Speaker Abstracts
 

Luke Arbogast

NIST-IBBR

Rockville, MD, United States

Welcome to the SIERRA, Where Have All the Large Peaks Gone?

2D 1H-13C methyl NMR has shown to be a highly reproducible and robust means to characterize higher order structure (HOS) of protein therapeutics at natural isotopic abundance [1]. Furthermore, when combined with chemometric analysis, 2D NMR has proven to be a highly sensitive means for detecting structural variation in monoclonal antibody (mAb) samples [2]. Thus 2D NMR holds great promise in the biopharmaceutical industry for establishing the consistency of protein folding for drug development and quality assessment, as well as for structural comparability studies of related drug products. However, there remains a major hindrance to recording 1H-13C methyl spectra on biotherapeutics when formulations contain aliphatic components with resonances that interfere with the protein methyl signals of interest, thus limiting the full potential of 2D NMR methods for biopharmaceutical characterization. Here we discuss our recently introduced SIERRA approach for selective attenuation of unwanted signals from formulation components [3]. The SIERRA (Selective Excipient Reduction/Removal) filter is a selective double resonance technique based upon weak-field cross-polarization [4] that allows for tenfold reduction of targeted signals while minimizing losses elsewhere in the spectrum. We describe the theory of the SIERRA approach and demonstrate its application on the NISTmAb reference monoclonal antibody [5] in several formulations. We further discuss improvements to the original design of SIERRA, enabling better performance and streamlined experimental setup. References (1) Brinson, R. G.; Marino, J. P.; Delaglio, F.; Arbogast, L. W.; Evans, R. M.; et.al. MAbs 2019, 11, 94-105. (2) Arbogast, L. W.; Delaglio, F.; Schiel, J. E.; Marino, J. P. Anal. Chem. 2017, 89, 11839-11845. (3) Arbogast, L. W.; Delaglio, F.; Tolman, J. R.; Marino, J. P. J. Biomol. NMR 2018, 72, 149-161. (4) Chiarparin, E.; Pelupessy, P.; Bodenhausen, G. Mol. Phys. 1998, 95, 759-767. (5) Schiel, J. E.; Turner, A.; Mouchahoir, T.; Yandrofski, K.; Telikepalli, S.; King, J.; DeRose, P.; Ripple, D.; Phinney, K. Anal. Bioanal. Chem. 2018, 410, 2127-2139.

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Tim Bays

Pacific Northwest National Laboratory

Richland, WA, United States

Using DOSY Spectroscopy to Understand the Origins of Fuel Properties

Evaporative processes associated with fuels, such as distillation, vapor pressure, and heat-of-vaporization, govern fuel behaviors ranging from evaporative emissions to combustion and driveability in an automobile. Although the fuel components appear to mix uniformly, their molecular-level interactions create heterogeneities that vary based on the component chemistries and concentrations, as well as the fuel's temperature. Molecular-level aggregations, such as clusters formed from hydrogen bonding, have been shown to affect azeotropic behavior in alcohol-water mixtures. In this work, diffusion-ordered spectroscopy (DOSY) and molecular dynamics simulations have been used to show the concentration and temperature-dependent behavior of ethanol, and other C1-C4 alcohols, in single-component gasoline surrogates and in gasoline. The size and number of ethanol clusters, resulting from hydrogen bonding among the ethanol molecules, have been associated with increases in Reid vapor pressure (RVP), a measure of gasoline volatility, up to about 22 mole % ethanol in the fuel. Above this value, the number and size of discrete ethanol clusters diminishes in favor of larger molecular aggregations or hydrogen-bonding networks. Observed decreases in RVP are related to the extent of the hydrogen-bonding networks and the balance of the intermolecular forces among the non-polar fuel molecules. Similar concentration and temperature-dependent shifts between alcohol clustering and the formation of larger aggregates are observed to be affected by the choice of alcohol and the choice of the gasoline surrogate or gasoline.

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Adam Barb

University of Georgia

Athens, GA, United States

The Structural Role of N-Glycosylation in interactions between Antibodies and Receptors

The recent expansion of biopharmaceuticals has transformed both patient care and the pharmaceutical industry itself. A substantial increase in molecular complexity accompanies the unparalleled benefits of therapeutic monoclonal antibodies (mAbs), a major class of biopharmaceuticals. This complexity creates major challenges for manufacture, regulation and fundamental efforts to define interaction mechanisms that are required to develop more potent drugs. Our laboratory is designed to define how a common complexity, the post-translation addition of a carbohydrate chain to an asparagine residue (an N-glycan), impacts the structure, function and efficacy of therapeutic monoclonal antibodies and other glycoproteins. N-glycans are required from proper mAb activity and proper folding and function of many other proteins. Solution NMR spectroscopy is particularly well suited to study N-glycosylated biopharmaceuticals because the carbohydrates form critical transient interactions with amino acid residues that affect mAb structure and receptor binding. Standard NMR approaches are not suited to investigate mAbs due to the requirement for a eukaryotic expression system and the concomitant inability to perdeuterate. Our laboratory has developed a suite of new approaches to overcome these limitations. Our studies have focused on the most common mAb scaffold, immunoglobulin G1 (IgG1), and the primary receptor responsible for the clearance of a mAb-coated target, Fc g receptor 3a (CD16a). Using NMR spectroscopy combined with a suite of new protein expression and purification technologies, we identified a link between motion of the conserved IgG1 N-glycan and CD16a-binding affinity. The IgG1 Fc N-glycan stabilizes a single Fc polypeptide loop through intramolecular interactions between carbohydrate and amino acid residues to preorganize the CD16a-binding interface for optimal affinity. The features that contribute to the capacity of the IgG1 Fc N-glycan to restrict protein conformation and tune binding affinity are conserved in other human antibodies including IgG2-4, IgD, IgE and IgM. We also found comparable intramolecular interactions formed between N-glycans and CD16a amino acid residues that impact affinity for IgG1. These NMR measurements and other glycoprotein studies from our lab indicate that carbohydrate-stabilizing interactions are potentially widespread and solution NMR spectroscopy is a promising tool for identification and definition.

 

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Frédéric Blanc

University of Liverpool

 Liverpool, United Kingdom

Zeolites Heterogeneous Catalysts Caught in the Act by (DNP) MAS NMR

The formation of carbocation intermediates plays a key role in reactivity, selectivity and deactivation in heterogeneous catalytic processes. However, their observation and determination remain a significant challenge due to the lack of selective techniques of sufficient sensitivity to detect their low concentrations (< 10 micromol/g).[1,2] We will show how an approach combining 13C isotopic enrichment with multinuclear NMR and, on selected occasions, efficient DNP MAS NMR at 9.4-14.1 T using bisnitroxide radicals as polarising agents, allows the fast detection of carbocations formed during catalysis. The approach is demonstrated in a range of zeolites with different topologies including in Mobil-type five (MFI, e.g. H-ZSM5),[3] Zeolite beta polymorph A (BEA, e.g. beta zeolite)[4] and chabazite (CHA, e.g. H-SSZ-13, H-SAPO-34).[5] We use two dimensional 13C-13C through-bond correlations to establish the carbon-carbon connectivity and unambiguously derive 5- and 6-membered ring cyclic carbocation and methylnaphthalenium ions as intermediates in the methanol to hydrocarbons catalytic reaction. We also showed that these species could be different even in zeolites with identical CHA topology.[5] These highlight that different catalytic routes exist for the formation of both targeted hydrocarbon products and coke exist.. We employ both 29Si-13C and 27Al-13C through-space experiments to quantitatively locate the confined carbocations with respect to the multiple surface sites of the zeolites, demonstrating that these species have strong van der Waals interaction with the frameworks and that their accumulation in the channels leads to deactivation. These results, obtained from multidimensional multinuclear (DNP) MAS NMR, enable understanding of deactivation pathways and open up opportunities for the design of catalysts with improved performances. We also show that introducing hierarchical pores into zeolites to form micro-meso-macroporous zeolite frameworks is a promising way to dramatically improve the overall DNP efficiency by a factor of ~ 4 on this type of materials[4] and may be a general method that could be applicable to other porous solids. [1] Kazanskii, V. B. Acc. Chem. Res. 1991, 12, 379. [2] Xu, S. et al. Angew. Chem. Int. Ed. 2013, 44, 11564. [3] Xiao, D. et al. Chem. Sci. 2017, 8, 8309. [4] Xiao, D. et al. Chem. Sci. 2018, 9, 8184. [5] Xiao, D. et al. RSC Adv. 2019, 9, 12415.

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Alexei Buevich

Merck and Co., Inc.

Kenilworth, NJ, United States

What can NMR analysis reveal about Acyl Glucuronides and their relative stability to inform on potential DILI risk?

Acyl Glucuronides (AGs) and their respective degradation products are one of the most common drug metabolites. They have been implicated in the cause of drug-induced liver injuries (DILI) - a major factor in the attrition of drug candidates. NMR and other analytical methods have previously been applied to study these degradation products and their transformations, though mainly to the purpose of evaluating the rates of AG degradation. It is clear now that a better understanding of the processes of AG transformations (transacylation, hydrolysis, mutarotation, glycation) would be of high benefit. Here we show that by using NMR spectroscopy, one can study both the kinetic mechanism of AG transformation and the structure of intermediates and degradation products. We present an example of such in-depth analysis using a tool compound, Ibufenac AG. We show that kinetic and structural NMR data analysis combined with theoretical DFT calculations can provide unprecedented clarity of details regarding the mechanism of AG transformations. These capabilities are of great help to propose new hypothesis and design new experiments which should ultimately lead to a better understanding of the mechanism of DILI and its prevention.

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Bradley Chmelka

Univ. of California, Santa Barbara

Santa Barbara, CA, United States

Analyzing zeolite catalysts at an atomic level by solid-state NMR

Recent advances in solid-state NMR characterization capabilities enable the compositions, structures, and properties of heterogeneous catalysts to be measured, understood, and correlated in ways that have not previously been possible. Examples include nanoporous aluminosilicate zeolites and supported metal clusters, whose industrially important reaction properties have been challenging to understand and control. This has been due in part to their non-stoichiometric compositions and heterogeneous surfaces, which result in complicated distributions of structural order and disorder that have important influences on their adsorption and reaction behaviors. These challenges can nevertheless be overcome by using powerful solid-state NMR spectroscopy techniques, notably dynamic-nuclear-polarization (DNP)-enhancement and two-dimensional (2D) NMR correlation methods. In combination with complementary scattering and bulk property measurements, they enable heterogeneous catalysts to be probed over multiple length scales to obtain and correlate insights on local bonding environments and interactions with macroscopic adsorption and reaction properties. Recent results will be presented on measuring and understanding at an atomic level the compositions and structures of zeolite catalysts, in particular the influences that heteroatom distributions and diverse adsorption sites have on catalyst reactivities. The analyses provide guidance for the rational design and syntheses of zeolite catalysts for applications in hydrocarbon conversion, automotive pollution mitigation, and separations. Our work in these areas is supported by and conducted with diverse industrial partners, whose crucial roles will be highlighted.

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Charlotte Corbett

US Drug Enforcement Administration

Dulles, VA, United States

Low Field qNMR of Methamphetamine HCl

Can the purity of methamphetamine HCl containing exhibits be quickly and easily determined in a reliable manner by low field NMR in a completely automated fashion? What experimental parameters are important to achieve less than 2% uncertainty? T1 values can be longer at low field, so proper internal calibrant selection can be important to increase sample throughput. Low field signals encompass a greater chemical shift region, so integral regions must be quite broad. For methamphetamine, only two integral regions are available. Can integration at specified set points yield reliable results and ensure the result is not too high due to overlapping signals? How can qNMR be simple and reliable at low field for further adoption of qNMR.

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Michael Dick

Green Imaging Technologies

Fredericton, NB, Canada

Reducing Sample Heating Employing A Variable Spaced Tau CPMG

The pore size (or T2) distribution is probably the most important measurement in NMR core analysis. The distributions are employed to derive the porosity of core samples as well as used as the basis of other measurements such as wettability determination, NMR log calibration and bound vs. free fluid determination. The T2 distribution is derived from the Carr-Purcell-Meiboom-Gill (CPMG) NMR pulse sequence. The CPMG sequence employs many RF pulses (linearly spaced), each of which deposit energy into the sample. This can lead heating of the sample, which can lead to a reduction in NMR signal, which can lead to inaccuracies in porosity determination. In addition, the T2 distribution itself is affected by sample temperature. One method to avoid sample heating by the CPMG pulse sequence is to employ a sequence where the RF pulses are spaced logarithmically. This maintains the accuracy needed to probe the T2 decay while reducing the number of RF pulses impinging on the sample. In a previous study, we implemented the logarithmically spaced CPMG sequence and have shown that this sequence still accurately reproduces the NMR-measurements for all core samples while eliminating sample heating. In the current study, we have expanded on these previous measurements to include measurements done at higher field (12 MHz vs. 2MHz) where unwanted sample heating can be amplified by the increase in in field. In addition, we have expanded the application of logarithmically spaced CPMG sequence to relaxation maps, such as a T1-T2 and T2-Diffusion maps. T1-T2 maps are often employed in NMR core analysis including for fluid typing in samples. Sample heating is particularly problematic for shale samples where it has been shown that the T1-T2 map are temperature dependant. We have replaced the linearly spaced CPMG pulse train with a logarthimic version in the T1-T2 pulse sequence. The results of this replacement and the subsequent reduction in sample heating will be discussed in this paper.

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Robert Evans

Aston University

Birmingham, West Midlands, United Kingdom

NMR Cryoporometry - Characterization of Porous Materials Using Liquid NMR

Porous material, such as silica, have become increasingly significant in a wide range of applications including heterogeneous catalysis. Reactions, such as the esterification of fatty acids, are enhanced when performed within the porous structures and these enhancements are found to be highly dependent on the size and structure of the pores. NMR cryoporometry, a direct measurement of the melting point depression of a liquid contained within a porous material, can be used to measure pore sizes of a wide range of different materials. Through appropriate choice of liquid, matched to both the likely range of pore sizes present and the surface chemistry of the materials being analysed, a wide range of pore sizes can be accurately measured. While water and cyclohexane are well-established for this technique, t-butanol and menthol have been identified as candidates for the study of hydrophilic materials with pore sizes > 50 nm. Appropriate choice of liquid allows the analysis of porous materials with markedly different surface chemistry.

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Hilary Fabich

ABQMR Inc.

Albuquerque, NM, United States

NMR of chemical reactions at elevated process conditions

Nuclear magnetic resonance (NMR) can be used to study chemical reactions in real time. However, this is not common practice at conditions of high temperature (up to 350 C) and high pressure (up to 7 MPa) as the design of an NMR compatible reactor is difficult. In this study we report reactions that are typical of refinery operations - hydrogenation, transfer dehydrogenation, methanol synthesis, and isomerization. We use sealed glass capillaries (ID = 2.6 mm, OD = 7 mm) to hold the pressurized sample plus the required oxygen or hydrogen and catalyst. To seal the capillaries, they are chilled in a liquid nitrogen bath meaning that any sample with a vapor pressure below 0.1 MPa (1 bar) at 77 K can be loaded and sealed. In addition to adding molecules that either freeze or condense at 77K (including oxygen), hydrogen gas is added by using a metal hydride, such as LiAlH4, that decomposes to release hydrogen at reaction conditions. The experiments discussed are performed using a low field (1 T, 43 MHz proton frequency), permanent magnet. The magnet has x, y, z, and z2 shims which produce a field homogeneity of approximately 3 Hz (0.07 ppm), full width half maximum (FWHM). Even with this modest resolution, it is possible to track formation of new species when new peaks appear as the reaction progresses. We report, as an example, the hydrogenation of benzene with H2 gas from LiAlH4 to produce cyclohexane. Initially, the spectrum shows one peak for benzene. As the sample is heated, a small cyclohexane peak appears as some of the benzene molecules react directly with the LiAlH4. At around 150 C the H2 is released from LiAlH4 and a third, broad peak (H2) appears in the spectrum. As the sample is heated, H2 reacts with benzene until all of the H2 is consumed and only two peaks remain, benzene and cyclohexane. As the sample is heated to drive the reaction, spectra are acquired sequentially. This gives insight into the degree and rate of reaction for a given set of conditions: reactant concentration, catalyst, temperature and pressure. Acquiring spectra at process conditions yields insight into the rate of reaction in real time. There is no need to cool the reaction or to sample portions of the material to measure the products at a given time. As the experiments can be performed at process conditions, it may be possible to detect intermediates that are not present after a sample is cooled because they condense, evaporate or further react. The idea of using NMR to study chemical reactions is certainly not new; however, the ability to do this easily at high temperature and high pressure process conditions offers the possibility of new information about a variety of industrially important reactions. For a wide range of refinery and commodity reactions, the low-field of 1 T provides sufficient resolution to monitor the reactions. This method brings us one step closer to the ultimate goal of online process monitoring.

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Roberto R. Gil

Carnegie Mellon University

Pittsburgh, PA, United States

3D Structure Elucidation of Small Organic Molecules Using MultiNMR Parameters

The combination of traditional NMR parameters such as NOE-derived distances, 3J scalar couplings and anisotropic NMR parameters (Residual Dipolar Couplings and Residual Chemical Shift Anisotropy), [1,2] allows the determination of the relative configuration together with the conformational preference of natural and synthetic organic compound with a high degree of confidence. In particular, the recently proposed CASE-3D (Computer Assisted 3D Structure Elucidation) methodology [3,4] includes the use of molecular modeling, quantum mechanical prediction of NMR parameters and the filtering of conformational models using statistical criteria. The strategies and protocols necessary for the use of this multi-parametric methodology will be discussed, as well as the validity of the different approaches and the use of the necessary software tools. Detailed application of (i) J-based analysis, (ii) use of stretched and compressed aligning gels, and lyotropic liquid crystals (LLC) solutions (PBLG) to measure anisotropic parameters, [1,5] and (ii) data analysis for determination of configuration/conformation [4] will be presented. 1. Liu, Y., Navarro-Vazquez, A., Gil, R.R.; Griesinger, C.; Martin, G.E.; Williamson, R.T. Nature Protocols2019,14,217-247. 2. Nath, N., Schmidt, M., Gil, R.R., Williamson, R. T., Martin, G.E., Navarro-Vazquez, A., Griesinger, C., Liu, Y. J. Am. Chem. Soc.2016,138,9548-9556. 3. Troche-Pesqueira, E.; Anklin, C.; Gil, R. R.; Navarro-Vằuez, A.;Angew. Chem. Int. Ed. 2017, 56, 3660. 4. Navarro-Vằuez, A.; Gil, R. R.; Blinov, K.;J. Nat. Prod. 2018, 81, 203. 5. Ndukwe, I.E.; Wang, X.; Pelczer, I.; Reibarkh, M.; Williamson, R. T.; Liu, Y.; Martin, G.E. Chem. Comm. 2019,55,4327-4330.

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Travis Gregar

3M

Maplewood, MN, United States

Implementation of a Benchtop NMR in a manufacturing Environment

Traditional analytical measurements in a manufacturing setting requires simple, repeatable, and easy to perform tests with minimal effort. With the introduction of low field NMR units, new analytical methods are now possible to both replace some of the traditional methods as well as implement new procedures to help increase throughput, reduce risk, and line down time in the factory setting. In this talk I will present the journey to qualify and deploy this new technology into one of our manufacturing locations.

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Marco Guerrini

Istituto di Ricerche Chimiche e Biochimiche G Ronzoni

Milan, Italy

Automated 2D quantitative NMR spectroscopy for complex drug analysis

The number of complex drug substances, including biologics, present in different pharmacopoeias has increased in recent decades. These drugs are distinct from chemically synthetized pharmaceutical products, since they do not represent a single entity, but a complex combination of substances. The quality control of these materials is more complex than the traditional small molecule pharmaceutical, and as a result, product quality assessment requires highly informative analytical approaches to validate that the product meets specifications. Among the different analytical approaches developed to assess the structural characteristics of complex drugs, NMR is one of the leading techniques1. An example of NMR used as a quality control tool for complex drugs is characterization of the life-saving anticoagulant heparin, included in the Essential Medicines List of the World Health Organization. Following the "heparin crisis" of 2007-2008, FDA and EMA introduced pharmacopeia monographs for 1H NMR as an identity test and to confirm the animal origin of the material2. Moreover, the introduction of quantitative 2D HSQC NMR spectroscopy enables both qualitative and quantitative characterization of heparins and impurities, and using this data with chemometric and statistical methods allows assessment of the quality of industrial production as well as comparison between a biosimilar drug with the originator product3. Performing heparin analysis reliably typically requires expertise in quantitative NMR as well as in the chemistry of glycosaminoglycans, especially for chemical shift assignment and analysis of the more complex 2D spectra. To expand the applicability of the method, we have worked to automate the NMR analysis to improve speed and reliability, and we conducted a round robin study to test the performance of our method when implemented in other laboratories. This allowed us to conclude that, automatization of the spectra processing and analysis allows this methodology to be a viable alternative for many laboratories, without the need for expertise in NMR of glycosaminoglycans. References [1] Guerrini, M., Rudd, T.R., Yates, EA. NMR in the characterization of complex mixture drugs. In: The Science and Regulations of Naturally Derived Complex Drugs. Sasisekharan, R., Lee, S.L., Rosenberg, A., Walker, L.A. Eds. Springer Heidelberg, Dordrecht, London, New York. 2019, Page:115-137. [2] A. Y. Szajek, E. Chess, C. Johansen, et al. Nat. Biotechn. Ed. 2016, 34, 625-630. [3] Mauri L., Boccardi G., Torri G., Karfunkle M., Macchi E., Muzi L., Keire D., Guerrini M. JPBA 2017, 136, 92-105

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Jian Zhi Hu

Pacific Northwest National Laboratory

Richland, WA, United States

In Situ (Operando) Magic Angle Spinning NMR for Harsh Experimental Conditions

High resolution magic angle spinning (MAS) NMR is a powerful technique for studying structure and dynamics in a heterogeneous system containing a mixture of e.g., solid, semi-solid, liquid, and gaseous phases. Due to its intrinsic advantage of probing local structure at molecular level, MAS NMR is an attractive tool for in situ (operando) investigations of reaction mechanisms, including but not limited to the identification of active centers, intermediates, and the reaction dynamics associated with material synthesis or chemical reactions using solid catalysts, the adsorption and desorption of molecules on surfaces of porous materials. However, the commercially available reusable-MAS rotors are rarely capable of achieving 100% seal under harsh experimental conditions of, e.g., significantly elevated temperature and pressure, or significantly cold temperature with high pressure. To address these challenges and advance the application of MAS NMR under harsh experimental conditions, we have developed a perfectly sealed, powerful, in situ MAS NMR rotor that is capable of sealing a heterogeneous sample under extreme experimental conditions of combined high pressure and high temperature.1 The same technology is equally applicable to low temperature and high pressure operations. The in situ MAS rotor is constructed using an integrated high mechanical strength zirconia rod except an O-ring and a spin tip. Herein, we will first report the latest advancements associated with high field and fast sample spinning where in situ MAS rotor with outside diameters of 4 and 3.2 mm have been successfully developed. To illustrate the power of in situ MAS NMR, examples of application in material synthesis and catalytic reactions, including but not limited to (a) Mechanisms of phenol-cyclohexanol alkylation in zeolite H-BEA studied by 13C and 1H NMR,2-3 (b) The crystallization of AlPO4-5 by 1H, 27Al, and 31P NMR,4 and (c) Genesis and stability of hydronium ions in zeolite channels by 1H and 1H-29Si CP NMR,5 and (d) Zeolite framework stability and degradation in hot water studied by high field and fast spinning 27Al MAS NMR. 

 References

1.         Hu, J. Z.; Hu, M. Y.; Zhao, Z. C.; Xu, S. C.; Vjunov, A.; Shi, H.; Camaioni, D. M.; Peden, C. H. F.; Lercher, J. A., Sealed rotors for in situ high temperature high pressure MAS NMR. Chem Commun 2015, 51 (70), 13458-13461.

2.         Zhao, Z. C.; Shi, H.; Wan, C.; Hu, M. Y.; Liu, Y. S.; Mei, D. H.; Camaioni, D. M.; Hu, J. Z.; Lercher, J. A., Mechanism of Phenol Alkylation in Zeolite H-BEA Using In Situ Solid-State NMR Spectroscopy. J Am Chem Soc 2017, 139 (27), 9178-9185.

3.         Liu, Y. S.; Barath, E.; Shi, H.; Hu, J. Z.; Camaioni, D. M.; Lercher, J. A., Solvent-determined mechanistic pathways in zeolite-H-BEA-catalysed phenol alkylation. Nature Catalysis 2018, 1 (2), 141-147.

4.         Zhao, Z. C.; Xu, S. C.; Hu, M. Y.; Bao, X. H.; Hu, J. Z., In Situ High Temperature High Pressure MAS NMR Study on the Crystallization of AlPO4-5. J Phys Chem C 2016, 120 (3), 1701-1708.

5.         Wang, M.; Jaegers, N. R.; Lee, M.-S.; Wan, C.; Hu, J. Z.; Shi, H.; Mei, D.; Burton, S. D.; Camaioni, D. M.; Gutiérrez, O. Y.; Glezakou, V.-A.; Rousseau, R.; Wang, Y.; Lercher, J. A., Genesis and Stability of Hydronium Ions in Zeolite Channels. J Am Chem Soc 2019, 141 (8), 3444-3455.

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Shaoying Huang

Singapore University of Technology and Design

Singapore

The Latest Progress of Low-field Permanent-Magnet-based MRI Head Imager

A magnetic resonance imaging (MRI) imager with portability may help this medical imaging modality to reach out areas that are remote or/and hard to access, and situations that the environment is dynamic, for example, for disaster rescues, in a field hospital, or in an ambulance. This talk reports the latest progress of the development of a low-field permanent-magnet-magnet (PMA) based MRI head imager in Singapore University of Technology and Design (SUTD). It includes two parts. The first part is the latest design of a hybrid inward-outward (IO) ring-pair magnet array, which supplies a strong longitudinal magnetic field with an electrically rotatable monotonical pattern. The second part is the recently proposed approaches to navigate in local k-spaces to improve image quality, based on an investigation of the encoding fields supplied by PMA's.

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Jinfeng Lai

Phillips 66 Company

Bartlesville, OK, United States

Elucidating Structural Characteristics of Renewable Products by NMR

A novel two-step ex situ hydropyrolysis vapor upgrading (HPVU) process can generate tailored oxygenated hydrocarbon products from lignocellulosic biomass by adjusting vapor upgrading temperature. Such products, containing varied oxygen contents (ranging from 2 to 17 wt%), are different from traditional petroleum hydrocarbons and conventional biomass-derived pyrolysis oil. Elucidation of the comprehensive structural motif of these unconventional products is critical for development and deployment of the HPVU process. To this end, two-dimensional heteronuclear 1H-13C NMR techniques were employed to first provide single- and multiple-bond correlations between proton and carbon characteristic of different organic functional groups. The distribution of carbon functional groups was further determined by quantitative 13C NMR spectroscopy with full relaxation and well-defined chemical shift regions obtained from diverse NMR methods. Phenols and ketones were identified as the main oxygenate species in these renewable products. For products obtained from higher vapor upgrading temperatures, a decrease in oxygenate species and an increase in alkylation on aromatic rings were observed. Of particular importance, compared to pyrolysis oil, the amounts of active oxygenate species, such as aldehyde, carboxylic acid, and carbohydrate, were found to be minimal, suggesting good stability of these renewable hydrocarbon products.

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Yizhou Liu

Pfizer Inc

Groton, CT, United States

Residual Chemical Shift Anisotropy (RCSA) in Small Molecule Structure and Dynamics: the nuts and bolts

In recently years, simple and robust methods have been developed to accurately measure residual chemical shift anisotropies (RCSAs) in small organic molecules in various types of alignment media. RCSAs provide valuable information especially for proton-deficient molecules in which proton-based NMR information is sparse. In this workshop, I will introduce to the audience the basic theory of RCSA, and then the practical strategy of measuring RCSA in dilute liquid crystal phases and using them for structure and dynamics characterization of small molecules. The goal is to equip the audience with the necessary knowledge to independently apply this technique in their research with the right tools in hand. Key references: 1. Liu, Y., Cohen, R.D., Gustafson, K.R., Martin, G.E., Williamson, R.T. Chem Commun. 2018, 54, 4254-4257. 2. Ndukwe, I.E., Wang, X., Pelczer, I., Reibarkh, M., Williamson, R.T., Liu, Y., Martin, G.E. Chem Commun. 2019, 55, 4327-4330. 3. Ndukwe, I.E., Brunskill, A., Gauthier, D.R. Jr, Zhong, Y.L., Martin, G.E., Williamson, R.T., Reibarkh, M., Liu, Y. Org. Lett. 2019, 21, 4072-4076.

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Joe Lubach

Genentech, Inc.

South San Francisco, CA, United States

Insights into Pharmaceutical Drug Products Using Multinuclear Solid-State NMR

High resolution characterization of pharmaceutical solid dosage forms represents an ever-challenging problem facing pharmaceutical scientists. Detailed solid-state analysis of a drug product should include active ingredients as well as excipients, and potential interactions among them. Historically, pharmaceutical solid-state NMR has primarily been applied to cases of relatively simple solid form determination, where it naturally excels, particularly in formulated products. This presentation will focus on modern applications of solid-state NMR spectroscopy to gain insights into complex solid dosage forms, including crystalline and amorphous materials, protonation states, drugs and excipients, and ubiquitous water. Historically for pharmaceutical solids, 13C is the most widely studied nucleus due to the information content available, its presence in nearly all pharmaceutical ingredients, and high resolution spectra that can be obtained. It is primarily accessed via cross polarization (CP) experiments due to its low natural abundance and relatively long relaxation times, which have hindered its use in quantitative analysis. We will illustrate practical examples of quantitative CP experiments in amorphous solid dispersion tablets, a commonly used modern dosage form. Additionally, direct 13C polarization experiments can provide particularly useful information in formulation development, and we will present a case where it was utilized in contrast with CP experiments to gain deeper understanding into a granulation process. 15N is particularly useful in protonation state investigations, but suffers from extremely poor sensitivity and sometimes prohibitively long experiment times. We will examine a case where it was utilized to show proton transfer from drug to excipient in an amorphous solid dispersion, and help to gain a better understanding of the overall landscape of these materials. 31P is a much friendlier nucleus from an NMR standpoint, though not nearly as widespread in pharmaceutical materials. Nonetheless, it is highly useful when present in materials such as phosphate salts and phosphoric acid cocrystals, and we will examine how it can be used for crystal form determination as well as protonation state investigations through chemical shift tensor measurements. Finally, water content is a frequently measured quantity is solid dosage forms, and is often considered a critical quality attribute. The methods used to quantitate water content, such as Karl Fischer titration, loss on drying, or thermogravimetric analysis, generally sample a bulk powder or tablet and report back the overall water percentage in a given quantity of material. However, the distribution of this water is far from homogeneous. We will demonstrate how 1H NMR relaxation times, detected through 13C CP experiments, can be used to ascertain how ubiquitous water is distributed among various ingredients in a formulation. This deeper understanding of the distribution of water molecules throughout a given formulation can provide valuable insight into dosage form design for more robust drug products and processes.

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Charlotte Martineau-Corcos

Institut Lavoisier de Versailles (ILV)

Versailles, France

A 3D-printed NMR insert to study porosity and gas diffusion in microporous order

NMR spectroscopy has proven capable of providing key information about adsorbed gases or molecules in microporous or mesoporous solids (zeolites, metal-organic frameworks (MOFs), etc), as well as self-diffusion and transport processes.1 However, NMR intrinsically suffers from low sensitivity, which requires using large quantity of expensive isotopically enriched gases like 13CO2 or C17O2, strongly restricting the applications of such methods on an industrial scale. Recently, we have developed a 3D-printed microfluidic insert,2 that greatly boosts the signal of insensitive and slowly relaxing nuclei, such as 13C, in solution-state NMR spectroscopy.3 We have adapted this insert to accommodate solids (powders, membranes or small crystals) over which a controlled flow of gas is sent. It requires the use of minute amount of gases and is versatile enough that it can be tuned to the resonance frequency of any nuclei (among which 1H, 19F, 129Xe, 13C, 15N). With the enhanced sensitivity provided by the designed insert and the micro-detection, it also becomes possible to perform new experiments like time-resolved NMR and localized spectroscopy. We will first detail the working principles of our fluidic system and show the validation of the proof-of-concept on various model microporous solids: investigation of the porosity (for example using hyperpolarized 129Xe NMR), of dynamic properties related to adsorption, diffusion and separation of single-component or gas mixtures of interest for medical application, for carbon capture or in catalysis. Finally, selected other applications of interest for MOFs will be illustrated.

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Yevgen Matviychuk

University of Canterbury

Christchurch, Canterbury, New Zealand

Automated Quantification with Benchtop NMR

Quantitative NMR is gaining traction as a fast non-destructive chemometric method that is well suited to a variety of industrial applications, owing to its almost universal detection capabilities. Recently developed user-friendly benchtop instruments reduce the capital and operational costs associated with conventional cryogenically cooled magnets and hence further promote NMR as a general analytical tool. Unfortunately, spectra acquired at the lower magnetic field strength of benchtop instruments often suffer from significant peak overlap. This prevents accurate quantification with established methods and motivated the development of alternative model-based algorithms. Instead of integrating areas under the peaks, these methods match an experimentally acquired spectrum with a set of adjustable signatures - one for each chemical species, which eventually determines their concentrations. Nevertheless, fitting a parametric model to low resolution spectra with potentially shifting peaks is a challenging problem: often multiple equally good solutions exist, yet only some of  parameter combinations have  physical meaning. However, many industrial applications of NMR are concerned with routinely analyzing sets of similar samples, which presents a route to developing fully automated processing methods. Here we present a novel data processing approach that extends the "one-click" philosophy of benchtop NMR from spectral acquisition to data analysis and allows us to immediately answer the question about the mixture composition.

 

Our algorithm takes into account any constraints on the model parameters (e.g. chemical shifts) imposed by underlying molecular interactions in the mixture. We leverage the prior knowledge about the studied system gleaned from existing (or purposefully collected) high field datasets and extrapolate it to previously unseen samples using machine learning techniques. Formulated in terms of the Bayesian framework, our approach seamlessly incorporates this prior information into the model fitting process, which enables automated analysis of benchtop data even with overlapping and moving peaks. Finally, our novel error correction mechanism improves the phasing of the spectra and brings the quantification accuracy of benchtop data to the level of traditional processing approaches applied to well-resolved high field spectra. We demonstrate its performance with several mixtures of industrially and biologically relevant species, including alcohols, acetates, organic acids, and sugars. Our fully automated analysis can achieve an average error in concentrations of below 0.6% in certain cases and surpasses manual processing in all tested examples.

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Evan McCarney

Korimako Chemical Limited

Wellington, WLG, New Zealand

Online monitoring of enzymatic hydrolysis of marine by-products

Nuclear magnetic resonance spectroscopy, in general, has much to offer regarding process monitoring and quality control. Unfortunately, high-field NMR instrumentation is often uneconomical or not suitable for remote locations where it is needed. Benchtop NMR spectroscopy can solve these two issues but forces a compromise on performance, such as sensitivity and chemical shift dispersion. Often, these are tolerable or can be overcome with advanced acquisition or processing methods. One promising application is monitoring of enzymatic hydrolysis, which is seeing increased usage to create high-value materials from traditionally low-value by-products and waste. The proteinaceous tissue from marine by-products, such as skin, entrails, head, tails, and fish frames, is broken down through enzymatic hydrolysis of peptide bonds releasing peptides, amino acids, oils, and metabolites into solution. These hydrolysate products are valued as pharmaceutical, nutraceutical, and value-added food components. However, the hydrolysis process is poorly understood and inconsistent due to the irregularity of raw materials and enzyme cultures, which results in inconsistent products. Development of this processing method is further hampered by a lack of tools to monitor the reaction in real-time under relevant conditions. Current analysis methods rely on taking offline samples, deactivating the enzymes, possibly freeze-drying the samples, and then performing offline analyses such as chromatography, amino acid analysis, and activity assays on the products after the reaction has already run its course. Here nuclear magnetic resonance benchtop spectroscopy and diffusion ordered spectroscopy (DOSY) were investigated as methods for online and at-line process monitoring of enzymatic hydrolysis. Two key characteristics that NMR can provide in real-time are the concentration of hydrolysate from the NMR signal intensity and the size of the peptides from their self-diffusion coefficients. These measurements allow the quantification of the efficiency and quality of the hydrolysate. In this study, NMR measurements were performed to monitor the enzymatic hydrolysis reactions on red cod, salmon, and shrimp. The reaction mixture was pumped through a benchtop NMR spectrometer, where proton NMR spectra were recorded. Samples were also collected in parallel for offline measurement comparisons. Diffusion measurements were measured at-line by transferring a sample to an NMR tube and immediately collecting DOSY spectra without deactivating the enzymes. Both the online and offline measurements were able to follow the product concentration during the reaction process. Offline measurements offer the benefit of a suite of established methods to characterize the reaction, however, they fail to capture the early stage hydrolysis due to the lengthy enzyme inactivation period relative to the overall process. At-line diffusion measurements were capable of estimating mean peptide size, which is a key factor in taste and the physical properties of hydrolysates used as emulsifiers in food additives. The molecular weight also directly relates to the degree of hydrolysis which is a standard measure of how completely hydrolyzed the sample is. In conclusion, online monitoring provides a more accurate description of the reaction progression than offline methods. Diffusion measurements are also able to characterize the average molecular size throughout the reaction as a key indicator of the hydrolysis progression and quality. Application of these methodologies to several types of raw materials indicates the technique is robust and is a promising tool for process monitoring and control of by-product hydrolysis.

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Vladimir Michaelis

University of Alberta

Edmonton, AB, Canada

Solid-state NMR Strategies to Decode Porous Materials

Nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful analytical methods available today to study atomic- and molecular-level structure and dynamics within the chemical sciences.  For example, dynamics within biomolecular solids or metal-organic frameworks may be monitored via 2H or 13C NMR providing insight into problems facing biophysics or carbon capture technologies, respectively.  Likewise, deciphering distinct coordination environments and medium-range structure has assisted in a variety of research areas, including catalytic zeolites, battery materials, and geochemical applications.  Over the last decade, solid-state NMR spectroscopy has experienced major engineering breakthroughs that are advancing research abilities to a new level ,such as the ability to boost detection limits through dynamic nuclear polarization (DNP), routinely performing experiments under low (<100 K) or high (800 K) temperatures, or pushing beyond "ultrahigh field" boundaries with magnetic fields now exceeding 1 GHz. These developments and others are reshaping materials science capabilities across the globe in industry, academic, and government research laboratories.

In this presentation, we will discuss recent adventures our team has experienced with porous catalytic materials that are critically important to the chemical and petrochemical industries. We have studied a new class of water-tolerant Lewis acid catalysts comprised of a silica-rich zeolite containing isolated catalytic metal centers.  Using emerging direct and indirect NMR methods that we and others have developed we will demonstrate how we can track and characterize these highly dispersed metal catalytic T-sites that are engulphed by a crystalline Si-rich porous framework material. Furthermore, discussion about specific labeling protocols to effectively track reaction intermediates and products using other strategies, including spectator molecules, dipolar-based recoupling methods, and DNP approaches, will be covered. The goal is to demonstrate some new approaches to further expand on the forefront of NMR and its role in characterizing next-generation materials.

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Nikolaus Nestle

BASF Advanced Materials and Systems Research

Ludwigshafen, RLP, Germany

Looking deeply into a turbid crystal ball: TD-NMR on liquid polymer dispersions

Aqueous polymer dispersions are widely used base products for many industrial products such as coatings and adhesives. From the chemical engineering point of view, they are especially attractive because they allow low-viscosity formulations with cross-linked polymers. Furthermore, the use of polymer dispersions instead of dissolved polymers allows to eliminate or greatly reduce the amount of organic solvents needed in these products. From a material characterization point of view, polymer dispersions with technically relevant solids contents are rather challenging due to their high turbidity, possible instabilities under shear forces and various changes upon dilution. One of the few noninvasive spectroscopic options to study such dispersions in their native state is time domain (TD) NMR. In the presentation, we provide a survey of information on the state of the polymer available from such measurements and how it can be correlated with micromechanics and application properties of films and composites obtained after drying of the polymer dispersions. Furthermore, the potential of unilateral NMR for film formation studies is shortly discussed.

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Matthew Nethercott

Kansas Analytical Services, LLC

Wellington, CO, United States

Opportunities and Challenges in 19F Detection of Fluorinated Pharmaceuticals

In 2018, 28% of new drugs approved by the FDA were fluorinated(1). 19F solid-state nuclear magnetic resonance (SSNMR) spectroscopy is an extremely powerful technique for the analysis of active pharmaceutical ingredients (API) in these new drugs. It has potentially lower limits of detection, quantitation, and faster experimental times without interference from excipients compared to 13C SSNMR experiments. The benefits of using 19F SSNMR spectroscopy to study pharmaceutical systems includes 19F being 100% natural abundance (vs. 1.1% for 13C), lack of interference from excipients in formulated drug product (DP), and short acquisition times (minutes to few hours) compared to 13C (hours to days). Some weaknesses may include the lack of spectral resolution between isotropic shifts and spinning sidebands, the potential for more than one peak for a 19F site, and spectral overlap between peaks. This talk will present the basics of 19F SSNMR, and use two case studies to demonstrate the opportunities and challenges of acquiring and interpreting 19F SSNMR data. Approaches highlighting the use of 19F SSNMR to analyze pharmaceuticals include the ability to perform spectral subtraction, 1H T1? filtering, and deconvolution to quantify the polymorphs present in both API and DP samples. Two case studies discussing the quantitation of fluorinated compounds in API and DP will be presented. Both case studies have multiple crystalline forms present in the DS and DP. (1) G. de la Torre, B.; Albericio, F. The Pharmaceutical Industry in 2018. An Analysis of FDA Drug Approvals from the Perspective of Molecules. Molecules 2019, 24, 809.

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Clark Ridge

US FDA

College Park, MD, United States

Complementary Small Molecule Mass Spectrometry and NMR: Food Applications

Small molecule analysis usually involves mass spectrometry (MS) and NMR spectroscopy performed in relative isolation. The NMR spectroscopist may get little more than the chemical formula from the MS analysis and in return provide a structure. There is usually far more information in both analyses that can be useful. In many other cases, more collaboration is needed to elucidate structure or solve the problem at hand. Instructive cases that highlight the complementary nature of the techniques will be presented here including the following: 1. Maitotoxin (MTX) is a large polyketide marine toxin. With a molecular weight upwards of 3000 Da, it is the largest known non-biopolymer marine toxin. Several compounds similar to MTX were isolated and found to be deficient in mass by either one sulfate group or a sulfate group, a methyl, and one degree of unsaturation. With only 50 nmol of material, a complete assignment was not possible. However, MTX has only two sulfate groups with one near the end of the molecular chain and accessible by NMR techniques. The combined high-resolution MS and NMR analysis were able to partially assign and distinguish which of these sulfate groups is missing in this new set of maitotoxins. [1] 2. Solid state NMR (SSNMR) has the ability to shed light on structure and composition without disrupting the sample. Analysis by liquid state NMR and LCMS techniques are limited by what can be brought into solution. There is also the possibility that bringing samples into solution can change them chemically or structurally. All ionization techniques necessarily change the sample in ways that can be hard to determine afterward. Most polymers used in food packaging or as food-contact-surfaces in other applications are difficult to observe without chemically changing the sample. SSNMR offers a way to look at these polymer samples directly. Several polymer samples were analyzed by LCMS to look for additives and try to confirm the structure of known additives before and after processing. The results of recent SSNMR experiments performed on these polymers will be discussed in relation to the complimentary information they provide on structure and composition. 3. There are several projects underway at the FDA involving a combined NMR/MS approach to look at FDA related consumer products that are inherently complex mixtures such as food syrups and edible oils. MS combined with separations (GC, LC, etc.) provides a rich dataset about the many minor components found in these mixtures. Sometimes hundreds of compounds can be identified and roughly quantified within a single sample run. In contrast NMR has the ability to better identify and quantify the major constituents of a mixture. In addition, NMR can be used to positively identify and quantify possible contaminants indicated by MS analyses. The combined data can be used to create a specific and detailed chemical profile of a product that includes both major and minor components. A few examples will be discussed. These examples and applications will show the advantage that can be gained by the combined analytical abilities of NMR and MS used together in a truly collaborative way. [1] E.P. Mazzola, J.R. Deeds, W.L.Stutts, C.D. Ridge, R.W. Dickey, K.D. White, R.T. Williamson, G.E. Martin, Elucidation and partial NMR assignment of monosulfated maitotoxins from the Caribbean, Toxicon 164 (2019) 44-50

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Dimitrios Sakellariou

KU Leuven - cMACS

Leuven, Belgium

Low-field NMR and MRI For In Operando NMR Studies.

High-field magnetic resonance is an extremely powerful characterization technique, which cannot however be trivially adapted to perform in in operando studies especially in an industrial environment. Limitations related to space, sample preparation, magnetic and radio-frequency compatibility, transportability, cost of operation and maintenance are some of the difficulties one has to fight against. On the other hand, low-field NMR and MRI makes use of permanent magnets, which are more versatile and solve a very large number of the above issues. In this contribution we present a variety of permanent magnet assemblies which offer new possibilities for material analysis and characterization in operando conditions. Small and large single-sided systems having unique field profiles simulating NMR logging tools is presented. These systems are used alone for the study of pore size distributions in rock plugs, or as imaging diagnostic systems in energy materials such as batteries. This effort can potentially offer great benefits especially in NMR of metals in materials such as batteries and catalysts. We also present new enclosed pseudo-Halbach magnets producing highly homogeneous field and offering excellent portability. These NMR systems can be used alone as desktop analyzers, but they have been used simultaneously with other characterization modalities (physisorption devices, neutron scattering and imaging devices) to provide hyphenated low-field NMR approaches for in operando physico-chemical characterization of materials. Applications on mesoporous materials (MOFs, zeolites) are presented where sorption of vapors of small molecules in natural abundance is monitored in operando and relaxometric information is recorded as a function of loading pressure. This information shows that NMR offers complementary information about the sorption dynamics in a direct manner which until today was missing. Last we show a high-resolution solid-state NMR analyzer with magic angle spinning capabilities offering the possibility to analyze intact agricultural products and eliminate sample preparation. This custom-made low-field (hyphenated or not) NMR instrumentation demonstrates sufficient generality and practicality, and seems a promising direction for dedicated industrial applications of magnetic resonance.

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Jacob Schaefer

Washington University

St. Louis, MO, United States

Molecular Basis of Secondary Relaxation in Stiff-Chain Glassy Polymers

Recent progress in establishing local order in polycarbonate-like glasses using rotational echo double resonance (REDOR) and centerband-only detection of exchange (CODEX) has stimulated a renewed attempt to connect molecular motion within a glassy polymer and the ductility of the glass. The resulting structural insights suggest that the chains of these polymers are simultaneously both Flory random coils and Vol'kenshtein bundles.

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Torsten Schoenberger

Bundeskriminalamt

Wiesbaden, Hesse, Germany

Adhesive Tapes in Magic Angle - NMR's Superior Discrimination Power

Adhesive tapes are often used in connection with a criminal offense, for example, for gagging victims, for making unconventional explosive and incendiary devices, or for closing drug packages. The connections between the materials used at different crime scenes or crime scenes and the materials seized from a suspect can provide important evidence. Ideally, matching cut edges will provide unambiguous proof. However, since this is very rarely found, the material of the adhesive tapes must be examined. The aim is to narrow down the source of the tape to a specific product or even a batch. In a study initiated by the FBI, 90 similar black insulating tapes were purchased and analyzed using several techniques. The study showed that NMR spectroscopy used at the Federal Criminal Police Office is clearly superior to all other techniques in terms of discrimination. The developed HR-MAS method is absolutely reproducible. The sample preparation is very simple. The 1D-1H NMR spectrum provides an excellent fingerprint of the material, reflecting the complex composition of different polymer types (including tacticities) and monomer additives at a glance. The excellent ability of NMR spectroscopy to discriminate bulk polymer products is not limited to adhesive tapes. Further forensically relevant examples which have already been successfully analyzed by the Federal Criminal Police Office will be shown.

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Yiqiao Song

Schlumberger-Doll Research

Cambridge, MA, United States

Pore connectivity by NMR-detected capillary pressure experiment

Porous media is ubiquitous in nature and porous sedimentary rocks are where energy (petroleum), water and minerals are extracted in order to support the contemporary life style of the world. They are complex materials of tortuous pore structures with a wide range of pore sizes and connectivity owing to geological processes. In particular, pore connectivity is critical to fluid flow in soil and rocks, however, our understanding remains simplistic (such as bundle-of-tubes) due to inadequate measurement techniques. This paper reports a method to decipher how pores are connected by measuring a pore size-throat correlation map using an NMR-detected capillary pressure experiment. Our data show that such map can identify a wide variety of pore connectivity types due to diagenesis processes. This method could be useful for petroleum engineering, understanding of diagenesis, soils and contaminant dispersion and the study of other engineering porous materials.

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Debra Sysyn

ExxonMobil Research & Engineering

Annandale, NJ, United States

Two-Dimensional NMR Applications for Structural Elucidation of Complex Petrochemical Mixtures

NMR is one of the best chemical diagnostic tools for the characterization of hydrocarbons. However, a major limitation of the NMR technique is its low sensitivity and difficulty resolving peaks in a mixture. Many advances have been made since its inception to overcome this limitation in both hardware and software; arguably the most important one in recent years is the development of cryogenically-cooled probes, giving up to a 4-fold increase in sensitivity. The increase in sensitivity (and, thus, 16x shorter acquisition time) and resolution at higher fields enables detailed characterization of complex hydrocarbon mixtures greatly enhancing the utility of two-dimensional NMR experiments. Detailed molecular characterization is performed using a variety of homonuclear and heteronuclear 2D NMR experiments that significantly enhance the structural characterization of complex mixtures. A proton-correlated TOCSY (Total Correlation Spectroscopy) experiment allows all the protons in a spin system to be mapped out. A selective TOCSY-HMBC (Total Correlation Spectroscopy - Heteronuclear Multiple Bond Correlation) experiment allows carbon signals of each component in a mixture to be identified. Furthermore, the cryoprobe capability enables advanced experiments such as Diffusion Ordered Spectroscopy (DOSY) to be applied to mixtures with ease. DOSY allows different components in a mixture to be differentiated based on their diffusion coefficients (functions of molecular motions and size of molecule). We are extending the application of DOSY by pairing with a variety of 2D NMR experiments to provide 3D experiments that further enhances the structural elucidation of complex mixtures. This allows for 2D NMR characterization of each component in the mixture without the need for chromatographic separations. An overview of 2D NMR techniques and their utility for characterizing complex petrochemical mixtures will be presented.

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Marc Taraban

University of Maryland, Baltimore

Rockville, MD, United States

1H2O NMR—From Solute Organization to Quality Control in Biomanufacturing

Strong water proton signal, which is often a nuisance in many NMR experiments, could be advantageously used to extract important information about the solute.  Water interactions with solute molecules result in sensitivity of water proton transverse relaxation rate, R2(1H2O), to the organization and/or structural transformations of solute molecules.  In this presentation, we discuss the applications of the analytical technology based on water proton NMR (wNMR) to many different areas, from conformation monitoring to manufacturing and formulation of biopharmaceuticals.

 wNMR was used to monitor conformational transitions of amphiphilic dendrimeric macromolecules mediated by water accessibility of hydrophilic protons.  Water protons are also sensitive to the clustering of nanoparticles.  The diffusive exchange between bulk water and water molecules inside the clusters' compartments affects R2(1H2O) which could be used as an analytical tool for nanoparticle products including drug products.  One of such examples of R2(1H2O) sensitivity is its capability to differentiate brand and generic drugs for iron-deficiency anemia formulated as iron-carbohydrate nanoparticles.  Another application to particulate formulations is the sensitivity of R2(1H2O) to concentration of aluminum hydroxide and aluminum phosphate microparticles used as aluminum adjuvants for many vaccine formulations.  wNMR could be used to access the filling level (concentration), and sedimentation and resuspension of the aluminum adjuvants in a quantitative fashion.  For the freeze-sensitive aluminum-adjuvanted vaccine products, we demonstrated that wNMR accurately detects prior freezing history—R2(1H2O) of the frozen and then thawed vaccines shows significant drop compared to the noncompromised ones.  Of note, all of the above wNMR measurements could be done in situ, noninvasively, in a sealed vials prior to their release or administration to patients.

Importantly, wNMR was found responsive to the changes in protein concentration and aggregate content in biopharmaceutical formulations.  We found that in the absence of aggregates, R2(1H2O) shows linear response to protein concentration.  For model proteins (bovine serum albumin and human IgGs) and for therapeutically-relevant mAb formulations, we found that wNMR is sensitive to the stress-induced formation of both soluble and insoluble protein aggregates within a wide size range, including subvisible particles.  In an exemplary application to the final product, we demonstrated the capability of wNMR to detect uneven filling of the drug substance into the injection devices (insulin pen).  The inspection could be done in a sealed pen, even without taking off a cap.

Since protein concentration and aggregation are important quality attributes of the product flow under continuous biomanufacturing settings, we have used the flow-wNMR to monitor concentration changes and aggregate content.  For the flow rates from 0 to 50 mL/min, we have found R2(1H2O) sensitive to variation of protein concentration and the content of protein aggregates.  As opposed to the existing analytical tools, flow-wNMR allows to analyze the product flow without contact with the product stream.

In conclusion, wNMR implemented using low-filed wide-bore time-domain benchtop NMR instruments has a potential to become a widely applied analytical tool in pharmaceutical industry—from formulation and manufacturing to pre- and post-release quality control of drug products.

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Elwin van der Cruijsen

DSM

Delft, Netherlands

Enzyme kinetics determined by rapid real-time NMR

Determination of enzyme kinetics is a laborious procedure, in which a series of reactions are followed in time by stop reactions and sequentially simple assays readouts to determine the kinetic parameters Vmax and the Km. Enzymatic reactions can also be followed by direct measurements via a spectrophotometer with often the limitation that it requires synthetic labeled substrates and adapted buffers. Via NMR experiments, we can combine the direct detection of the conversion of the natural substrate and product in an environment that mimic the application condition. For these real-time NMR experiments, we have developed a sequence that allows fast repetitive acquisition, below 1 second, this resulted that fast conversions could be followed as well. NMR based monitoring in combination with automation can speed up enzyme characterization and development. As case study, the conversion of asparagine to aspartate by DSM enzyme asparaginase is followed by automated real-time NMR measurements. The ability to determine enzyme kinetics in an automated real-time NMR measurement makes it possible to do a fast screening of enzymes with respect to their performance with better quality.

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Manfred Wilhelm

KIT

Karlsruhe, Ba-Wü, Germany

Low-field RheoNMR

While Rheo-NMR is a long-established method to investigate the flow behaviour of soft matter using high-field NMR (both spectroscopically and via flow imaging), the lack of a possibility for in-line rheological measurement and need for high-end NMR systems using cryomagnets has limited its audience to mostly academic specialists.

The advent of miniaturised Halbach magnets has allowed to integrate a time-domain NMR system  into a commercially available high-end rheometer (Ratzsch, Friedrich, Wilhelm, Journal of Rheology 2017). We would like to present our latest version of this novel instrument combination, and applications for the investigation of polyolefin crystallistion (Räntzsch, Wilhelm et al, Macromolecular Materials and Engineering, 2019) and rubber curing.

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Robert “Thomas” Williamson

University of North Carolina at Wilmington

Wilmington, NC, United States

3D Structure Elucidation of Small Organic Molecules Using Multi-NMR Parameters

The combination of traditional NMR parameters such as NOE-derived distances, 3J scalar couplings and anisotropic NMR parameters (Residual Dipolar Couplings and Residual Chemical Shift Anisotropy), [1,2] allows the determination of the relative configuration together with the conformational preference of natural and synthetic organic compound with a high degree of confidence. In particular, the recently proposed CASE-3D (Computer Assisted 3D Structure Elucidation) methodology [3,4] includes the use of molecular modeling, quantum mechanical prediction of NMR parameters and the filtering of conformational models using statistical criteria. The strategies and protocols necessary for the use of this multi-parametric methodology will be discussed, as well as the validity of the different approaches and the use of the necessary software tools. Detailed application of (i) J-based analysis, (ii) use of stretched and compressed aligning gels, and lyotropic liquid crystals (LLC) solutions (PBLG) to measure anisotropic parameters, [1,5] and (ii) data analysis for determination of configuration/conformation [4] will be presented. - Overview, measurement, calculation (DFT), and application of various J-based parameters (3JHH, 1JCH, nJCH, 1JCC, nJCC) - Kishi's NMR database - Bioinformatics as a tool for NMR studies of natural products 1. Liu, Y., Navarro-Vazquez, A., Gil, R.R.; Griesinger, C.; Martin, G.E.; Williamson, R.T. Nature Protocols 2019, 14, 217-247. 2. Nath, N., Schmidt, M., Gil, R.R., Williamson, R. T., Martin, G.E., Navarro-Vazquez, A., Griesinger, C., Liu, Y. J. Am. Chem. Soc. 2016, 138, 9548-9556. 3. Troche-Pesqueira, E.; Anklin, C.; Gil, R. R.; Navarro-Vằuez, A.; Angew. Chem. Int. Ed. 2017, 56, 3660. 4. Navarro-Vằuez, A.; Gil, R. R.; Blinov, K.; J. Nat. Prod. 2018, 81, 203. 5. Ndukwe, I.E.; Wang, X.; Pelczer, I.; Reibarkh, M.; Williamson, R. T.; Liu, Y.; Martin, G.E. Chem. Comm. 2019, 55, 4327-4330.

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