Summary of Core Content on Silica Gel Column Chromatography

1. Separation Principle

Silica gel column chromatography achieves separation based on the difference in adsorption capacity of substances on silica gel. Substances with higher polarity are easily adsorbed by silica gel, while those with lower polarity are not. The entire process is a cycle of "adsorption → desorption → re-adsorption → re-desorption".

2. Mobile Phase Selection

Different solvent systems are matched according to the polarity of substances, and tailing problems can be solved in a targeted manner:

•For substances with low polarity: Ethyl acetate : petroleum ether system is used.

•For substances with relatively high polarity: Methanol : chloroform system is used.

•For substances with high polarity: Methanol : water : n-butanol : acetic acid system is used.

•When tailing occurs: A small amount of ammonia water or glacial acetic acid is added.

3. Core Operating Steps (8 Steps)

1.Weighing: Select 200-300 mesh silica gel, and the dosage is 30-70 times the sample loading volume. For extremely difficult separation, 100 times the amount of silica gel H is used (the apparent density of dry silica gel is about 0.4; for example, 40g of silica gel can be measured as 100ml with a measuring cup).

2.Making Slurry: Add a solvent with the same volume as dry silica gel and stir (e.g., if the eluent is a petroleum ether/ethyl acetate/acetone system, mix with petroleum ether; if it is a chloroform/alcohol system, mix with chloroform). The solvent must be dry (anhydrous sodium sulfate is used for ethyl acetate/acetone, and anhydrous calcium chloride is used for chloroform). For acid-sensitive samples, avoid using the chloroform system.

3.Column Packing: Tightly plug the bottom of the column with cotton, add 1/3 volume of petroleum ether (or chloroform), install a liquid storage bulb, open the piston, pour the slurry into the column at one time, and rinse the residual silica gel in the liquid storage bulb with solvent.

4.Compacting: After the silica gel settles, add more petroleum ether, and pressurize with a double bulb or air pump until the flow rate is constant. The column bed is compressed to 9/10 of its original volume (this can improve resolution and prevent column bed cracking).

5.Sample Loading: Both dry and wet methods can be used, and sea sand is not required. After sample loading, add a small amount of eluent, and then plug a ball of absorbent cotton close to the surface of the silica gel to prevent the silica gel from being damaged by the subsequent addition of eluent.

6.Column Passing and Collection: Gradient elution is recommended (to balance diffusion and separation). Example: For a sample loading of 10mg, 0.5ml per fraction is used; for a sample loading of 1-2g, 20-50ml per fraction is used.

7.Detection: Special spray developing agents are preferred (their sensitivity is 1-2 orders of magnitude higher than that of UV lamps, reducing product loss).

8.Spectroscopy Submission: Collect the product, spin it to dryness, and recrystallize it before submitting for spectroscopy. When the sample is small or in liquid form, pass it through a small gel column to remove the "silica gel peak" around 1.5ppm in the hydrogen spectrum.

4. Operational Precautions

•Column Packing: Do not apply lubricant to the piston (to avoid product contamination). The column must be "appropriately tight and uniform", and cracking is prohibited (as it affects separation or even renders the column useless). Small air bubbles can be eliminated by pressurization, and more practice is needed to avoid frequent bubble generation.

•Sample Adding: Dissolve the sample with a small amount of solvent before loading. Wait until the solvent layer drops to the surface of the quartz sand, and repeat the process 2-3 times with a small amount of low-polarity solvent. When adding the eluent, do not apply pressure first; wait until the distance between the solvent and the sample layer reaches 2-4cm before pressurizing to prevent the sample from being quickly carried downward.

•Eluent Selection: The Rf value of the target substance should be 0.2-0.3. When Rf = 0.6, even a difference of 0.2 makes separation difficult. This is because the column undergoes multiple plate climbing processes, and a lower Rf value results in better separation efficiency.

•Sample Collection: Substances with strong adsorption may elute later (while those with weak adsorption elute first). Alumina can be used as the stationary phase instead. The size of the test tube should match the sample volume (use small test tubes for small samples to avoid collecting multiple samples at once).

•Final Treatment: Column-separated products need to be washed with a small amount of solvent (to remove impurities dissolved in the solvent), and recrystallization is necessary if required. Volatile solvents (ether, dichloromethane) or high room temperatures are prone to bubble generation; high-boiling solvents can be used instead. For mixed solvents, those with similar boiling points should be selected (e.g., ethyl acetate + 60-90℃ petroleum ether). Pressurize to exhaust air completely before column packing.

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