Guide to Use Flash Chromatography Column
Flash column chromatography is a fast and (usually) easy way to separate complex mixtures. Perform a relatively large amount of flash chromatographic separation. The principle of column chromatography 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 column chromatography. This not only makes the separation effect better, but also shortens the column time.
Preparation and operation of flash column chromatography
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, gradient elution may be needed. Simply put, it is to continuously increase the polarity of the solvent during the purification and elution process. This technique is described in more detail later. But in TLC analysis, you must determine which solvent system will make the different spots in the Rf range of 0.2 ~ 0.3.
3. Determine the method used to load the sample onto the column. You have three choices: the clean sample method, the solution method or the silica gel adsorption method.
a. Clean sample method
If the sample is a non-viscous oil, it is easiest to use the clean 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 components into the 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 dichloromethane in a round bottom flask and add silica gel (the mass of silica gel is approximately twice the mass of the compound). The solution was concentrated on a rotary evaporator.
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 dry), remove the flask from the rotary evaporator, and then use a vacuum pump to exhaust the solvent (assuming that there are no volatile substances in the mixture).
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 part of the solid to the top of the separation column, and then rinse with the eluent (1.5 mL each time).
4. Determine the appropriate ratio of silica gel compounds. For simple separation, the ratio between 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 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 has not yet been determined.
6. Select the appropriate test tube for collection. The simpler method: divide the volume of silica gel by 4, and then select a test tube that can hold this volume.
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.
8. Pack the separation column in a fume hood.
9. Close the piston and add a few inches of eluent.
10. Use a funnel to add some sand (dry and washed) to the 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 and appropriate amount of silica gel, the safest way is to measure in a fume hood
12. Add at least 1.5 times the volume of solvent to the silica gel just measured, make it into a slurry, vigorously shake and stir to make it fully mixed, and remove the gas in the silica gel
13. Use the powder funnel to carefully and slowly move the slurry into the 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 to the top edge of the column into the solvent layer
15. When all the silica gel has been washed away from the 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 flat, you must re-bid and then settle down. Under pressure, add excess eluent and tap the column gently with a pencil tip or rubber stopper, which will make the silica particles packed more tightly.
16. When the column is filled, add sand on top of the silica gel as a protective layer. The thickness is about 2cm.
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 column.
19. Carefully add your compound to the separation column. When rinsing the flask containing the mixture, carefully add one dropper of eluent to the separation column at a time. 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.
20. Carefully fill the separation column with eluent.
21. After filling the separation column with the eluent, you can start “passing the column”.
22. Use TCL to track the separation process of the column.
23. When operating gradient elution, first use a solvent to ensure that compounds with larger Rf are eluted from the column first. After they are safely eluted into the cell phone flask, they can be eluted with a more polar solvent.
24. When you are sure that all target compounds have been eluted from the column, you can pack everything up.
25. When the separation column is dry, you can start to combine the components.
26. When the solvent is completely removed, the resulting compound can be analyzed by NMR.