressure In situ Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy

The molecular structures and dynamics of solids, liquids, gases, and mixtures are of critical interest to diverse scientific fields. High-temperature, high-pressure in situ MAS NMR enables detection of the chemical environment of constituents in mixed phase systems under tightly controlled chemical environments.

Nuclear magnetic resonance (NMR) spectroscopy represents an important technique to understand the structure and bonding environments of molecules. There ...

This non-destructive method can be used to analyze complex systems at controlled temperatures and pressures over time, and is therefore highly useful for investigating key scientific questions. These methods allow NMR experiments to be conducted under tightly controlled and specialized conditions, to solve problems under environments relevant to the scientific hypothesis being explored. The multi-nuclear nature of NMR makes it suitable for numerous samples.Coupled with this method, it can provide insight into an array of systems, spanning fields such as ketolysis, geochemistry, and biology. For pre-treatment of the sample solid, weigh approximately twice the mass of the solid sample that is desired for the NMR experiment, and place the solid sample into a quartz sample tube plugged with quartz wool. Connect the isolation valves to the tube for the pretreatment of interest, and fix the end of the tube onto the gas isolation valve in the open position.Then, place the tube into the cool furnace and begin the treatment. After the treatment, stop the flow and vacuum, and turn off the temperature controller. Disconnect the quartz tube from the treatment system, and quickly seal the sample with the isolation valves to maintain the desired sample environment.Then, transfer the tubes and closed valves to the antechamber. To load the samples into an NMR rotor, first, place the rotor into the holder to maintain directionality, and place the sample funnel into the bore of the rotor. Remove the isolation valve from the sample tube, and pour a small quantity of solid material into the funnel.Tap the powder down into the funnel using a packing rod to lightly direct the sample into the rotor as necessary. When the desired quantity of sample has been loaded, a microsyringe can be used to slowly inject the desired volume of a liquid sample into the center of the rotor before placing the cap onto the top of the rotor, and turning the cap counterclockwise with the rotor cap bit to engage the O-ring between the rotor and cap. To charge the NMR rotor with the chemicals of interest, place the sealed rotor onto the rotor stage and tighten the nut by hand to secure the rotor in place.Lower the rotor stage into the lower section of the high-pressure exposure device, and use an Allen wrench to turn one of the screws 90 degrees to secure the rotor stage into the bottom of the exposure device. Place the top section of the NMR loading device into and on top of the bottom section, with the NMR cap bit aligned with the top of the cap head of the NMR rotor. Place the two clamps over the top of the lip where the upper and lower sections of the exposure device meet, and latch the clamps into place.Tighten the six bolts on the top of the upper section of the exposure device to engage the ceiling surface between the upper and lower sections, and connect the thermocouple on the upper section of the NMR exposure device to the gas line inlet and outlets. Apply vacuum to the system to purge the high-pressure loading chamber of ambient gases. Open the gas source valves on the high pressure syringe pump, and run the program set on the pump while monitoring the real pressure inside of the exposure device.To open the NMR rotor, rotate the external screw mechanism, which is coupled to the interior NMR cap bit in the clockwise direction to allow the gas of the desired pressure to enter the NMR rotor and equilibrate. To reseal the NMR rotor, rotate the external screw mechanism counter-clockwise. A viewing window will assist in determining when the rotor is closed.Then, open the exposure device gas outlet valve to slowly depressurize the system. To conduct a magic angle spinning NMR experiment, place the NMR rotor into the NMR coil on the NMR probe, and raise and lock the probe into place in the magnet bore. Use the magic angle spinning control box to adjust the sample to the desired rotor spinning rate, and initiate sample spinning.Use the computer to begin the tuning match sequence on the desired channel, and adjust the tuning match settings on the probe to optimize the probe electronics, then, collect the magic angle spinning NMR data. In this representative analysis, some insight into the operational reaction mechanism for the conversion of ethanol to butenes can be observed. Butyraldehyde formation, coupled with the simultaneous disappearance of peaks characteristic of crotonaldehyde, followed by butyraldehyde subsequent consumption and the concomitant appearance of peaks characteristic of n-butenes, suggests that butyraldehyde is an intermediate in the formation of n-butene.In situ, high-temperature, high-pressure magic angle spinning NMR can also be used to better understand the evolution of chemical species for biological applications. For example, as this representative carbon-13 magic angle spinning NMR spectrum of vape juice solution shows, parent glycerol is present at 63 and 73 parts per million. As time progresses at 130 degrees Celsius in an oxygen environment, the toxins acrylic acid and formic acid with formaldehyde appear at 175 and 164 parts per million respectively.The oxidation product carbon dioxide is observed at 125 parts per million. And most importantly, even at such low temperatures, acetyl species of formaldehyde and acetaldehyde are observed between 50 and 112 parts per million. The addition of parent glycerol to formaldehyde and acetylaldehyde generates new hemi-acetyl species at 105 and 112 parts per million, which act as aldehyde carriers and can self-interact and dehydrate to generate new acetyl species.Numerous other peaks between 50 and 80 parts per million correspond to the many other chemical environments of the hemi-acetyls and acetyls. In addition to working safely, it is important to maintain the environment within the rotor during transfers, particularly when removing the chamber top, as the rotor cap will still be engaged. Since NMR is non-destructive, additional characterizations, such as x-ray diffraction, gas chromatography/mass spectrometry, and x-ray photoelectron spectroscopy, can be pursued, enabling comparison of the NMR results with complimentary information on the same sample.These measurements obtained at elevated temperatures and pressures have enabled researchers to address challenging problems in a way not previously possible, by looking at complex systems under relevant conditions.

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Unit 1

Interpreting Nuclear Magnetic Resonance Spectra

SCIENTISTS IN ACTION

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3]2+ (bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of novel materials. Coupled with analysis by in situ secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS), soft landing provides unprecedented insights into the interactions of well-defined species with surfaces.

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with nitroxide spin probe 3-(2-Iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IPSL) as a guest inside the periodic mesoporous organosilica (PMO) aerogel UKON1-GEL as a host and ethanol as a solvent is used as an example to describe the protocol. Data is shown from a previous publication, where the protocol has been applied to both IPSL and Tris(8-carboxy-2,2,6,6-perdeutero-tetramethyl-benzo[1,2-d:4,5-d&#8242;]bis(1,3)dithiole) methyl (Trityl) as guest molecules and UKON1-GEL and SILICA-GEL as host systems. 
A method is shown to prepare aerogel samples that cannot be synthesized directly in the sample tube for measurement due to a size change during synthesis. The aerogel is attached to sample tubes using heat shrink tubing and a pressure cooker to reach the necessary temperature without evaporating the solvent in the process. The method does not assume a clearly defined initial distribution of guest molecules at the start of the measurement. Instead, it requires a reservoir on top of the aerogel and experimentally determines the influx rate during data analysis.
The diffusion is monitored continually over a period of 20 hr by recording the 1d spin density profile within the sample. The spectrometer settings for the imaging experiment are described quantitatively. Data analysis software is provided to take the resonator sensitivity profile into account and to numerically solve the diffusion equation. The software determines the macroscopic translational diffusion coefficient by least square minimization of the difference between the experiment and the numerical solution of the diffusion equation.

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

A protocol for the in situ monitoring of the diffusion of guest molecules in porous media using electron paramagnetic resonance (EPR) imaging is presented.

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with ...

High Pressure Single Crystal Diffraction at PX^2

In this report we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell (DAC) at the GSECARS 13-BM-C beamline at the Advanced Photon Source. The DAC program at 13-BM-C is part of the Partnership for Extreme Xtallography (PX^2) project. BX-90 type DACs with conical-type diamond anvils and backing plates are recommended for these experiments. The sample chamber should be loaded with noble gas to maintain a hydrostatic pressure environment. The sample is aligned to the rotation center of the diffraction goniometer. The MARCCD area detector is calibrated with a powder diffraction pattern from LaB6. The sample diffraction peaks are analyzed with the ATREX software program, and are then indexed with the RSV software program. RSV is used to refine the UB matrix of the single crystal, and with this information and the peak prediction function, more diffraction peaks can be located. Representative single crystal diffraction data from an omphacite (Ca0.51Na0.48)(Mg0.44Al0.44Fe2+0.14Fe3+0.02)Si2O6 sample were collected. Analysis of the data gave a monoclinic lattice with P2/n space group at 0.35 GPa, and the lattice parameters were found to be: a = 9.496 &#177;0.006 &#197;, b = 8.761 &#177;0.004 &#197;, c = 5.248 &#177;0.001 &#197;, &#946; = 105.06 &#177;0.03&#186;, &#945; = &#947; = 90&#186;.

In this report, we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell at the GSECARS 13-BM-C beamline at the Advanced Photon Source. ATREX and RSV programs are used to analyze the data.

In this report we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell (DAC) at the ...

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance imaging (MRI). Their purpose is to enhance the relaxation rate of water protons in tissue, thus increasing the MR image contrast and the specificity of the MRI measurements. Current clinically approved contrast agents are low molecular weight molecules that are rapidly cleared from the body. The use of dendrimers as carriers of paramagnetic chelators can play an important role in the future development of more efficient MRI contrast agents. Specifically, the increase in local concentration of the paramagnetic species results in a higher signal contrast. Furthermore, this CA provides a longer tissue retention time due to its high molecular weight and size. Here, we demonstrate a convenient procedure for the preparation of macromolecular MRI contrast agents based on poly(amidoamine) (PAMAM) dendrimers with monomacrocyclic DOTA-type chelators (DOTA &#8211; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate). The chelating unit was appended by exploiting the reactivity of the isothiocyanate (NCS) group towards the amine surface groups of the PAMAM dendrimer to form thiourea bridges. Dendrimeric products were purified and analyzed by means of nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Finally, high resolution MR images were recorded and the signal contrasts obtained from the prepared dendrimeric and the commercially available monomeric agents were compared.

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

This protocol describes the preparation and characterization of a dendrimeric magnetic resonance imaging (MRI) contrast agent that carries cyclen-based macrocyclic chelates coordinating paramagnetic gadolinium ions. In a series of MRI experiments in vitro, this agent produced an amplified MRI signal when compared to the commercially available monomeric analogue.

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance ...

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. They offer new possibilities for the characterization of living systems and are particularly appropriate for detecting, localizing and quantifying the presence of metal oxide nanoparticles both in biological specimens and the environment. Indeed, these techniques all meet relevant requirements in terms of (i) sensitivity (from 1 up to 10 &#181;g.g-1 of dry mass), (ii) micrometer range spatial resolution, and (iii) multi-element detection. Given these characteristics, microbeam chemical element imaging can powerfully complement routine imaging techniques such as optical and fluorescence microscopy. This protocol describes how to perform a nuclear microprobe analysis on cultured cells (U2OS) exposed to titanium dioxide nanoparticles. Cells must grow on and be exposed directly in a specially designed sample holder used on the optical microscope and in the nuclear microprobe analysis stages. Plunge-freeze cryogenic fixation of the samples preserves both the cellular organization and the chemical element distribution. Simultaneous nuclear microprobe analysis (scanning transmission ion microscopy, Rutherford backscattering spectrometry and particle induced X-ray emission) performed on the sample provides information about the cellular density, the local distribution of the chemical elements, as well as the cellular content of nanoparticles. There is a growing need for such analytical tools within biology, especially in the emerging context of Nanotoxicology and Nanomedicine for which our comprehension of the interactions between nanoparticles and biological samples must be deepened. In particular, as nuclear microprobe analysis does not require nanoparticles to be labelled, nanoparticle abundances are quantifiable down to the individual cell level in a cell population, independently of their surface state.

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

We describe a procedure for the detection of chemical elements present in situ in human cells as well as their in vitro quantification. The method is well-suited to any cell type and is particularly useful for quantitative chemical analyses in single cells following in vitro metal oxide nanoparticles exposure.

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. ...

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables the silk proteins to be compact and ordered by shearing and elongation forces. Here, a biomimetic microfluidic channel was designed to mimic the specific geometry of the spinning duct of the silkworm. Regenerated silk fibroin (RSF) spinning doped with high concentration, was extruded through the microchannel to dry-spin fibers at ambient temperature and pressure. In the post-treated process, the as-spun fibers were drawn and stored in ethanol aqueous solution. Synchrotron radiation wide-angle X-ray diffraction (SR-WAXD) technology was used to investigate the microstructure of single RSF fibers, which were fixed to a sample holder with the RSF fiber axis normal to the microbeam of the X-ray. The crystallinity, crystallite size, and crystalline orientation of the fiber were calculated from the WAXD data. The diffraction arcs near the equator of the two-dimensional WAXD pattern indicate that the post-treated RSF fiber has a high orientation degree.

A protocol for the microfluidic spinning and microstructure characterization of regenerated silk fibroin monofilament is presented.

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables ...

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. 
Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

In Situ Lithiated Reference Electrode: Four Electrode Design for In-operando Impedance Spectroscopy

Extending operating voltage of Li-ion batteries results in higher energy output from these devices. High voltages, however, may trigger or accelerate multiple processes responsible for long-term performance decay. Given the complexity of physical processes occurring inside the cell, it is often challenging to achieve a full understanding of the root causes of this performance degradation. This difficulty arises in part from the fact that any electrochemical measurement of a battery will return the combined contributions of all components in the cell. Incorporation of a reference electrode can solve part of the problem, as it allows the electrochemical reactions of the cathode and the anode to be individually probed. A variation in the voltage range experienced by the cathode, for example, can indicate alterations in the pool of cyclable lithium ions in the full-cell. The structural evolution of the many interphases existing in the battery can also be monitored, by measuring the contributions of each electrode to the overall cell impedance. Such wealth of information amplifies the reach of diagnostic analysis in Li-ion batteries and provides valuable input to the optimization of individual cell components. In this work, we introduce the design of a test cell able to accommodate multiple reference electrodes, and present reference electrodes that are appropriate for each specific type of measurement, detailing the assembly process in order to maximize the accuracy of the experimental results.

The incorporation of reference electrodes in a Li-ion battery provides valuable information to elucidate degradation mechanisms at high voltages. In this article, we present a cell design that accommodates multiple reference electrodes, along with the assembly steps to assure maximum accuracy of the data obtained in electrochemical measurements.

Extending operating voltage of Li-ion batteries results in higher energy output from these devices. High voltages, however, may trigger or accelerate ...

Characterizing Lewis Pairs Using Titration Coupled with In Situ Infrared Spectroscopy

Lewis acid-activation of carbonyl-containing substrates is a fundamental basis for facilitating transformations in organic chemistry. Historically, characterization of these interactions has been limited to models equivalent to stoichiometric reactions. Here, we report a method utilizing in situ infrared spectroscopy to probe the solution interactions between Lewis acids and carbonyls under synthetically relevant conditions. Using this method, we were able to identify 1:1 complexation between GaCl3 and acetone and a highly ligated complex for FeCl3 and acetone. The impact of this technique on mechanistic understanding is illustrated by application to the mechanism of Lewis acid-mediated carbonyl-olefin metathesis in which we were able to observe competitive binding interactions between substrate carbonyl and product carbonyl with the catalyst.

Here, we present a method for the observation of solution interactions between Lewis acids and bases by employing in situ infrared spectroscopy as a detector for titration under synthetically relevant conditions. By examining solution interactions, this method represents a complement to X-ray crystallography, and provides an alternative to NMR spectroscopy.

Lewis acid-activation of carbonyl-containing substrates is a fundamental basis for facilitating transformations in organic chemistry. Historically, ...