Flash chromatography column (Flash Column) is a fast and (usually) easy method to separate complex mixtures. In some experiments, we need to perform a relatively large amount of flash chromatography column to separate about 1g of material.
The principle of chromatography column and thin layer chromatography is the same, but it can be used for the separation of preparative substances. Because we use compressed air to push the solvent through the column, it is called flash chromatography column. This not only makes the separation effect better but also shortens the column time.
Preparation and operation of flash chromatography column:
1. Determine the weight of the dry, solvent-free mixture be separated.
2. Use thin layer chromatography to select the solvent system so that the value of Rf is between 0.2 and 0.3, but if the mixture is complex, this may be unrealistic. In more complicated cases, you may need to use gradient elution. Simply put, it is to continuously increase the polarity of the solvent during the purification and elution process. However, in thin layer chromatography, you must determine which solvent system Will make the different spots in the range of Rf 0.2 ~ 0.3.
3. Determine the method used to load the sample onto the column. You have three choices: clean sample method, solution method, or silica gel adsorption method.
a.Net sample method: If the sample is a non-viscous oil, it is easiest to use the net sample method. You can use a long dropper filter to introduce the liquid into the column, and then rinse with a predetermined solvent system to wash all the components into the chromatography column.
b.Solution method: The clean sample method may sometimes cause the separation column to break. Therefore, for liquids and solids, the more common method is to dissolve the sample in a solvent and then add the solution to the separation column.
The most ideal state is that the Rf of all components in the mixture in the solvent system (usually pentane or hexane) is zero. This is difficult to achieve in most cases, so you can choose a solvent that moves only one compound in the mixture, or you can simply use the eluent of your choice. Remember: the latter two options are risky for difficult separation and purification.
c.Silica gel adsorption method: The last technique is to deposit (adsorb) compounds onto silica gel, which is useful for some liquids and all solids.
Note: Silica gel is acidic, so this step will destroy some acid-sensitive compounds, which usually need to be regenerated on the silica column.
First, dissolve the mixture in methylene chloride in a round bottom flask and add silica gel (the mass of silica gel is about twice the mass of the compound). The solution was concentrated on a rotary evaporator.
Note: Silica gel is a very fine powder and can easily be sucked into the rotary evaporator.
Plug the connector or pump protection device with glass wool to prevent solids from being sucked into the pump. Fast turning can also avoid this problem. When the solids are basically dry (when most of the solids fall off the wall of the container, the solids are already dry), remove the flask from the rotary evaporator, and then use a vacuum pump to exhaust the solvent (assuming there are no volatile substances in the mixture).
Note: Use glass wool to plug the vacuum pump connector, otherwise you may find silica gel (and your compound) enter the vacuum tube and deposit there. Once it is completely dry (no more bubbles in the solid), remove the flask from the vacuum system and scrape the solid from the wall with a clean spatula. Now, you can simply use the powder funnel to add this solid to the top of the separation column, and then rinse with the eluent (1.5mL each time).
4. Determine the appropriate ratio of silica gel to the compound. For simple separation, the ratio of the two is usually 30~50:1 (weight ratio); but for the more difficult separation, the ratio is as high as 120:1.
5. Choose a suitable flash separation column. The amount of silica you need determines the size of the separation column. Whether to use a short and thick or long and thin separation column.
6. Select the appropriate test tube for collection. This is also a good opportunity to consult experienced colleagues. But there is also a simple method: divide the volume of silica gel by 4, and then select a test tube that can hold this volume. (200mL silica gel corresponds to 50mL component).
7. Once you have selected the separation column, you need to block the bottom end of the piston to avoid the loss of silica gel. Usually, it can be done with a small ball of cotton or glass wool plus a long stick or glass rod.
8. Pack the flash separation column in the fume hood. Considering the use of a large number of volatile solvents and the health hazards of dry silica gel, it is not allowed to operate the flash column outside the fume hood. Check to make sure that the flash column is completely vertical. The inclined column is not conducive to separation.
9. Close the piston and add a few inches of eluent.
10. Use the funnel to add some sand (dry and washed) to the flash separation column. The purpose is to spread a thin layer of sand (no more than 1cm) on the plug, so as to prevent silica gel from falling into the collection bottle.
11. Measure the appropriate amount of silica gel. The safest way is to measure it in a fume hood. The density of silica gel is about 0.5 g/mL, so it can be measured directly in an Erlenmeyer flask (100g=200mL). Do not let the volume of silica gel exceed 1/3 of the flask, because we have to add solvent to it.
12. Add at least 1.5 times the volume of solvent to the newly measured silica gel, make it into a slurry, vigorously shake and stir it to make it fully mixed, and remove the gas in the silica gel (the presence of bubbles will make the flash separation column, the efficiency is greatly reduced).
13. Use the powder funnel to carefully and slowly move the slurry into the flash separation column, taking care not to damage the sand layer below. Pay attention to stop and shake the slurry from time to time during the grouting process to ensure that the silica gel is evenly mixed. After grouting, rinse the flask several times with the eluent and add the remaining solvent silica gel mixture to the separation column.
14. Use a dropper and eluent to rinse the silica gel stuck on the top edge of the flash column into the solvent layer.
15. When all the silica gel has been washed away from the flash column wall, open the piston and pressurize the column with compressed air. The silica gel in the column will be compressed to about half of its original height. Check to make sure that the top section of the column is flat. If it is not, it must be stirred again and then settled down. Under pressure, add excess eluent and tap the column gently with a pencil tip or rubber stopper. This will make the silica particles packed more tightly.
Collect all the eluate from the flash column and reuse it after adding the compound.
Note: Remember not to let the solvent level be lower than the filling layer.
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.