快速柱色谱纯化操作 | Flash Column Chromatography

1. 安全规范

• 注意： 硅胶和氧化铝粉末吸入后对呼吸道有毒性——所有称量和装柱操作须在通风橱内进行。
• 废弃硅胶应倒入指定废固容器；收集全部目标组分后方可处理废弃组分，切勿在确认目标产品已回收前丢弃任何馏分。

2. 溶剂体系的选择（TLC 预实验）

• 配制少量待分离样品溶液，在不同溶剂体系下点板，筛选合适的洗脱体系。TLC 板所用固定相须与色谱柱保持一致（硅胶或氧化铝）。
• 调节溶剂极性，使目标组分的 Rf ≈ 0.3（此值在分离速度与分辨率之间取得最佳平衡——Rf 过低意味着极性不足，洗脱时间过长；Rf 过高则极性过强，组分过早洗出、分离度差）。
  • 分离两个及以上组分时，以各组分 Rf 的中点位于 0.3 为目标调节溶剂极性。
  • 若各组分极性相差悬殊，可先以使极性最低的组分 Rf ≈ 0.3 的溶剂体系开始洗脱，待该组分完全洗出后，再切换至使次极性组分 Rf ≈ 0.3 的溶剂体系继续洗脱（等度切换，非梯度洗脱）。
  • 常用溶剂体系：乙酸乙酯/石油醚适用于大多数化合物；甲醇/二氯甲烷适用于高极性化合物；乙醚/戊烷适用于需低沸点溶剂的挥发性化合物。
• 注意（溶剂极性调节）： 若目标组分仅需极低比例极性溶剂（如 ≤ 2% 乙酸乙酯）即可达到 Rf = 0.3，应进一步降低极性（如使用 0.5% 乙酸乙酯），以避免柱上过快洗脱。

3. 柱型与硅胶用量的选择

• 根据样品规模选择柱径与收集每份馏分体积，可以参照下表的推荐值。该表改编自Zakarian Group教程，结合了实验室常用的柱色谱的内径大小，对不同内径的柱子所适用的样品量与馏分体积进行了粗略估算。开展实验时，可以根据实际情况，对馏分体积等参数进行一些调整：

• 硅胶用量以硅胶质量：样品质量 = 20:1 为基准（分离难度大时可提高至 50:1 或 100:1）；柱高固定为 6 英尺（约 15 cm），以保证足够的分离行程（柱过高会导致谱带扩散重叠，柱过短则比表面积不足）。
• 硅胶规格：40–63 μm（230–400 目）硅胶 60——该粒径在快速柱条件下分辨率最优；粒径 > 63 μm（如 63–200 μm）分辨率显著下降，粒径 < 40 μm 则无进一步提升（Still et al., 1978）。

4. 装柱

• 在柱底放入少量棉花，轻轻压实（不可过紧，否则阻碍溶剂流通）；铺上约 2 mm 厚石英砂，使底部平整。
• 将硅胶与洗脱溶剂混合成流动性良好的浆料，经漏斗一次性倒入色谱柱（避免分批加入产生分层）；用少量溶剂冲洗漏斗和柱壁残余硅胶。
• 对柱体施加压力并开启旋塞，同时轻敲柱壁，使硅胶层均匀沉降、表面平整——气泡和不均匀的硅胶床是分离失败的最常见原因，务必在加样前消除。
• 硅胶沉降均匀后，用移液管冲洗柱壁残余硅胶，再铺一层薄沙（约 1–2 mm）保护硅胶表面；将溶剂液面降至刚好高于硅胶表面，关闭旋塞。
• 注意： 自始至终保持溶剂液面高于硅胶顶部，严禁让柱子跑干。

5. 样品上样

• 溶液上样（首选）： 将样品溶于尽量少量的洗脱溶剂（若溶解困难，可加数滴二氯甲烷助溶，但所用溶剂极性不得高于洗脱溶剂，否则样品带会扩散变宽，分离度下降）。沿柱壁缓缓滴加样品溶液，勿扰动沙层；将液面降至刚高于硅胶表面，用少量溶剂润洗上样瓶，同法加入；重复润洗 1–2 次。正确上样后，样品应以紧密细带的形式集中在沙层正下方。
• 干法上样（样品难溶时）： 将样品溶于二氯甲烷，加入少量硅胶，旋蒸除去溶剂至得到自由流动的粉末（若仍结块，需补加硅胶重复操作）；将粉末铺在柱顶沙层上，再盖一层薄沙后加入洗脱溶剂。
• 样品上柱后须立即开始洗脱，中途不应停止。

6. 加压洗脱与馏分收集

• 安装加压装置，将洗脱速率控制在溶剂液面以 2.0 in/min（约 5 cm/min）的速率下降（此流速经 Still et al. 系统优化，为 40–63 μm 硅胶的最佳流速；过慢流速会明显降低分辨率）；调节压力至液面匀速下降，避免过慢（逐滴）或过快（失控喷流）。
• 按对应柱径的馏分体积连续收集；随时补充洗脱溶剂，保持液面不低于硅胶顶部；全程勿离开，专注监控液面。
• 通常于 10–15 min 内完成洗脱；目标组分一般集中在第 5–15 管馏分中（Zakarian Group）。

7. TLC 点板检测与馏分合并

• 每隔数管取一个馏分点 TLC（可在一块板上同时点 5–6 个馏分），用与分析柱分离时相同或分离效果更好的溶剂体系展开，以确认组分分布。有色组分可直接目视；无色组分需紫外灯或显色剂辅助检测。
• 将含有同一纯净组分的馏分合并于圆底烧瓶中（含有多个组分的混合馏分单独收集留作进一步处理）；用少量溶剂润洗试管 2–3 次，洗涤液并入主馏分。
• 旋蒸除去溶剂，完成纯化。

8. 色谱柱的清洗与处理

• 确认目标产品已完全收集后，向柱中加入乙醇，施加气压将其推过整柱，至硅胶呈干燥自由流动状态，再倒入指定废固容器。
• 也可直接将柱中的液体加压压干（需要长时间加压直到液体完全排干）并收集废液。然后，将压干后的硅胶粉倒入指定容器。
• 若样品中无极性基线杂质残留，可用 100% 乙酸乙酯冲洗一遍，再用 100% 正己烷冲洗一遍（溶剂体积约为硅胶体积的 110–120%）后重复使用该柱（Zakarian Group）；若有基线杂质或不确定柱的状态，应重新装柱。

注（洗脱方式差异）： MIT 视频教程建议在分离极性差异较大的混合物时可逐步提高溶剂极性（梯度洗脱）；Zakarian Group 教程明确要求仅使用等度体系（单一浓度），通过切换至不同等度体系代替连续梯度。本 SOP 采用等度/等度切换方式，原因是连续梯度洗脱易引起谱带扩散、重现性差，不利于标准化操作；如遇极性跨度极大的混合物，可在前一组分完全洗出后切换溶剂体系。

1. Safety

• Caution: Silica and alumina are highly toxic when inhaled — all weighing and column-packing steps must be performed in the fume hood.
• Dispose of spent silica in a designated solid waste container. Never discard any fraction until the desired material has been confirmed and recovered.

2. Solvent System Selection (TLC Screening)

• Dissolve a small amount of the sample and spot several TLC plates developed in different solvent systems. The adsorbent on the TLC plates must match that used in the column (silica or alumina).
• Adjust solvent polarity so that the target compound has Rf ≈ 0.3 — this value balances speed and resolution; a lower Rf indicates insufficient polarity and excessively long elution, while a higher Rf indicates overly polar conditions that cause rapid, poorly resolved elution.
  • When separating two or more compounds, adjust solvent polarity so that the midpoint between spots is at Rf ≈ 0.3.
  • For mixtures with large polarity differences, begin with a solvent system that places the least polar compound at Rf ≈ 0.3, then switch to a new isocratic system targeting the next compound once the first has fully eluted (isocratic switching, not gradient elution).
  • Common solvent systems: ethyl acetate/petroleum ether for most compounds; methanol/DCM for highly polar compounds; diethyl ether/pentane for volatile compounds requiring low-boiling solvents.
• Caution (solvent polarity): If the target compound reaches Rf = 0.3 with ≤ 2% of the polar component (e.g., 1% EtOAc/petroleum ether), further reduce polarity (e.g., use 0.5% EtOAc) to prevent premature elution from the column.

3. Column and Silica Selection

• Select column diameter and recommended fraction volume based on sample scale。 The table below provides general guidelines adapted from the Zakarian Group tutorial, which is based on common column sizes used in our lab. Adjust fraction volumes as needed based on actual flow rates and resolution observed during the run:

• Use a silica-to-sample mass ratio of 20:1 as a baseline (increase to 50:1 or 100:1 for difficult separations); pack to a fixed bed height of 6 in (~15 cm) — a taller column causes band dispersion and overlap, while a shorter column lacks sufficient surface area for adequate resolution.
• Silica specification: 40–63 μm (230–400 mesh) silica gel 60 — this particle size gives optimal resolution under flash conditions; coarser grades (63–200 μm) give significantly poorer resolution, while finer grades (<40 μm) offer no improvement (Still et al., 1978).

4. Column Packing

• Place a small plug of cotton at the column outlet and press it gently into place (do not overpack, as this will impede solvent flow); add a thin layer of sand (~1–2 mm) to level the base.
• Prepare a mobile slurry of silica in the chosen eluent and pour it into the column in a single portion through a funnel (adding silica in batches causes stratification); rinse residual silica from the funnel and column walls with a small amount of solvent.
• Apply pressure and open the stopcock, tapping the column walls gently to achieve a flat, uniform silica bed — bubbles and inhomogeneities in the silica bed are the leading cause of failed separations and must be eliminated before sample loading.
• Once the bed is uniform, rinse residual silica from the column walls with a pipette, add a protective sand layer (~1–2 mm) on top, then drain the solvent to just above the silica surface and close the stopcock.
• Caution: Maintain the solvent level above the top of the silica at all times. Never allow the column to run dry.

5. Sample Loading

• Solution loading (preferred): Dissolve the sample in the smallest possible volume of the chosen eluent (a few drops of DCM may be added to aid dissolution if needed, but the loading solvent must be no more polar than the eluent — a more polar loading solvent causes band broadening and poor resolution). Drip the solution slowly down the column wall without disturbing the sand layer; drain to just above the silica surface, then rinse the sample vial with a small volume of fresh eluent and add in the same manner; repeat 1–2 times. When loaded correctly, the sample should appear as a compact, narrow band just below the sand layer.
• Dry loading (for poorly soluble samples): Dissolve the sample in DCM, add a small quantity of silica, and remove solvent by rotary evaporation until a free-flowing powder is obtained (if the mixture remains clumpy, redissolve and add more silica); apply the powder to the top of the sand layer, cover with a thin sand layer, then add eluent carefully.
• Once the sample is loaded, begin elution immediately without interruption.

6. Pressurized Elution and Fraction Collection

• Apply pressure and regulate flow so that the solvent surface drops at 2.0 in/min (~5 cm/min) — this flow rate was established by systematic optimization by Still et al. (1978) as optimal for 40–63 μm silica; slower flow rates demonstrably reduce resolution. Adjust pressure to achieve steady, controlled flow — neither a slow drip nor an uncontrolled stream.
• Collect fractions continuously at the volume specified for the column diameter; replenish eluent frequently to keep the solvent level above the silica surface; remain attentive throughout the run.
• Elution is typically complete within 10–15 min; the target compound generally elutes in fractions 5–15 (Zakarian Group).

7. TLC Monitoring and Fraction Pooling

• Spot TLC plates at regular intervals (5–6 fractions per plate); develop with a solvent system that spreads spots well (need not be the same as the column eluent). Colored compounds can be tracked visually; colorless compounds require UV lamp or staining reagents.
• Pool fractions containing the same pure compound into a round-bottom flask (set aside mixed fractions for further purification); rinse each test tube 2–3 times with a small volume of clean solvent and combine the rinses with the pooled fractions.
• Remove solvent by rotary evaporation to complete the purification.

8. Column Cleanup and Disposal

• Once all desired material has been collected, fill the column with ethanol and push air through until the silica is dry and free-flowing, then pour into the designated solid waste container.
• You can also directly pressurize the column to dry the liquid (requires prolonged pressurization until the liquid is completely removed) and collect the waste liquid. Then, pour the dried silica powder into the designated container.
• If no baseline impurities were present in the original sample, the column may be reused after sequential washes with 100% ethyl acetate then 100% hexanes (each wash volume ~110–120% of silica volume) (Zakarian Group); if the history of the column is uncertain or baseline impurities were loaded, repack with fresh silica.

Note (elution mode): The MIT laboratory video recommends stepwise increases in solvent polarity (gradient elution) for mixtures with widely differing polarities. The Zakarian Group protocol explicitly requires isocratic systems only, substituting continuous gradients with discrete isocratic switches. This SOP follows the isocratic/isocratic-switching approach, as continuous gradient elution promotes band broadening and poor reproducibility. For mixtures with extreme polarity differences, switch to a new isocratic system only after the preceding compound has fully eluted.

Ref:

• Still, W. C.; Kahn, M.; Mitra, A. Rapid chromatographic technique for preparative separations with moderate resolution. J. Org. Chem. 1978, 43, 2923–2925. DOI: 10.1021/jo00408a041.
• Thompson, W. J.; Hanson, B. A. An inexpensive, foolproof apparatus for flash chromatography. J. Chem. Educ. 1984, 61, 645.
• Zakarian Group. How to Do Flash Column Chromatography in 15 Minutes; UCSB Technical Notes.
• MIT OpenCourseWare. Column Chromatography. MIT Digital Lab Techniques Manual. https://ocw.mit.edu.