Flash column chromatography, also known as a flash column, is a fast separation method, but it does not sacrifice resolution as others have said. In the general column chromatography process, it is usually accompanied by lateral diffusion, which will greatly reduce the resolution, so when walking the column, you should try to shorten the column chromatography time, especially to avoid the column overnight. The purpose of the flash column, that is, without sacrificing exchange efficiency (adsorption and desorption), use the fastest possible flow rate to reduce this lateral diffusion.
Flash column chromatography is a fast and usually easy method to separate complex mixtures. The principle of flash 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 passing time.
Development of Flash Column
Flash chromatography is a kind of fast chromatographic separation mode, which separates substances by using optimized pre-packaged medium and low-pressure columns. Flash chromatography, which was developed in 1978, is considered to separate by medium and low pressure. And it is a fast and inexpensive technology compared with traditional chromatography, which is widely used in drug research, sample purification, and purification of natural products currently. The characteristics of low-cost and simplicity result in its irreplaceable in the separation and purification.
The most used common filler for flash columns is silica gel. But why silica gel is chosen to be the filler? One of the reasons is the limited choice of fillers using for the chromatographic columns at that time. And another reason is other bonds, such as C8 and C18, are too expensive.
As a consequence, most methods of flash chromatographic application in the literature are developed using silica gel as the separation substrate. Flash chromatography usually is a normal-phase liquid chromatography technology. The size of the sample is usually in the milligram level to a hundred-gram. The flow rate is 10 mL/min to 300 mL/min. And organic solvents with low polarities, such as hexane and ethyl acetate, are used as the mobile phases.
Flash column is not blindly pressurized to achieve the fastest flow rate; the flow rate has certain requirements. Regardless of the thickness of the column, the flow rate is generally adjusted to the rate of eluent reduction in the column is 2cm/min. This is an experience speed, adjustments to it should consider the balance of exchange efficiency and lateral diffusion.
Similarly, VLC (Vacuum Column Chromatography) is also a very good column chromatography technique. It uses TLC’s “multiple unfolding” techniques so that the two samples to be separated immediately overlap (on TLC). Get better separation. Unfortunately, such good technology, some people think that it is used for rough separation.
For synthetic and medicinal chemistry, it is often necessary to screen a large number of potential compounds in the early stage. Once the relevant active compounds are found, the compounds need to be mass-produced. At this time, the previous milligram-level synthetic preparation methods need to be modified and upgraded to develop a new set of synthetic purification preparation process to meet the clinical testing and application of a large number of later compounds. In this process, the process development of compound purification is a very important link; on the other hand, in the field of natural products, when high value-added monomers are found, how to obtain the target monomers in large quantities through purification means has been These are the core problems that need to be solved.
The process development mentioned above for purification and preparation, whether in the field of medicinal chemistry of natural products, is a problem that requires scientists to spend a lot of manpower and material resources to explore and solve. Generally, improvements are made in two aspects: 1. Change the synthetic route and try to use the recrystallization method to reduce the cost; 2. Use column chromatography above 100 grams to prepare purification technology.
In addition to the transformation of the synthetic route of the process, efficient column chromatography amplification technology is also a very critical step. In terms of the linear gradient method used, more solvent is consumed in practical applications. Therefore, the optimization of the method is urgently needed and very necessary. The success of the project is often closely related to this. Through the optimization of the method, we can isolate the most samples with the least time and solvent consumption.
Isocratic or step gradient methods are used for optimization. On the other hand, a column with higher column efficiency can help increase the sample load and speed up the separation.
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 Rf value is between 0.2 and 0.3, but if the mixture is complex, this may not be realistic. In more complicated situations, gradient elution may be required. Simply put, the polarity of the solvent is continuously increased during purification and elution. This technique is described in more detail later. However, in TLC analysis, you must determine which solvent system will make the different spots within the range of 0.2 to 0.3. 3) Determine the method used to apply the sample to the column. You have three choices: net sample method, solution method, or silica gel adsorption method.
a. Net sample method: If the sample is a non-sticky oil, it is easiest to use the net sample method. You can use a long dropper filter to introduce liquid into the column and then rinse with a predetermined solvent system to wash all the 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 all components of the mixture have an Rf of 0 in the solvent system (usually pentane or hexane).
This is difficult to achieve in most cases, so choose a solvent that only moves one compound in the mixture, or you can simply use the eluent of your choice. Remember: the latter two options are risky for more difficult separations and purifications.
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. They usually need to be regenerated on the silica gel column.
First, dissolve the mixture in dichloromethane 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 be easily drawn into the rotary evaporator.
Use glass wool to plug the connector or the pump’s protective device to prevent solids from being drawn into the pump. Quick rotation can also avoid this problem. When the solids are substantially dry (when most solids fall off the wall of the container, indicating that the solids have dried), remove the flask from the rotary evaporator and use a vacuum pump to exhaust the solvent (assuming there are no volatile substances in the mixture).
Note: Plug the vacuum pump connector with glass wool, 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 off the wall with a clean spatula. Now, you can simply use a powder funnel to add this part of the solid to the top of the separation column, and then rinse with eluent (1.5mL each time).
Contains 5 simple steps
TLC spot plate to test various mobile phase combinations; use a column with higher column efficiency; create a linear gradient method based on the TLC spot plate information; based on the results of the previous gradient method, use the gradient method to optimize; gradually increase the loading volume, to find the best sample loading range, while ensuring that the linear velocity of the method does not change.
This method can greatly reduce the time and solvent consumption, thereby reducing the cost of the final product. The use of the step gradient method greatly increases the efficiency of scale-up production, while reducing the consumption of solvent and time costs. On the other hand, for reversed-phase preparation, the optimization of the five-step method is also applicable.