NMR Sample Preparation | 核磁样品制备标准操作

1. 样品准备与浓度

• 使用优质 5 mm 核磁管，控制氘代溶剂体积在 0.5–0.6 mL（液面高度约 4–5 cm）。
• 氢谱 (¹H NMR)：纯净样品用量通常为 2–10 mg。避免浓度过高导致匀场困难及峰形变宽。
• 碳谱 (¹³C NMR)：由于碳的天然丰度低，样品用量建议在 10–50 mg。
• 配样时，必须在小瓶中完全溶解后再转移至核磁管中，严禁在核磁管内直接混合。

2. 浑浊体系过滤

• 核磁样品必须是完全均相的透明溶液，固体悬浮颗粒会严重破坏磁场均匀性。
• 若样品溶解后出现浑浊或有微量不溶物，严禁直接上机。
• 过滤操作：在短巴斯德吸管底部塞入一小团脱脂棉或玻璃棉，将样品溶液通过该简易过滤器缓慢滤入核磁管中。

3. 定量核磁操作 (qNMR)

• 内标选择：在惰性、不反应的前提下，精确称量已知质量和纯度的内标物。确保内标物的特征峰与目标产物峰完全不重叠。
• 样品与内标物必须完全溶解于选定的氘代溶剂中，保证体系绝对均匀。
• 参数设置：为了获得准确的积分面积比，必须设置充足的弛豫延迟时间 (D1)。通常需要将 D1 设定为体系中最慢质子 T₁ 弛豫时间的 5 倍以上。

4. 上机与维护

• 将核磁管插入转子（Spinner），并使用**深度规（Depth Gauge）**严格校准样品高度。
• 永远不要将核磁管推至超过深度规所示的最大深度，否则进入仪器时可能会撞碎并损坏探头。
• 进样前，必须使用擦镜纸（Kimwipe）将核磁管外壁及转子彻底擦拭干净，去除指纹与污渍。

1. Sample Preparation and Concentration

• Use high-quality 5 mm NMR tubes, and control the deuterated solvent volume to 0.5–0.6 mL (sample depth approx. 4–5 cm).
• Proton NMR (¹H NMR): The typical amount of pure sample is 2–10 mg. Avoid over-concentration, which causes shimming difficulties and line broadening.
• Carbon NMR (¹³C NMR): Due to low natural abundance, a sample amount of 10–50 mg is recommended.
• Always prepare the sample in a vial first and ensure it is fully dissolved before transferring it into the NMR tube. Never mix components directly inside the NMR tube.

2. Filtration of Turbid Samples

• The NMR sample must be a completely homogeneous, clear solution. Suspended solid particles will severely distort the magnetic field homogeneity.
• If the solution appears turbid or contains insoluble matter, never insert it directly into the instrument.
• Filtration Procedure: Insert a small plug of cotton wool or glass wool into a short Pasteur pipette, and filter the dissolved sample through this setup directly into the NMR tube.

3. Quantitative NMR Procedure (qNMR)

• Internal Standard: Accurately weigh a known amount of an internal standard that is chemically inert and highly pure. Ensure its signals do not overlap with the analyte peaks.
• Both the analyte and the standard must be completely dissolved in the deuterated solvent to ensure a perfectly homogeneous mixture.
• Parameter Acquisition: For accurate peak integration, an adequate relaxation delay (D1) must be set. Typically, D1 should be at least 5 times the longest T₁ relaxation time in the system.

4. Insertion and Maintenance

• Insert the NMR tube into the spinner, and strictly adjust the sample height using the depth gauge.
• Never push the tube below the maximum depth indicated on the depth gauge, as it could hit and break the probe inside the magnet.
• Before inserting the tube into the spectrometer, thoroughly wipe the outside of the tube and the spinner with a Kimwipe to remove fingerprints or contamination.

Ref: Yale Chemical and Biophysical Instrumentation Center (CBIC). Basic NMR Sample Preparation.