16. When the flash chromatography column is filled, add sand on top of the silica gel as a protective layer. The sand layer needs to be filled relatively flat, with a thickness of about 2cm. This acts as a protection for the flash column when the solvent is added-when the solvent is added too fast, if there is no protection from the sand layer, the solvent may damage the flat surface of the filled silica gel (thus affecting the separation effect).
17. Before the solvent reaches the sand layer, compressed air can be used to push the solution layer down.
18. Close the piston and place the first test tube under the outlet of the flash column.
19.Carefully add your compound to the separation flash chromatography column-when adding liquid, make sure to add it along the wall of the flash column, not directly on the top of the flash column. When rinsing the flask containing the mixture, carefully add a dropper of eluent to the separation flash column all at once.
Then open the piston and turn off the piston when the liquid drops to the top section of the filling. Rinse the flask three times in this way. For the mixture deposited on the silica gel, a 2cm thick protective sand layer is also added.
20.Carefully fill the separation flash chromatography column with eluent. The solvent can be added using Pasteur bulbs at the beginning. After adding 1cm of solvent, it is best to open the piston. Continue to drop the solvent with a dropper until the solvent is several centimeters above the packed layer in the flash column.
Now, the solvent can be added from the Erlenmeyer flask through a powder funnel-slowly let it add along the flash chromatography column wall. Be patient and don’t damage the top section of the flash column packing.
21. After filling the separation flash chromatography column with the eluent, you can start “passing the flash column”. Remember that a fast flow rate will make the separation better. Adjust the air pressure to achieve a fast flow rate-but not as fast as a fire bridle!
Maintain the pressure and replace the collection tube with a new tube after it is full. Pay attention to add solvent to the flash column at any time.
22. Use TCL to track the separation process of the flash chromatography column. Collect the sample while performing thin layer chromatography analysis. This may cause some confusion, so if you want to observe the progress of the flash column, you can reduce the pressure (or even completely remove it) from the beginning.
23. When operating gradient elution, first use a solvent to ensure that compounds with larger Rf are eluted from the flash chromatography column first. After they are safely eluted into the collection flask, they can be replaced with a more polar solvent to continue elution.
Note: Gradually increase the polarity of the solvent. Too fast a polarity change may cause the silica gel to split-just like the terrible earthquake scene in the movie, the filling layer inside the flash chromatography column cracks. This will be very detrimental to your separation!
Therefore, increase the polarity by about 5% per 100 mL (or more) of the solvent until the desired solvent is reached. Then, elute with this eluent, Until the target compound is eluted. At this time, you can continue to change the eluent or proceed directly to the next step.
24.When you are sure that all target compounds have been eluted from the flash column, you can pack everything up.
Place a large flask at the bottom of the separation flash column, and then use a clamp to cut off the compressed gas path that communicates with the separation flash chromatography column. Let the gas push the remaining solvent out of the flash column, and then dry the silica gel (it is more difficult to remove silica gel from the separation flash chromatography column unless it is completely dry). For large separation flash columns, this process takes almost an hour.
25.When the separation flash chromatography column is dry, start to combine the components. Use thin layer chromatography to determine which test tube contains the pure sample you want. Combine the components of similar purity in a large round-bottomed flask and concentrate on a rotary evaporator.
For flash chromatography columns that are time-consuming and longer, the eluted components can be combined during the flash column separation process to speed up the process.
26. When the solvent is completely removed, the resulting compound can be analyzed by NMR.