根据DE-AC05-RL01830和FWP-47319合同，美国能源部、科学办公室、基础能源科学办公室、化学科学、生物科学和地球科学催化计划支持对催化剂应用的审查。国家卫生研究所、国家环境卫生科学研究所根据R21ES029778赠款支持对生物医学应用的审查。实验在EMSL（网格.436923.9）进行，EMSL是能源部科学用户设施办公室，由生物和环境研究办公室赞助，位于太平洋西北国家实验室（PNNL）。PNNL 是巴特尔根据 DE-AC05-RL01830 和 FWP-47319 合同为美国能源部运营的多项目国家实验室。

协议分为四个部分，其中规定1）制备系统中使用的任何固体材料或激活或清除不受欢迎的受体物种，2）在NMR转子中添加固体和液体材料，3）在转子中添加气体，4）在光谱仪中进行NMR实验。该过程代表一个典型的序列，但可以修改以适应实验的具体需要。
1. 预处理固体样品
<ol><li>重量大约是 NMR 实验所需的固体样品质量的两倍（对于 7.5 毫米转子，~250 毫克），并将固体样品放入用于处理炉系统材料的石英样品管中，用石英羊毛堵塞管子以保持材料到位。</li>
	<li>通过将管子放入冷却炉并拧紧连接，将隔离阀连接到固体处理 1） 流或 2） 真空系统。</li>
	<li>将石英管端贴在开阔位置的气体隔离阀上。</li>
	<li>开始治疗。<ol>
<li>对于流系统:<ol>
<li>将热电偶贴在管子外部，用耐热材料将其固定到位。</li>
			<li>开始处理气体的流动（例如，N 2 在 100 sccm），以清除固体表面或激活材料。</li>
		</ol>
</li>
		<li>或者，对于真空系统:<ol>
<li>关闭真空系统的隔离阀并启动真空泵。</li>
			<li>当完全真空建立时，非常缓慢地打开隔离阀将真空涂抹在样品上，定期暂停，使系统保持平衡。继续，直到阀门打开。</li>
		</ol>
</li>
	</ol>
</li>
	<li>打开炉子控制器，将温度坡道程序设置为所需的条件（例如，以 5 °C/min 的坡道速率在 4 小时内 300 °C）。</li>
	<li>启动温度程序并让它运行。</li>
	<li>完成后，允许样品冷却到可行的温度。</li>
	<li>关闭温度控制器并停止流量/真空。</li>
	<li>使用隔离阀快速密封样品，以保持所需的样品环境。</li>
	<li>将石英管与处理系统断开，并将管子和封闭的阀门转移到干燥的 N2净化手套箱的安泰坎伯。</li>
	<li>清空并重新填充安泰坎伯至少4次，并将管子转移到手套箱内。</li>
</ol>2. 将固体样品加载到 NMR 转子中
<ol><li>用转子盖称空和干净的高压、高温 NMR 转子。</li>
	<li>将 NMR 转子放在支架中以保持方向性。</li>
	<li>将样品漏斗放入转子孔中。</li>
	<li>从样品管中取出隔离阀，将少量固体材料倒入漏斗中。</li>
	<li>将粉末向下敲入漏斗中，必要时用包装棒轻轻引导粉末进入转子。</li>
	<li>重复逐步添加固体材料，直到达到所需的数量（例如 1/2 转子）。</li>
	<li>将 NMR 转子（和盖子）与样品进行称重，以确定添加的样本数量。</li>
	<li>如果需要，绘制任何液体样品的指定数量，然后用微型注射器缓慢地将液体注入 NMR 转子的中心。</li>
	<li>将盖子放在顶部并逆时针与转子帽位对接以接合转子，使转子和盖之间的 O 形环接合，从而密封转子。请注意，可能需要定期使用新的 O 环以防止泄漏，特别是使用化学磨蚀性混合物或氢气等小气体时。</li>
	<li>称量 NMR 转子以确定添加样本的总质量。</li>
</ol>3. 在所需条件下向 NMR 转子充电，使用所需的化学品
<ol><li>将密封的 NMR 转子放入转子阶段，确保舞台插入的大小与转子大小兼容，并用手拧紧螺母以固定到位。请注意，在此步骤中，支架中转子的紧度将决定盖封的紧度。</li>
	<li>将转子阶段降低到高压暴露装置的下部。</li>
	<li>使用 Allen 扳手将其中一个螺钉 90° 将转子阶段固定到暴露设备的底部。</li>
	<li>将 NMR 加载设备的顶部部分放入底部部分并放在底部部分的顶部，将 NMR 盖位排列到 NMR 转子的盖头顶部，以确保其接合。</li>
	<li>将 2 个夹子放在唇部顶部，暴露装置的上部和下部在上面和下部相遇并将其锁定到位。</li>
	<li>拧紧暴露装置上部顶部的 6 个螺栓，使上部和下部之间的密封表面接合。</li>
	<li>将 NMR 暴露装置的上部连接到煤气管入口和插座。</li>
	<li>将 NMR 暴露装置上部的热电偶连接到温度传感器。</li>
	<li>如有需要，将加热胶带缠绕在燃气管线和暴露装置的上部，以便与相应的控制器进行加热。热板也可以接合。</li>
	<li>确保暴露室插座打开并关闭气源阀，打开真空泵以清除暴露装置和相关线路中的空气。</li>
	<li>用所需的气体或惰性气体清除线路，在真空和大气压力之间循环三次，以确保线路清除空气。</li>
	<li>从 1） 高压输送系统或 2） 流动系统中准备所需的气体组成，以便在指定压力下引入蒸汽。<ol>
<li>用于高压或真空样品制备:<ol>
<li>关闭暴露装置气体出口，设置气体歧管阀，绕过液体喷射管。</li>
			<li>在高压输送系统的高压注射器泵上设置所需的压力。</li>
			<li>打开高压注射器泵上的气源阀门，运行泵上设置的程序，监控暴露装置内部的实际压力。</li>
			<li>当暴露装置内达到所需的压力时，请停止注射器泵并关闭源气阀。</li>
			<li>通过顺时针旋转外部螺杆机制打开 NMR 转子，该机制与内部 NMR 盖位耦合。</li>
			<li>允许所需压力的气体进入 NMR 转子并平衡。</li>
			<li>通过逆时针旋转外部螺杆机制重新塞入 NMR 转子。查看窗口将帮助确定转子何时关闭。</li>
			<li>通过打开暴露装置气体出口阀，缓慢地压低系统。</li>
		</ol>
</li>
		<li>用于流动气体或蒸汽样本制备:<ol>
<li>确保暴露装置气体出口打开，防止压力过大。</li>
			<li>在质量流量控制器上设置所需的气体流速，并开始气流。</li>
			<li>将液体供应线从液体注射器泵连接到气体歧管。</li>
			<li>设置气体歧管阀，使液体注入线能够流动。</li>
			<li>设置液体注射器泵上的液体流速，以达到所需的蒸汽压力，并开始液体注入。</li>
			<li>通过顺时针旋转与内部 NMR 盖位耦合的外部螺钉机制打开 NMR 转子。</li>
			<li>允许系统平衡到 NMR 转子内部所需的气体压力，并通过逆时针旋转外部螺杆机制重新塞入 NMR 转子。查看窗口将帮助确定转子何时关闭。</li>
			<li>停止液体注射器泵喷射并配置阀门以绕过液体喷射管，使泵与系统断开连接。</li>
			<li>停止流动的气体。</li>
		</ol>
</li>
	</ol></li>
	<li>用惰性气体清除系统，以清除任何潜在的有毒或易燃气体。</li>
	<li>停止任何加热，让系统冷却。</li>
	<li>断开任何加热胶带和热电偶。</li>
	<li>断开输气管和出口煤气管道。</li>
	<li>松开暴露装置顶部的 6 个螺栓，以损坏密封件。</li>
	<li>拆开 2 个夹紧部分，并将它们从曝光设备中删除。</li>
	<li>小心地向上和向下抬起上部。</li>
	<li>使用艾伦扳手松开转子阶段，并用螺纹杆绘制。</li>
	<li>松开转子阶段上的螺母，从设备组件中取下转子。</li>
	<li>称量转子，以确保所需的气体量。</li>
</ol>4. 进行 MAS NMR 实验
<ol><li>将 NMR 转子放入 NMR 探头上的 NMR 线圈中。</li>
	<li>将探头举到磁铁孔中并锁定到位。</li>
	<li>使用 MAS 控制框启动样品旋转，并调整到所需的转子旋转速率。</li>
	<li>使用计算机在所需的通道上开始调整/匹配序列。</li>
	<li>调整探头上的调谐/匹配设置，以优化探头电子设备。</li>
	<li>退出计算机上的调谐/匹配序列，并设置所需的实验参数（如脉冲序列、实验阵列、温度等）。</li>
	<li>收集 MAS NMR 数据。</li>
</ol>

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<li>Hu, J. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+large+sample+volume+magic+angle+spinning+nuclear+magnetic+resonance+probe+for+in+situ+investigations+with+constant+flow+of+reactants%5BTitle%5D)+AND+%22Physical+Chemistry+Chemical+Physics%22%5BJournal%5D)">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/pubmed?term=((In+situ+MAS+NMR-UV%2FVis+investigation+of+H-SAPO-34+catalysts+partially+coked+in+the+methanol-to-olefin+conversion+under+continuous-flow+conditions+and+of+their+regeneration%5BTitle%5D)+AND+%22Microporous+and+Mesoporous+Materials%22%5BJournal%5D)">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/pubmed?term=((Enhanced+In+situ+Continuous-Flow+MAS+NMR+for+Reaction+Kinetics+in+the+Nanocages%5BTitle%5D)+AND+%22Journal+of+the+American+Chemical+Society%22%5BJournal%5D)">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/pubmed?term=((In+situ+Al-27+NMR+Spectroscopy+of+Aluminate+in+Sodium+Hydroxide+Solutions+above+and+below+Saturation+with+Respect+to+Gibbsite%5BTitle%5D)+AND+%22Inorganic+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Boehmite+and+Gibbsite+Nanoplates+for+the+Synthesis+of+Advanced+Alumina+Products%5BTitle%5D)+AND+%22ACS+Applied+Nano+Materials%22%5BJournal%5D)">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/pubmed?term=((Transformation+of+Gibbsite+to+Boehmite+in+Caustic+Aqueous+Solution+at+Hydrothermal+Conditions%5BTitle%5D)+AND+%22Crystal+Growth+%26+Design%22%5BJournal%5D)">Transformation of Gibbsite to Boehmite in Caustic Aqueous Solution at Hydrothermal Conditions.</a> Crystal Growth & Design. 19 (10), 5557-5567 (2019).</li>
<li>Hu, J. Z., Jaegers, N. R., Hu, M. Y., Mueller, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+situ+and+ex+situ+NMR+for+battery+research%5BTitle%5D)+AND+%22Journal+of+Physics%3A+Condensed+Matter%22%5BJournal%5D)">In situ and ex situ NMR for battery research.</a> Journal of Physics: Condensed Matter. 30 (46), (2018).</li>
<li>Hu, J. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adsorption+and+Thermal+Decomposition+of+Electrolytes+on+Nanometer+Magnesium+Oxide%3A+An+in+situ+C-13+MAS+NMR+Study%5BTitle%5D)+AND+%22ACS+Applied+Materials+%26+Interfaces%22%5BJournal%5D)">Adsorption and Thermal Decomposition of Electrolytes on Nanometer Magnesium Oxide: An in situ C-13 MAS NMR Study.</a> ACS Applied Materials & Interfaces. 11 (42), 38689-38696 (2019).</li>
</ol>

作者宣布以下相互竞争的经济利益。J.Z.H和他的同事拥有转子设计（US9151813B2）的专利。J.Z.H.、N.R.J.等人已经就暴露装置提出了临时专利申请。

此处概述的 MAS NMR 光谱测量方法代表了进行高温、高压 MAS NMR 的最先进的技术状态。这些方法能够以可靠、可重复的方式观察在真空大气中发生的相互作用，以及从低温（远低于0°C至250°C）。在灵活的化学环境中探测含有固体、液体和气体混合物的系统的能力，使各种兴趣的实验得以进行。
虽然以前的许多努力都集中在低磁场（300 MHz）中相对较大的（7.5毫米）NMR转子的利用...

样品光谱分析是一种分析工具，用于获取有关感兴趣的材料（如化学状态、结构或反应性）的宝贵信息。从简单的角度来看，核磁共振（NMR）是利用强磁场操纵原子核自旋状态以更好地了解感兴趣物种的化学环境的技术之一。核自旋状态是指由旋转核运动引起的磁力矩的相对方向，这是一种带正电荷的粒子。在没有磁场的情况下，核自旋是随机方向的，但在磁场存在的情况下，核自旋优先与处于低能量自旋状态的磁体外部磁场对齐。将自旋状态拆分为离散能量值称为 Zeeman 效应。这些能量水平（ΔE）之间的差额按方程 1 建模: <img alt="Equation 1" src="/files/ftp_upload/61794/61794equ01.jpg"> h是浮力的常数，B0 是外部磁场的强度，γ是原子核的陀螺磁比。这些自旋的化学环境也对这些能量水平施加轻微的扰动。相应频率的无线电波可用于激发核，随着纵向磁化（基于平行和反平行状态的自旋群）的减少，由于自旋获得相干性，核产生横向磁化。当原子核继续在磁场轴上预切时，旋转磁运动产生一个磁场，该磁场也在旋转并产生电场。该字段调节 NMR 检测线圈中的电子，生成 NMR 信号。样品中核化学环境的微小差异会影响线圈中检测到的频率。
对固体样品的NMR分析引入了流体中未发现的复杂性。在流体中，分子以快速的速度下降，平均原子核周围的化学环境。在固体样品中，没有出现这种平均效应，在NMR信号中引入了方向依赖的化学环境和宽广的光谱线。为了减轻这些挑战，一种称为魔角旋转（MAS）的技术被采用1，2。在 MAS NMR 中，样品在 54.7356° 的角度快速旋转（几千赫），外部磁场使用外部旋转机制处理 NMR 的定向依赖（非热带）相互作用。这大大缩小了 NMR 特征，并通过平均化学移位单极、二极相互作用和四极相互作用的定向依赖条件来增强光谱分辨率。两个值得注意的例外确实妨碍了MAS NMR的线路缩小能力。第一种是强同核耦合，有时存在于1H NMR 中，需要高旋转速度 （~70 kHz） 才能去除。然而，高温应用温度的显著升高将通过传授增强的热运动来大大抑制1H同核相互作用，从而大大降低样品旋转率，从而显著提高光谱分辨率。此外，随着技术的不断发展，现在可以制造直径较小的转子，以达到远超5 kHz的旋转速率，这有助于进一步抑制1H同核二极相互作用。第二个例外是自旋超过一半的核的残余二阶四极相互作用，因为只有第一个顺序术语在神奇的角度被消除，留下更复杂的线形，只有更强的外部磁场才能改进。需要强调的是，2D MQMAS技术可以轻易地融入到目前的技术中，从而以与标准MQMAS实验3类似的方式获得真正的同位素化学移位光谱。
MAS NMR 实现了固体材料的详细定性，加强了观测质量。然而，在NMR转子（样品持有人）中以高速率旋转样品的必要性，也给在高温和压力下进行实验带来了挑战，这些实验可能与感兴趣的条件更相关。有时，在对NMR转子相对苛刻的条件下检查材料是可取的。多项工作已成功改造液态NMR技术，进行高温、高压NMR 4、5、6、7:但是，用于固态 MAS NMR 的商用转子盖可能会在高压下从转子中排出，对设备造成重大损坏。通过检查大大增加样品持有人压力的分解反应，可以加剧这种影响。因此，需要新的设计来有效和安全地进行原位NMR实验。例如，转子必须坚持多种品质才能在 MAS NMR 中有效使用，即非磁性、轻量级、耐用性、耐温性、低 NMR 背景材料、可密封性、高强度和耐化学性。转子必须承受的压力相当大。转子不仅要承受样品中所含的压力（例如高压气体），而且设备的旋转还传递离心力，这种离心力对系统总压力有其自身的贡献8，PT，方程 2: <img alt="Equation 2" src="/files/ftp_upload/61794/61794equ02.jpg"> RI 和RO 分别是内转子和外转子半径，ω是每秒半径的旋转频率，P是 样品压力。
已制定多项策略，以解决这些问题。早期的例子类似于火焰密封管10，11，12或聚合物插入13，14，这不足以在高温和压力下延长，精细控制操作。转子设计的迭代由于使用环氧树脂或陶瓷插入物8、15、16的样品体积减少而受制于最高工作温度的限制。最近的一项技术通过在商用转子套管中采用简单的捕捉功能来降低单位生产成本，但相对较少地控制其可操作17的条件。这里采用的设计是一个全氧化锌，洞穴风格的转子袖子磨与螺纹顶部18。盖子也螺纹，以允许安全密封。反向螺纹可防止样品旋转松开氧化锌帽，O 形环构成密封表面。这种转子设计可见图1和类似的转子和说明，使他们已获得专利19。这种策略具有较高的机械强度、耐化学性和耐温性。
这些设计适用于温度和压力至少 250 °C 和 100 bar，受现成的 NMR 探针技术限制温度。与专业的样品制备设备相结合后，它代表了一种真正强大的技术，用于碳固存、催化、储能和生物医学20等影响深远的应用。此类设备包括一种预处理固体材料的方法，以去除不需要的表面物种，如水。这一步骤通常使用熔炉。干盒通常用于将固体样品加载到 NMR 转子中。从那里，转子被转移到一个曝光装置，使转子可以在严格控制的大气层下打开，将所需的气体或混合物装载到转子中。图 2中描绘了这样的设备。

<table><tbody><tr><td>1) Preparation of Solids Samples</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>Tube Furnace</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>Quartz Tube</td><td></td><td></td><td></td></tr><tr><td>Isolation Valves</td><td></td><td></td><td></td></tr><tr><td>Quartz Wool</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>2) Loading solid samples into the rotor</td><td></td><td></td><td></td></tr><tr><td>Dry glove box</td><td></td><td></td><td></td></tr><tr><td>High-temperature, high-pressure NMR rotor</td><td></td><td></td><td></td></tr><tr><td>Sample funnel</td><td></td><td></td><td></td></tr><tr><td>Sample packing rod</td><td></td><td></td><td></td></tr><tr><td>Rotor holder</td><td></td><td></td><td></td></tr><tr><td>Analytical Balance</td><td></td><td></td><td></td></tr><tr><td>Microsyringe</td><td></td><td></td><td></td></tr><tr><td>Rotor cap bit</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>3) Addition of gases to the rotor</td><td></td><td></td><td></td></tr><tr><td>NMR loading chamber</td><td></td><td></td><td></td></tr><tr><td>Rotor stage and appropriately sized inserts</td><td></td><td></td><td></td></tr><tr><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

核磁共振（NMR）光谱是了解分子结构和粘合环境的重要技术。存在一种驱动，即在与感兴趣的化学过程相关的条件下对材料进行定性。为了解决这个问题，已经开发了原位高温、高压 MAS NMR 方法，以便能够观察一系列压力（真空到几百柱）和温度（远低于 0 °C 至 250 °C）的化学相互作用。此外，样品的化学标识可以由固体、液体、气体或三者的混合物组成。该方法集成了全硅NMR转子（MAS NMR的样品持有人），可以使用螺纹帽密封以压缩O环。该转子具有极大的耐化学性、温度兼容性、低NMR背景，可承受高压。这些综合因素使它能够在广泛的系统组合中加以利用，这反过来又允许它在碳固存、催化、材料科学、地球化学和生物学等不同领域使用。这项技术的灵活性使它成为来自众多学科的科学家的一个有吸引力的选择。

NMR 光谱仪的输出采用自由感应衰减 （FID） 的形式，即兴奋旋转在放松回到热力学平衡时发出的时间域信号。这样的FID类似于图3。当 Fourier 从时间域转换为频率域（频率到 PPM 的方程 3，其中差绝对频率和参考值除以 NMR 光谱仪的载体频率）时，它表示 NMR 频谱，每个峰值表示在独特的化学环境中的核（图 3）。 <img alt="Equation 3" src="/files/ftp_upload/61794/6...

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

固体、液体、气体和混合物的分子结构和动力学对不同的科学领域至关重要。高温、高压原位MAS NMR使在严格控制的化学环境中，能够检测混合相系统中成分的化学环境。

高温高压原位魔角旋转核磁共振光谱

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

high-temperature-high-pressure-situ-magic-angle-spinning-nuclear

Research

Education

JoVE Core

JoVE Journal

Analytical Chemistry

Chemistry

Unit 1

Interpreting Nuclear Magnetic Resonance Spectra

SCIENTISTS IN ACTION

5.5K 次观看.  Pacific Northwest National Laboratory. 固体、液体、气体和混合物的分子结构和动力学对不同的科学领域至关重要。高温、高压原位MAS NMR使在严格控制的化学环境中，能够检测混合相系统中成分的化学环境。

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

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

固体核磁共振波谱法研究高分子组件的原子尺度结构

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

科研

生物化学

High-Sensitivity Nuclear Magnetic Resonance at Giga-Pascal Pressures: A New Tool for Probing Electronic and Chemical Properties of Condensed Matter under Extreme Conditions

Nuclear Magnetic Resonance (NMR) is one of the most important techniques for the study of condensed matter systems, their chemical structure, and their electronic properties. The application of high pressure enables one to synthesize new materials, but the response of known materials to high pressure is a very useful tool for studying their electronic structure and developing theories. For example, high-pressure synthesis might be at the origin of life; and understanding the behavior of small molecules under extreme pressure will tell us more about fundamental processes in our universe. It is no wonder that there has always been great interest in having NMR available at high pressures. Unfortunately, the desired pressures are often well into the Giga-Pascal (GPa) range and require special anvil cell devices where only very small, secluded volumes are available. This has restricted the use of NMR almost entirely in the past, and only recently, a new approach to high-sensitivity GPa NMR, which has a resonating micro-coil inside the sample chamber, was put forward. This approach enables us to achieve high sensitivity with experiments that bring the power of NMR to Giga-Pascal pressure condensed matter research. First applications, the detection of a topological electronic transition in ordinary aluminum metal and the closing of the pseudo-gap in high-temperature superconductivity, show the power of such an approach. Meanwhile, the range of achievable pressures was increased tremendously with a new generation of anvil cells (up to 10.1 GPa), that fit standard-bore NMR magnets. This approach might become a new, important tool for the investigation of many condensed matter systems, in chemistry, geochemistry, and in physics, since we can now watch structural changes with the eyes of a very versatile probe.

Nuclear magnetic resonance is one of the most important spectroscopic tools. Here, the development of a new approach under high pressure, currently up to 10.1 GPa, is presented. This opens a new window into condensed matter physics and chemistry, where high-pressure research is of great importance.

高灵敏度核磁共振的千兆帕斯卡压力:一种新的工具，用于极端条件下探测电子和化工凝聚态的性质

Nuclear Magnetic Resonance (NMR) is one of the most important techniques for the study of condensed matter systems, their chemical structure, and their ...

工程学

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer. The high NMR signal obtained can be used to monitor chemical reactions in real time. The downside of hyperpolarized NMR resides in the limited time window available for signal acquisition, which is usually on the order of the nuclear spin longitudinal relaxation time constant, T1, or, in favorable cases, on the order of the relaxation time constant associated with the singlet-state of coupled nuclei, TLLS. Cellular uptake of endogenous molecules and metabolic rates can provide essential information on tumor development and drug response. Numerous previous hyperpolarized NMR studies have demonstrated the relevancy of pyruvate as a metabolic substrate for monitoring enzymatic activity in vivo. This work provides a detailed description of the experimental setup and methods required for the study of enzymatic reactions, in particular the pyruvate-to-lactate conversion rate in presence of lactate dehydrogenase (LDH), by hyperpolarized NMR.

The sensitivity enhancement provided by dissolution dynamic nuclear polarization (DNP) enables following metabolic processes in real time by NMR and MRI. The characteristics and performances of a dedicated dissolution DNP setup designed for study enzymatic reactions are discussed.

溶出动态核极化仪器用NMR实时酶反应速率测量

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of ...

化学

Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR and sensitivity-enhanced DNP experiments. The sample processing procedure of plant biomass is also detailed. This method allows the measurement of a series of 1D and 2D 13C-13C/15N correlations spectra, which enables high-resolution structural elucidation of complex biomaterials in their native state, with minimal perturbation. The isotope-labeling can be examined by quantifying the intensity in 1D spectra and the polarization transfer efficiency in 2D correlation spectra. The success of dynamic nuclear polarization (DNP) sample preparation can be evaluated by the sensitivity enhancement factor. Further experiments examining the structural aspects of the polysaccharides and proteins will lead to a model of the three-dimensional architecture. These methods can be modified and adapted to investigate a wide range of carbohydrate-rich materials, including the natural cell walls of plants, fungi, algae and bacteria, as well as synthesized or designed carbohydrate polymers and their complex with other molecules.

A protocol for preparing 13C,15N-labeled fungal and plant samples for multidimensional solid-state NMR spectroscopy and dynamic nuclear polarization (DNP) investigations is presented.

动态核极化固态核磁共振法制备结构澄清用真菌和植物材料

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR ...

Methods of Ex Situ and In Situ Investigations of Structural Transformations: The Case of Crystallization of Metallic Glasses

We demonstrate the use of two nuclear-based analytical methods that can follow the modifications of microstructural arrangement of iron-based metallic glasses (MGs). Despite their amorphous nature, the identification of hyperfine interactions unveils faint structural modifications. For this purpose, we have employed two techniques that utilize nuclear resonance among nuclear levels of a stable 57Fe isotope, namely M&#246;ssbauer spectrometry and nuclear forward scattering (NFS) of synchrotron radiation. The effects of heat treatment upon (Fe2.85Co1)77Mo8Cu1B14 MG are discussed using the results of ex situ and in situ experiments, respectively. As both methods are sensitive to hyperfine interactions, information on structural arrangement as well as on magnetic microstructure is readily available. M&#246;ssbauer spectrometry performed ex situ describes how the structural arrangement and magnetic microstructure appears at room temperature after the annealing under certain conditions (temperature, time), and thus this technique inspects steady states. On the other hand, NFS data are recorded in situ during dynamically changing temperature and NFS examines transient states. The use of both techniques provides complementary information. In general, they can be applied to any suitable system in which it is important to know its steady state but also transient states.

Methods of Ex Situ and In Situ Investigations of Structural Transformations: The Case of Crystallization of Metallic Glasses

Here, we present a protocol to describe ex situ and in situ investigations of structural transformations in metallic glasses. We employed nuclear-based analytical methods which inspect hyperfine interactions. We demonstrate the applicability of M&#246;ssbauer spectrometry and nuclear forward scattering of synchrotron radiation during temperature-driven experiments.

结构转换的原位和原位研究方法: 金属玻璃结晶的实例

We demonstrate the use of two nuclear-based analytical methods that can follow the modifications of microstructural arrangement of iron-based metallic ...

High-Pressure NMR Experiments for Detecting Protein Low-Lying Conformational States

High-pressure is a well-known perturbation method that can be used to destabilize globular proteins and dissociate protein complexes in a reversible manner. Hydrostatic pressure drives thermodynamical equilibria toward the state(s) with the lower molar volume. Increasing pressure offers, therefore, the opportunities to finely tune the stability of globular proteins and the oligomerization equilibria of protein complexes. High-pressure NMR experiments allow a detailed characterization of the factors governing the stability of globular proteins, their folding mechanisms, and oligomerization mechanisms by combining the fine stability tuning ability of pressure perturbation and the site resolution offered by solution NMR spectroscopy. Here we present a protocol to probe the local folding stability of a protein via a set of 2D 1H-15N experiments recorded from 1 bar to 2.5 kbar. The steps required for the acquisition and analysis of such experiments are illustrated with data acquired on the RRM2 domain of hnRNPA1.

We provide a detailed description of the steps required to assemble a high-pressure cell, set up and record high-pressure NMR experiments, and finally analyze both peak intensity and chemical shift changes under pressure. These experiments can provide valuable insights into the folding pathways and structural stability of proteins.

检测蛋白质低洼构象状态的高压NMR实验

High-pressure is a well-known perturbation method that can be used to destabilize globular proteins and dissociate protein complexes in a reversible ...

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-(1H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

<meta charset="utf8"></head><body>通过固体 NMR 对天然膜中的蛋白质进行高分辨率研究

This work details robust basic routines on how to prepare isotope-labeled membrane protein samples and analyze them at high-resolution with modern solid-state NMR spectroscopy methods.

通过固体 NMR 对天然膜中的蛋白质进行高分辨率研究

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy ...

<meta charset="utf8"></head><body>蛋白质动力学研究中 NMR 15N 弛豫和 hetNOE 实验的方案

NMR 15N Relaxation Experiments for the Investigation of Picosecond to Nanoseconds Structural Dynamics of Proteins

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide groups of the protein backbone or the methyl groups in side chains are used as reporters of structural dynamics in proteins. A structural dynamics study of the protein backbone of globular proteins on 15N labeled and fully protonated samples usually works well for proteins with a molecular weight of up to 50 kDa. When side chain deuteration in combination with transverse relaxation optimized spectroscopy (TROSY) is applied, this limit can be extended up to 200 kDa for globular proteins and up to 1 MDa when the focus is on the side chains. When intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs) are investigated, these weight limitations do not apply but can go well beyond. The reason is that IDPs or IDRs, characterized by high internal flexibility, are frequently dynamically decoupled. Various NMR methods offer atomic-resolution insights into structural protein dynamics across a wide range of time scales, from picoseconds up to hours. Standard 15N relaxation measurements overview a protein&#39;s internal flexibility and characterize the protein backbone dynamics experienced on the fast pico- to nanosecond timescale. This article presents a hands-on protocol for setting up and recording NMR 15N R1, R2, and heteronuclear Overhauser effect (hetNOE) experiments. We show exemplary data and explain how to interpret them simply qualitatively before any more sophisticated analysis.

<meta charset="utf8"></head><body>作者聚焦：通过 NMR 弛豫实验探索内在无序的蛋白质动力学

Nuclear magnetic resonance (NMR) spectroscopy can characterize structural protein dynamics in a residue-specific manner. We provide a hands-on protocol for recording NMR 15N R1 and R2 relaxation and {1H}-15N heteronuclear Overhauser effect (hetNOE) experiments, sensitive to the picoseconds to nanoseconds timescale.

NMR 15N 弛豫实验，用于研究蛋白质皮秒至纳秒的结构动力学

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide ...

高温高压原位魔角旋转核磁共振光谱

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NMR ¹⁵N 弛豫实验，用于研究蛋白质皮秒至纳秒的结构动力学

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